Acta Neuropathologica

, Volume 121, Issue 4, pp 431–443 | Cite as

Perivascular drainage of solutes is impaired in the ageing mouse brain and in the presence of cerebral amyloid angiopathy

  • Cheryl A. Hawkes
  • Wolfgang Härtig
  • Johannes Kacza
  • Reinhard Schliebs
  • Roy O. Weller
  • James A. Nicoll
  • Roxana O. Carare
Original Paper

Abstract

The deposition of amyloid-β (Aβ) peptides in the walls of leptomeningeal and cortical blood vessels as cerebral amyloid angiopathy (CAA) is present in normal ageing and the majority of Alzheimer’s disease (AD) brains. The failure of clearance mechanisms to eliminate Aβ from the brain contributes to the development of sporadic CAA and AD. Here, we investigated the effects of CAA and ageing on the pattern of perivascular drainage of solutes in the brains of naïve mice and in the Tg2576 mouse model of AD. We report that drainage of small molecular weight dextran along cerebrovascular basement membranes is impaired in the hippocampal capillaries and arteries of 22-month-old wild-type mice compared to 3- and 7-month-old animals, which was associated with age-dependent changes in capillary density. Age-related alterations in the levels of laminin, fibronectin and perlecan in vascular basement membranes were also noted in wild-type mice. Furthermore, dextran was observed in the walls of veins of Tg2576 mice in the presence of CAA, suggesting that deposition of Aβ in vessel walls disrupts the normal route of elimination of solutes from the brain parenchyma. These data support the hypothesis that perivascular solute drainage from the brain is altered both in the ageing brain and as a consequence of CAA. These findings have implications for the success of therapeutic strategies for the treatment of AD that rely upon the health of the ageing cerebral vasculature.

Keywords

Alzheimer’s disease Amyloid-β Cerebral vasculature Perivascular drainage Basement membranes Cerebral amyloid angiopathy 

Introduction

Cerebral amyloid angiopathy (CAA) results from the deposition of amyloid-β (Aβ) peptides in the walls of leptomeningeal and cortical arteries and capillaries and is present in the majority of patients with Alzheimer’s disease (AD) [28]. The vascular Aβ deposits, which are also found in an estimated 30–40% of non-demented elderly individuals, are associated with capillary thinning and vessel tortuosity, vasoconstriction, intraluminal thickening, inhibition of angiogenesis and the death of pericytes, endothelial and smooth muscle cells [9, 22, 39, 49, 61]. CAA is also associated with cerebral hypoperfusion, microhaemorrhages and cognitive impairment [15, 44, 50, 51, 56].

While the aetiology of sporadic CAA is unclear, it is likely caused by the failure of Aβ elimination from the brain parenchyma. Aβ is degraded by enzymes and removed from the brain by multiple mechanisms, including receptor-mediated transport across the endothelium, uptake by microglia and macrophages and, from its distribution in CAA, by bulk flow along the basement membranes of capillary and artery walls [4, 69]. With ageing and in AD, enzymatic degradation, absorption of Aβ into the blood and microglial uptake have been reported to be reduced or dysfunctional [19, 35, 41]. However, to date, the effects of ageing on the perivascular drainage of solutes, and the implications for the development and progression of CAA, have not been examined.

Basement membranes are thin sheets of highly specialized extracellular matrix that, outside the brain, are typically found at the epithelial-mesenchymal interface and regulate cell growth, differentiation and migration [3, 18, 62]. Cerebrovascular basement membranes are composed of laminins, nidogens and collagen IV, as well as heparan sulphate proteoglycans such as perlecan, agrin and fibronectin [21]. Multiple lines of evidence suggest that basement membranes may play a role in the aetiology of CAA. Kinetic and biophysical studies have shown that laminin, collagen IV and nidogen interact directly with Aβ to prevent its aggregation and promote disaggregation of pre-formed fibrils, whereas agrin and perlecan induce and stabilize Aβ fibrillization [7, 8, 13, 17, 31, 32, 43]. Transgenic mice overexpressing transforming growth factor-β1 (TGF-β1) have thickened basement membranes preceding the development of murine CAA [45, 72]. Furthermore, thickening, reduplication and vacuolization of basement membranes, along with upregulation of heparan sulphate proteoglycans, have been reported in the AD brain [49, 55, 67]. Collectively, these findings indicate that alterations in the composition, level of expression or morphology of cerebrovascular basement membranes may disturb perivascular drainage of Aβ from the parenchyma and initiate deposition of Aβ within the walls of the cerebral vasculature. Moreover, clearance of Aβ and other toxic metabolites from the brain may be further impeded by the accumulation of vascular Aβ deposits as CAA.

