Alzheimer's disease is the commonest form of dementia. It is likely that a lack of clearance of amyloid beta (Aβ) results in its accumulation in the parenchyma as Aβ oligomers and insoluble plaques, and within the walls of blood vessels as cerebral amyloid angiopathy (CAA). The drainage of Aβ along the basement membranes of blood vessels as intramural periarterial drainage (IPAD), could be improved if the driving force behind IPAD could be augmented, therefore reducing Aβ accumulation. There are alterations in the composition of the vascular basement membrane in Alzheimer's disease. Lysyl oxidase (LOX) is an enzyme involved in the remodelling of the extracellular matrix and its expression and function is altered in various disease states. The expression of LOX is increased in Alzheimer's disease, but it is unclear whether this is a contributory factor in the impairment of IPAD in Alzheimer's disease. The pharmacological inhibition of LOX may be a strategy to improve IPAD and reduce the accumulation of Aβ in the parenchyma and within the walls of blood vessels.

α-Dystrobrevin (α-DB) is a major component of the dystrophin-associated protein complex (DAPC). Knockout (KO) of α-DB in the brain is associated with astrocytic abnormalities and loss of neuronal GABA receptor clustering. Mutations in DAPC proteins are associated with altered dopamine signaling and cognitive and psychiatric disorders, including schizophrenia. This study tested the hypothesis that motivation and associated underlying biological pathways are altered in the absence of α-DB expression. Male wildtype and α-DB KO mice were tested for measures of motivation, executive function and extinction in the rodent touchscreen apparatus. Subsequently, brain tissues were evaluated for mRNA and/or protein levels of dysbindin-1, dopamine transporter and receptor 1 and 2, mu opioid receptor 1 (mOR1) and cannabinoid receptor 1 (CB1). α-DB KO mice had significantly increased motivation for the appetitive reward, while measures of executive function and extinction were unaffected. No differences were observed between wildtype and KO animals on mRNA levels of dysbindin-1 or any of the dopamine markers. mRNA levels of mOR1were significantly decreased in the caudate-putamen and nucleus accumbens of α-DB KO compared to WT animals, but protein levels were unaltered. However, CB1 protein levels were significantly increased in the prefrontal cortex and decreased in the nucleus accumbens of α-DB KO mice. Triple-labelling immunohistochemistry confirmed that changes in CB1 were not specific to astrocytes. These results highlight a novel role for α-DB in the regulation of appetitive motivation that may have implications for other behaviours that involve the dopaminergic and endocannabinoid systems.

The extracellular matrix (ECM) of the cerebral vasculature provides a pathway for the flow of interstitial fluid (ISF) and solutes out of the brain by intramural periarterial drainage (IPAD). Failure of IPAD leads to protein elimination failure arteriopathies such as cerebral amyloid angiopathy (CAA). The ECM consists of a complex network of glycoproteins and proteoglycans that form distinct basement membranes (BM) around different vascular cell types. Astrocyte endfeet that are localised against the walls of blood vessels are tethered to these BMs by dystrophin associated protein complex (DPC). Alpha-dystrobrevin (α-DB) is a key dystrophin associated protein within perivascular astrocyte endfeet; its deficiency leads to a reduction in other dystrophin associated proteins, loss of AQP4 and altered ECM. In human dementia cohorts there is a positive correlation between dystrobrevin gene expression and CAA. In the present study, we test the hypotheses that (a) the positive correlation between dystrobrevin gene expression and CAA is associated with elevated expression of α-DB at glial-vascular endfeet and (b) a deficiency in α-DB results in changes to the ECM and failure of IPAD. We used human post-mortem brain tissue with different severities of CAA and transgenic α-DB deficient mice. In human post-mortem tissue we observed a significant increase in vascular α-DB with CAA (CAA vrs. Old p < 0.005, CAA vrs. Young p < 0.005). In the mouse model of α-DB deficiency, there was early modifications to vascular ECM (collagen IV and BM thickening) that translated into reduced IPAD efficiency. Our findings highlight the important role of α-DB in maintaining structure and function of ECM, particularly as a pathway for the flow of ISF and solutes out of the brain by IPAD.