In the present study, we tested the hypothesis that ageing of cerebral blood vessels and the development of CAA alter the pattern of perivascular drainage of solutes from the brain in naïve mice and in the Tg2576 mouse model of AD. We report that clearance of small molecular weight dextran is impaired in the hippocampal microvasculature of 22-month-old wild-type mice compared to 3- and 7-month-old animals. This was associated with age-dependent changes in both capillary density and basement membrane protein composition. The presence of CAA affected the drainage of dextran mainly in the walls of veins of 22-month-old Tg2576 mice, compared to younger mice without vascular Aβ deposits. Dextran also bound directly to parenchymal and vascular Aβ in aged Tg2576 mice. These data support the hypothesis that perivascular drainage is altered both in the ageing brain and as a consequence of the development of CAA. Our findings have implications for the success of therapies that rely on the diffusion and clearance of parenchymal Aβ via bulk flow movement along perivascular drainage pathways in the elderly brain. There are also wider consequences related to the kinetics of drug distribution within the elderly and AD brain.

Materials and methods

Animals

Male and female Tg2576 mice expressing the human Swedish (K670N, M671L) APP mutations were kindly provided by Dr. Karen Ashe (University of Minnesota, USA) and maintained on an outbred C57BL6 background. For perivascular drainage experiments, 3-, 7- and 22-month-old Tg2576 mice or age-matched wild-type littermates were used (n = 6/group). A separate group of 3-, 8- and 16-month-old BalbC mice (n = 5/group) was used for immunoblotting experiments. All experiments were performed in accordance with animal care guidelines stipulated by the Animal Care and Use Committees at the Universities of Leipzig and Southampton and conformed to UK Home Office regulations and to the European Communities Council Directive (86/609/EEC).

Intracerebral injections

Tg2576 mice and wild-type littermates were anaesthetized with an intraperitoneal injection of etomidate (Hypnomidate; 33 mg/kg body weight; Janssen-Cilag, Neuss, Germany). Additionally, local anaesthesia of the skull was achieved with a subcutaneous injection of lidocaine hydrochloride (Licain; 1%; 17.5 mg/kg body weight; DeltaSelect, Pfullingen, Germany). All animals received a stereotaxic injection of 0.7 μL FITC-conjugated 10 kDa dextran amine (Sigma-Aldrich, Dorset, UK; 1 mg/ml in sterile PBS) into the left dorsal hippocampus over a 4-min period (coordinates from Bregma: AP = −2 mm; ML = 1.8 mm and DV = 1.6 mm). Injection capillaries were left in situ for 2 min to allow for diffusion of dextran in the brain parenchyma and mice were killed 15 min post-injection.

Immunohistochemistry

Mice were deeply anaesthetised with an overdose of sodium pentobarbital, intracardially perfused with 0.1 M phosphate buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde, brains were post-fixed overnight, cryoprotected in 30% sucrose and sectioned at 10 μm. For thioflavin S staining, sections were treated with 1% thioflavin S for 5 min, differentiated twice in 70% ethanol, washed in PBS and coverslipped with anti-fading solution (Vector Labs, Peterborough, UK). For Aβ immunolabelling, tissue sections were incubated for 15 min with 3% hydrogen peroxide, treated with 70% formic acid (5 min), blocked with 15% normal goat serum and incubated overnight at 4°C with anti-Aβ17–24 (clone 4G8; 1:500; Millipore, Watford, UK). For glucose transporter-1 (glut-1) immunostaining, sections were blocked in 3% hydrogen peroxide, followed by 15% normal goat serum and incubated overnight with anti-glut-1 (1:750; Merck, Nottingham, UK). Sections were washed with PBS, incubated with anti-mouse or anti-rabbit horseradish peroxidase conjugates (1:400; Vector Labs, Peterborough, UK) and developed with nickel-enhanced diaminobenzidine as chromogen. For double immunofluorescence labelling, sections were blocked in 15% goat serum and incubated overnight at 4°C with rabbit anti-laminin (1:500; Sigma-Aldrich, Dorset, UK) and anti-α smooth muscle actin (1:300; Sigma-Aldrich). The next day, sections were rinsed in PBS, developed with the appropriate fluorescently conjugated secondary antibodies (1:200; Invitrogen, Paisley, UK) and coverslipped with anti-fading solution (Vector Labs). For double labelling of Aβ and laminin, brain tissue sections were incubated overnight with anti-laminin (1:500), developed with AlexaFluor 546-conjugated anti-rabbit (1:200), treated for 2 min with 70% formic acid and washed in PBS before being incubated overnight with anti-Aβ17–24 IgG (1:100). Sections were developed the following day with AlexaFluor 633-conjugated anti-mouse (1:200). Photomicrographs were captured using a Lexica SP2 confocal laser scanning microscope and exported to Photoshop CS or Image J imaging software (NIH, Maryland, USA).

Isolation of cortical blood vessels

Mice were deeply anaesthetised with an overdose of sodium pentobarbital, intracardially perfused with 0.1 M PBS (pH 7.4), and the brains rapidly removed and the cerebral cortex removed. To isolate blood vessels, cortical tissues were mechanically disrupted in 500 μL of 0.1 M NH4CO3 + 7% sodium dodecyl sulphate (SDS) (plus protease inhibitor cocktail set III, Merck) and agitated for approximately 4 h. Capillaries were separated from large-diameter vessels by passing the vascular tufts through a 10 μm nylon mesh filter and collected in the filtrate. Large-diameter vessels were suspended in RIPA lysis buffer [20 mM Tris–HCl (pH 8.0), 150 mM NaCl 1 mM EDTA, 1% Igepal, 0.1% SDS, 50 mM NaF, 1 mM NaVO3] sonicated, aliquoted and stored at −80°C.

Immunoblotting

For laminin and fibronectin blots, samples were diluted in NuPAGE® LDS sample buffer (Invitrogen) containing 2.5% β-mercaptoethanol and heated at 75°C for 5 min. Collagen samples were prepared without β-mercaptoethanol or heat. For the detection of perlecan, samples (30 μg) were digested with 5 mU heparinase III (Sigma-Aldrich) in the presence of protease inhibitor cocktail set for 2 h at 37°C before loading. Proteins were separated by gel electrophoresis on 3–8% Tris–acetate gels (15 μg/lane; Invitrogen) and transferred to nitrocellulose membranes. Membranes were blocked for 1 h at room temperature with 8% non-fat milk powder and incubated overnight at 4°C with anti-collagen IV (1:2,500; Abcam, Cambridge, UK), anti-laminin (1:500), anti-fibronectin (1:3,000; AbD Serotec, Kidlington, UK) or anti-perlecan (1:750; Millipore) antibodies. Membranes were developed using an enhanced chemiluminescence detection kit, stripped and reproved with anti-glyceraldehyde-3-PDH (GAPDH) antibody (1:50,000; Sigma-Aldrich) to ensure equal protein loading.

Quantification and statistical analysis

The number of blood vessels containing dextran was quantified from five images per mouse throughout the hippocampus using Zeiss Image Analysis KS-400 software. The number and diameter of dextran-positive blood vessels in a 574 μm × 689 μm area were documented manually. Vessels were categorized as: (1) capillaries if their diameters were less than 10 μm, (2) arteries if their diameters were greater than 10 μm and positive for α smooth muscle actin or (3) veins if the diameters were greater than 10 μm and the vessels lacked α-smooth muscle actin immunoreactivity [63]. Histograms were compiled using mean ± SEM values and analysed using one-way analysis of variance (ANOVA) with Newman–Keuls post hoc test, significance set at p < 0.05. For quantification of glut-1, micrographs were converted to binary images (4 sections/mouse), evaluated by densitometry using Image J software and evaluated using one-way ANOVA with Newman–Keuls post hoc test (significance p < 0.05). Immunoblots were quantified by densitometry as an optical density ratio of protein levels normalized to GAPDH levels and analysed using repeated measures two-way ANOVA with Bonferroni post hoc test (significance set at p < 0.05).