INTRODUCTION:Accumulation of amyloid beta (Aβ), α-synuclein (αSyn), and tau in dementias indicates their age-related failure of elimination from the brain. Aβ is eliminated along basement membranes in walls of cerebral arterioles and leptomeningeal arteries (intramural peri-arterial drainage [IPAD]); IPAD is impaired with age. We test the hypothesis that αSyn and tau are also eliminated from the normal brain along IPAD pathways.METHODS:Soluble αSyn or tau was injected into mouse hippocampus. Animals were perfused 5 minutes to 7 days post-injection. Blood vessels were identified by ROX-SE for light-sheet and immunolabeling for confocal microscopy. IPAD was quantified by measuring the proportion of arterioles with αSyn/tau.RESULTS:αSyn and tau are eliminated from the brain by IPAD but with different dynamics.DISCUSSION:Age-related failure of IPAD may play a role in the pathogenesis of synucleinopathies and tauopathies. αSyn persists within IPAD at 24 hours, which may affect immunotherapy for αSyn.

White matter hyperintensities (WMH) occur in association with dementia but the aetiology is unclear. Here we test the hypothesis that there is a combination of impaired elimination of interstitial fluid from the white matter together with a degree of hypoxia in WMH. One of the mechanisms for the elimination of amyloid-β (Aβ) from the brain is along the basement membranes in the walls of capillaries and arteries (Intramural Peri-Arterial Drainage – IPAD). We compared the dynamics of IPAD in the grey matter of the hippocampus and in the white matter of the corpus callosum in 10 week old C57/B16 mice by injecting soluble Aβ as a tracer. The dynamics of IPAD in the white matter were significantly slower compared with the grey matter and this was associated with a lower density of capillaries in the white matter. Exposing cultures of smooth muscle cells to hypercapnia as a model of cerebral hypoperfusion resulted in a reduction in fibronectin and an increase in laminin in the extracellular matrix. Similar changes were detected in the white matter in human WMH suggesting that hypercapnia/hypoxia may play a role in WMH. Employing therapies to enhance both IPAD and blood flow in the white matter may reduce WMH in patients with dementia.

The accumulation of amyloid-β (Aβ) in the walls of capillaries and arteries as cerebral amyloid angiopathy (CAA) is part of the small vessel disease spectrum, related to a failure of elimination of Aβ from the brain. Aβ is eliminated along basement membranes in walls of cerebral capillaries and arteries (Intramural Peri-Arterial Drainage-IPAD), a pathway that fails with age and ApolipoproteinEε4 (ApoE4) genotype. IPAD is along basement membranes formed by capillary endothelial cells and surrounding astrocytes. Here, we examine (1) the composition of basement membranes synthesised by ApoE4 astrocytes; (2) structural differences between ApoE4 and ApoE3 astrocytes, and (3) how flow of Aβ affects Apo3/4 astrocytes. Using cultured astrocytes expressing ApoE3 or ApoE4, immunofluorescence, confocal, correlative light and electron microscopy (CLEM), and a millifluidic flow system, we show that ApoE4 astrocytes synthesise more fibronectin, possess smaller processes, and become rarefied when Aβ flows over them, as compared to ApoE3 astrocytes. Our results suggest that basement membranes synthesised by ApoE4 astrocytes favour the aggregation of Aβ, its reduced clearance via IPAD, thus promoting cerebral amyloid angiopathy.