Results

CAA development in Tg2576 mice

The profile of Aβ deposition in the parenchyma and vasculature of the Tg2576 mice has been well described and begins around 2–3 months of age with a rise in the levels of soluble Aβ that increase rapidly around 7 months. Thioflavin S-positive deposits can be detected from 9 months onwards [14, 20, 30]. To confirm the presence or absence of CAA in our tissues, brain sections from 3-, 7- and 22-month old Tg2576 mice were stained with thioflavin S and an antibody against Aβ17–24. No Aβ deposits were noted in either the brain or vasculature of 3-month-old Tg2576 mice (Fig. 1a, d). Some Aβ immunoreactivity was observed in leptomeningeal arteries in 7-month-old brains (Fig. 1b, e), while 22-month-old mice showed both thioflavin S- and Aβ-positive plaques in the parenchyma and vasculature (Fig. 1c, f). No Aβ deposits were found in wild-type mice at any age (data not shown).
Fig. 1

Age-dependent development of cerebral amyloid angiopathy (CAA) in Tg2576 mice. Brain tissue sections from 3- (a and d), 7- (b and e) and 22-month-old (c and f) Tg2576 mice showing progressive CAA in anterior cerebral arteries in the cingulate and motor cortices above the corpus callosum. Thioflavin S staining (ac) detected fibrillar Aβ in the parenchyma and cerebral vasculature of 22-month-old mice, while Aβ17–24 immunohistochemical labelling (df) showed Aβ in the blood vessel walls of 7- and 22-month-old mice. Scale barsac 100 μm; df 200 μm

Effect of age and CAA on perivascular drainage of solutes

To determine whether perivascular drainage is altered in the ageing brain and in the presence of CAA, FITC-conjugated soluble fixable dextran was injected into the hippocampi of 3-, 7- and 22-month-old Tg2576 mice and wild-type littermates. In agreement with previous reports [12], dextran co-localized with laminin-positive basement membranes in both wild-type and transgenic mice at all ages (Fig. 2a, d). In accordance with their respective roles in perivascular drainage of solutes, dextran was identified primarily in the walls of capillaries and arteries, with minimal amounts of dextran labelling of veins in wild-type mice. High power examination showed a smooth, even distribution of dextran within cortical and leptomeningeal arteries of 22-month-old wild-type mice following its diffusion from the parenchyma (Fig. 2b). By contrast, in the blood vessel walls of age-matched Tg2576 mice, dextran appeared patchy and fragmented, similar to the pattern of Aβ deposition observed in human CAA (Fig. 2c). Dextran also bound to parenchymal deposits in the hippocampus of 22-month-old Tg2576 mice (Fig. 2d). Double immunolabelling confirmed co-localization of dextran with Aβ-positive plaques in the brain parenchyma and in cortical and leptomeningeal vessels (Fig. 2e, f).
Fig. 2

Distribution of dextran within the cerebrovasculature of the ageing brain and in the presence of cerebral amyloid angiopathy (CAA). a and b Photomicrographs of dextran (green) distribution along laminin-positive (red) basement membranes in hippocampal and leptomeningeal arteries after injection into the hippocampus of a 22-month-old wild-type mouse. Following a 15-min diffusion period, dextran had a smooth, even distribution within the media and adventitia of leptomeningeal arteries (blue α-smooth muscle actin). c By contrast, dextran (green) localization within the wall of a leptomeningeal artery of a 22-month-old Tg2576 mouse had a patchy, band-like appearance and distributed between α-smooth muscle actin-positive cells (blue; red laminin). d Dextran (green) injected into the hippocampus of a 22-month-old Tg2576 mouse also bound to parenchymal deposits (red laminin). e and f Double fluorescence labelling demonstrated co-localization of dextran (green) with Aβ (blue) in the cerebrovasculature (e) and brain parenchyma (f) of 22-month-old Tg2576 mice (red laminin). Scale barsa and d 180 μm, b and c 120 μm, e and f 24 μm

To quantify perivascular drainage, the number of hippocampal capillaries, arteries and veins containing dextran in their walls after the 15-min diffusion period were counted (Fig. 3a–f) [12]. Within the wild-type group, the number of labelled capillaries increased with age, such that 22-month-old mice demonstrated significantly higher numbers of dextran-positive capillaries than both 3- and 7-month-old mice (Fig. 3g, 3 vs. 22 months p < 0.001; 7 vs. 22 months p < 0.001, one-way ANOVA). Labelling of arteries showed a significant rise between 3 and 7 months of age, which declined subsequently in the 22-month-old mice (Fig. 3h, 3 vs. 7 months p < 0.001; 7 vs. 22 months p < 0.05, one-way ANOVA). No differences in the labelling of veins were noted between wild-type age groups (Fig. 3i, p > 0.05, one-way ANOVA).
Fig. 3

Quantification of the number of cerebral blood vessel walls containing dextran in the ageing brain and in the presence of cerebral amyloid angiopathy (CAA). af Photomicrographs of dextran (green) distribution along laminin-positive (red) basement membranes of capillaries (arrows), arteries (arrowheads) and veins (asterisks) in the hippocampi of 3- (a, d), 7- (b, e) and 22-month-old (c, f) wild-type (ac) and Tg2576 mice (df). Within the wild-type group, the number of hippocampal capillary walls containing dextran was significantly increased in 22-month-old mice, compared to both 3- and 7-month-old mice (g). Significantly more arteries were labelled in the brains of 7-month-old mice, versus 3- and 22-month-old mice (h). No differences were noted in the labelling of veins between wild-type age groups (i). Among the Tg2576 mice, the number of capillaries or arteries containing dextran in their walls did not differ between young, adult and aged Tg2576 mice (g and i). However, 22-month-old Tg2576 mice had significantly fewer dextran-positive capillaries compared to wild-type littermates (g). A significantly lower number of labelled arteries were also counted in 7-month-old Tg2576 mice compared to age-matched controls (h). Dextran labelling of veins in the Tg2576 mice was significantly higher in 22-month-old mice, compared with both 3- and 7-month-old Tg2576 mice, as well as 22-month-old wild-type animals (i). Histograms were compiled from analysis of 5 brain sections per mouse and represent mean values ± SEM. Green dextran, red laminin, blue α-smooth muscle actin. Scale bar 50 μm

Among the Tg2576 mice, no differences were observed in the number of capillaries or arteries containing dextran in their walls between young, adult and aged mice (Fig. 3g, h, p > 0.05, one-way ANOVA). However, 22-month-old Tg2576 mice had significantly fewer dextran-positive capillaries compared to wild-type littermates (Fig. 3g, p < 0.001, one-way ANOVA). Similarly, a significantly lower number of labelled arteries were counted in 7-month-old Tg2576 mice compared to age-matched controls (Fig. 3h, p < 0.05, one-way ANOVA). Dextran labelling of veins in the Tg2576 mice was increased proportional to CAA severity, such that 22-month-old mice demonstrated significantly more labelling of veins compared with both 3- and 7-month-old Tg2576 mice (Fig. 3i, p < 0.001 and p < 0.05, respectively, one-way ANOVA), as well as age-matched controls (Fig. 3i, p < 0.01, one-way ANOVA). Taken together, these data suggest that perivascular drainage of solutes from the brain is disrupted by age and CAA, but with differential effects on the micro- and macrovasculature, respectively.

Changes in vessel density in wild-type and Tg2576 mice

Reduction of blood vessel density related to Aβ plaques deposition has been reported in Tg2576 mice, but is less clear in human studies [29, 34, 53]. To determine if changes in the number of dextran-labelled vessels were due to altered vessel density, glut-1 staining of capillaries and large-diameter vessels was quantified in the hippocampi of wild-type and Tg2576 mice (Fig. 4a–f). In agreement with previous reports [34], capillary density increased significantly between 3 and 7 months of age in wild-type mice (p < 0.01, one-way ANOVA), and was maintained thereafter in 22-month-old animals (Fig. 4h, 3 vs. 22 months p < 0.05; 7 vs. 22 months, p > 0.05, one-way ANOVA). A similar, but non-significant trend was also observed in the Tg2576 mice. Notably, hippocampal capillary glut1 density was significantly lower in 22-month-old Tg2576 mice compared to wild-type littermates (Fig. 4g, p < 0.05, one-way ANOVA). No differences were noted in the mean grey value of glut-1 staining per capillary between wild-type and Tg2576 mice (data not shown), indicating that the observed decrease was not due to decreased glut-1 expression [25]. Large-diameter vessel density did not differ between age groups or genotype (Fig. 4h, p > 0.05, one-way ANOVA).
Fig. 4

Quantification of vessel density in the hippocampi of wild-type and Tg2576 mice, as detected by glucose transporter-1 (glut-1) immunoreactivity. af Photomicrographs of glut-1-positive capillaries and arteries in the hippocampi of 3- (a, d), 7- (b, e) and 22-month-old (c, f) wild-type (ac) and Tg2576 mice (df). g Capillary density increased significantly between 3 and 7 months in wild-type mice, and was maintained thereafter in 22-month-old animals. A similar trend was also observed in the Tg2576 mice, but hippocampal capillary density was significantly lower in 22-month-old Tg2576 mice compared to wild-type littermates. h Large-diameter vessel density did not differ between age groups or genotype. Histograms were compiled from analysis of 4 brain sections/mouse and represent mean values ± SEM. Scale bar 100 μm

Changes in basement membrane protein levels with age

Thickening of basement membranes is consistently reported in the aged and AD brain [49], but the nature of this change is not known. To determine whether the expression of specific cerebrovascular basement membrane proteins is altered with age, levels of collagen IV, laminin, fibronectin and perlecan were assessed by immunoblotting in a separate cohort of wild-type mice. As demonstrated in Fig. 5a, b, no significant changes were detected in the expression of collagen IV in either capillaries or large-diameter blood vessels isolated from the cortices of 3-, 8- and 16-month-old mice (p > 0.05, repeated measures two-way ANOVA). By contrast, levels of laminin were significantly decreased in the capillaries (p < 0.05, repeated measures two-way ANOVA), but not in large-diameter vessels, of 16-month-old mice compared to young mice (Fig. 5c, d). Blotting for fibronectin detected two specific bands of approximately 250 and 60 kDa in size, corresponding to the full-length protein and the cleaved N-terminal portion, respectively [57, 59]. Although no changes were detected in the expression level of the 250 kDa band between age groups (data not shown), significantly higher levels of 60 kDa fibronectin were observed in the capillaries at 16 months compared with 3 months of age (Fig. 5e, p < 0.01, repeated measures two-way ANOVA). A small but significant increase in the levels of the 60 kDa fibronectin fragment was also noted in the large-diameter vessels of 16-month-old mice compared to 8-month-old animals (Fig. 5f, p < 0.05, repeated measures two-way ANOVA). Analysis of perlecan levels in the capillary samples revealed a U-shaped pattern of expression, whereby protein levels decreased significantly between 3 and 8 months of age (p < 0.05, repeated measures two-way ANOVA), rising again in 16-month-old mice (Fig. 5g, 8 vs. 16 months p < 0.05, repeated measures two-way ANOVA). No changes were observed in perlecan levels in large-diameter vessels between young, adult and aged mice (Fig. 5h). These results indicate that there is an age-related alteration in the protein composition of cerebrovascular basement membranes.
Fig. 5

Basement membrane protein levels in capillaries and large-diameter vessels of a separate cohort of wild-type mice. Quantification of immunoblots revealed no changes in collagen IV expression in either capillaries (a) or large-diameter blood vessels (b) isolated from the cortices of 3-, 8- and 16-month-old wild-type mice. By contrast, levels of laminin were significantly decreased in the capillaries (c), but not in large-diameter vessels (d), of 16-month-old mice, compared to young mice. Significantly higher levels of the 60 kDa fragment of fibronectin were observed in capillary samples between 3 and 16 months of age (e). A small, but significant increase in fibronectin levels was also noted in the large-diameter vessels of 16-month-old mice compared to 8-month-old animals (f). Capillary levels of perlecan decreased significantly between 3 and 8 months of age, rising again in 16-month-old mice (g), whereas no differences where noted in large-diameter vessels between young, adult and aged mice (h). Optic density ratios of protein to GAPDH levels from 3 blots were analysed by repeated measures two-way ANOVA with Bonferroni post hoc test. Histograms represent mean optic density ratio values ± SEM from a representative blot for each antibody

Discussion

There is now a strong body of evidence suggesting that failure of clearance mechanisms for the elimination of Aβ from the brain contributes to the development of CAA and AD [38, 70]. We report here the first experimental data demonstrating that drainage of intracerebrally injected dextran along the basement membranes of capillaries and arteries is altered in the ageing mouse brain. Furthermore, the presence Aβ deposits within the walls of blood vessels disrupts the normal route of elimination of solutes from the brain parenchyma. The disturbance in the pattern of perivascular drainage of tracer is associated with changes in blood vessel density related to increasing age and the development of CAA. The expression levels of specific cerebrovascular basement membrane proteins are also altered in the ageing brain.

Unlike other organs, the brain has no conventional lymphatics for the removal of interstitial fluid (ISF). Instead, ISF and solutes are eliminated by bulk flow along the basement membranes of capillary and artery walls [6, 27, 58]. Several lines of evidence support the hypothesis that dysfunction in the perivascular drainage of neuronally produced Aβ leads to the development of CAA: (1) transgenic mice expressing mutant human Aβ only in neurons develop vascular Aβ deposits [11, 65]; (2) tracers injected into the interstitial fluid of the adult mouse brain distribute along basement membranes in exactly the same pattern as Aβ deposition in human CAA [12]; (3) basement membrane abnormalities are observed in the ageing and AD brain and precede CAA development in the TGF-β1 transgenic mice [45, 49, 72], (4) Aβ deposits in the AD brain have been identified in perivascular compartments [52, 60] and (5) anti-Aβ immunotherapy experiments in mice and humans have demonstrated increased severity of CAA associated with decreased cerebral plaque load [5, 48, 51, 71]. The current findings provide strong experimental support for the hypothesis that perivascular drainage of solutes from the brain is altered with age and presence of CAA.

Abnormalities in the ageing cerebrovasculature, particularly within capillaries, have previously been described in both animal and human studies. These include morphologic (e.g., vessel looping, tortuosity and twisting), physiological (e.g., loss of smooth muscle and basement membrane thickening) and functional changes (e.g., decreased cerebral blood flow) [2, 10, 24, 26, 29, 36, 42, 53, 63]. In correspondence with these reports, we found that perivascular drainage was affected primarily in the small vessels of the aged mouse brain, with significant increases in the number of dextran-labelled capillaries between 3-, 7- and 22-months of age. Hippocampal capillary density was also significantly higher in 22-month-old mice compared to 3-month-old, but not 7-month-old wild-type mice, indicating that the number of capillaries increases in the maturing mouse brain but remains constant after 7 months of age. The stability of capillary density between 7- and 22-months of age, suggests that the increase in dextran labelling of capillaries observed in 22-month-old animals is due to a decline in the rate of perivascular drainage of solutes from the extracellular spaces of the parenchyma. This may be accompanied by a reduction in the rate of drainage of dextran from capillaries to arteries, as a decrease in the number of arteries labelled with dextran was also noted in the 22-month-old mice.

Basement membrane proteins play a critical role in the drainage of ISF from the brain. We found that intracerebrally injected dextran co-localized with laminin in hippocampal blood vessels, suggesting that basement membranes may interact directly with solutes to mediate their clearance along perivascular pathways. Therefore, alterations to basement membrane morphology and/or up- or downregulation of specific basement membrane proteins could influence Aβ aggregation, alter the rate of drainage and influence the development of CAA. Our immunoblotting results showed that the levels of laminin within capillary walls are significantly lower in the aged brain, while expression of fibronectin and perlecan is increased compared to young and adult mice, respectively. Considering that laminin and perlecan are known to have opposing effects on the prevention and promotion of Aβ aggregation, respectively, a corresponding shift in their expression levels in the aged human brain could promote vascular Aβ deposition [1, 8, 13, 43, 66]. Perlecan has also been shown to regulate the growth of smooth muscle and endothelial cells, and is thought to provide stability to the basement membrane scaffold [16, 33]. Although further experiments are needed, we suggest that the observed decrease in perlecan expression between 3 and 8 months may serve to prevent excess blood vessel formation and to stabilize the structure of established vessels in the adult brain. In the ageing brain, perlecan levels may be upregulated to compensate for the destabilization of the basement membrane induced by the decreased expression of laminin and other basement membrane proteins. Although little is known about the interaction between fibronectin and Aβ, a recent report has shown an association between fibronectin and the Aβ-degrading angiotensin converting enzyme-1 in the artery walls of human brains with CAA, suggesting a putative role for fibronectin in vascular Aβ processing [40]. Collectively, these results suggest that ageing may be accompanied by a change in the biochemical composition of the cerebrovascular basement membranes, towards the promotion of Aβ deposition. Interestingly, although increased immunohistochemical staining for collagen IV has been reported in the ageing human brain [61, 64], we did not observe any changes in collagen IV expression between age groups in the mouse by Western blotting. Although further investigation is needed to account for this discrepancy, it is possible that mouse collagen IV is less susceptible to age-related changes or that other collagen isoforms are predominantly affected by ageing.

We have previously established that tracers injected into the extracellular spaces of the naïve adult mouse brain drain predominantly along the basement membranes of capillaries and arteries, with little or no labelling of veins [12]. In the current study, we found that the number of capillaries labelled with dextran was significantly lower in 22-month-old mice Tg2576 mice compared to wild-type littermates. This finding likely correlates with the decrease in hippocampal capillary density in these mice, as reported here and previously [34, 46]. However, the most striking effect of CAA on perivascular drainage was the significant increase in the number of veins that were positive for dextran, which was not associated with alterations in total vessel number. This suggests that the deposition of vascular amyloid in capillary and artery walls alters the normal route of elimination of solutes from the brain. In addition, the reported decrease in vasodilatory response and cerebral blood flow induced by Aβ in artery walls of both young and aged Tg2576 mice may further disrupt the drainage of interstitial fluid (ISF) [23, 37, 47, 56]. Indeed, if the motile force for elimination of solutes along perivascular drainage pathways is generated from arterial pulsations [54], it possible that reductions in pulse amplitude related to alterations in cerebral blood pressure or flow and altered compliance of the aged vessel walls (i.e. stiffening), would slow the rate of drainage and encourage precipitation of Aβ in vessel walls [68].

Finally, our data indicate that dextran within the ISF co-localizes with the insoluble deposits of Aβ in the parenchyma and vasculature. This suggests that solutes, including soluble Aβ, brain metabolites or drugs that are contained in parenchymal ISF, may bind to Aβ plaques and thus affect their distribution within the brain, particularly in areas of high plaque and CAA load. Furthermore, the design of AD therapies and therapeutic agents that target parenchymal plaque removal should take into consideration the decreased efficiency of Aβ clearance mechanisms in the elderly and AD brain.

Notes

Acknowledgments

This work is funded by the Alzheimer’s Research Trust UK (C.A.H, J.N, R.O.C) and the German Alzheimer Forschung Initiative (R.S.). The authors would like to thank the Biomedical Imaging Unit (Southampton General Hospital), as well as Ute Bauer and Dr. Anke Hoffmann for excellent technical assistance. We would also like to express our gratitude to Dr. Karen Hsiao Ashe, Department of Neurology, University of Minnesota, USA, for kindly providing Tg2576 founder mice.

Conflict of interest

The authors declare they have no conflict of interest.

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Cheryl A. Hawkes
    • 1
  • Wolfgang Härtig
    • 2
  • Johannes Kacza
    • 3
  • Reinhard Schliebs
    • 2
  • Roy O. Weller
    • 1
  • James A. Nicoll
    • 1
  • Roxana O. Carare
    • 1
  1. 1.Division of Clinical Neurosciences, Southampton General HospitalUniversity of SouthamptonSouthamptonUK
  2. 2.Paul Flechsig Institute for Brain ResearchUniversity of LeipzigLeipzigGermany
  3. 3.Department of Anatomy, Histology and Embryology, Faculty of Veterinary MedicineUniversity of LeipzigLeipzigGermany

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