Aims: Cerebral amyloid angiopathy (CAA) is the accumulation of amyloid beta (Aβ) in the walls of cerebral arterioles, arteries and capillaries. Changes in the white matter in CAA are observed as hyperintensities and dilated perivascular spaces on MRI suggesting impairment of fluid drainage but the pathophysiology behind these changes is poorly understood. We tested the hypothesis that proteins associated with clearance of Aβ peptides are upregulated in the white matter in cases of CAA. Methods: In this study, we compare the quantitative proteomic profile of white matter from post-mortem brains of patients with CAA and age-matched controls in order to gain insight into the cellular processes and key molecules involved in the pathophysiology of CAA. Results: Our proteomic analysis resulted in the profiling of 3,734 proteins (peptide FDR p<0.05). Of these, 189 were differentially expressed in CAA vs. control. Bioinformatics analysis of these proteins showed significant enrichment of proteins related to cell adhesion | cell-matrix interaction, mitochondrial dysfunction and hypoxia. Upregulated proteins in CAA included EMILIN2, COL4A2, TLN1, CLU, HSPG2. Downregulated proteins included DSP, IDE, HBG1. Conclusions: The present study reports an in-depth quantitative proteomic profiling of white matter from patients with CAA, highlighting extracellular matrix proteins and clusterin as key molecules in the pathophysiology of white matter changes in cases of CAA.

In the absence of lymphatics, fluid and solutes such as amyloid-β (Aβ) are eliminated from the brain along basement membranes in the walls of cerebral capillaries and arteries—the Intramural Peri-Arterial Drainage (IPAD) pathway. IPAD fails with age and insoluble Aβ is deposited as plaques in the brain and in IPAD pathways as cerebral amyloid angiopathy (CAA); fluid accumulates in the white matter as reflected by hyperintensities (WMH) on MRI. Within the brain, fluid uptake by astrocytes is regulated by aquaporin 4 (AQP4). We test the hypothesis that expression of astrocytic AQP4 increases in grey matter and decreases in white matter with onset of CAA. AQP4 expression was quantitated by immunocytochemistry and confocal microscopy in post-mortem occipital grey and white matter from young and old non-demented human brains, in CAA and in WMH. Results: AQP4 expression tended to increase with normal ageing but AQP4 expression in severe CAA was significantly reduced when compared to moderate CAA (p = 0.018). AQP4 expression tended to decline in the white matter with CAA and WMH, both of which are associated with impaired IPAD. Adjusting the level of AQP4 activity may be a valid therapeutic target for restoring homoeostasis in the brain as IPAD fails with age and CAA.

There are very few reliable methods in the literature to discern with certainty between cerebral arterioles and venules. Smooth muscle cells (SMC) and pericytes are present in both arterioles and venules, so immunocytochemistry for markers specific to intramural cells (IMC) is unreliable. This study employed transmission electron microscopy (TEM) and a canine brain to produce robust criteria for the correct identification of cerebral arterioles and venules based on lumen:vessel wall area, tested against the less accurate lumen diameter:vessel wall thickness. We first used morphology of IMC to identify two distinct groups of vessels; group 1 with morphology akin to venules and group 2 with morphology akin to arterioles. We then quantitatively assessed these vessels for lumen:vessel wall area ratio and lumen diameter:wall thickness ratio. After assessing 112 vessels, we show two distinct groups of vessels that can be separated using lumen:vessel wall area (group 1, 1.89 -10.96 vs. group 2, 0.27-1.57; p < 0.001) but not using lumen diameter:vessel wall thickness where a substantial overlap in ranges between groups occurred (group 1, 1.58-22.66 vs. group 2, 1.40-11.63). We, therefore, conclude that lumen:vessel wall area is a more sensitive and preferred method for distinguishing cerebral arterioles from venules. The significance of this study is wide, as cerebral small vessel disease is a key feature of vascular dementia and understanding the pathogenesis relies on correct identification of vessels.

Tracers injected into CSF pass into the brain alongside arteries and out again. This has been recently termed the “glymphatic system” that proposes tracers enter the brain along periarterial “spaces” and leave the brain along the walls of veins. The object of the present study is to test the hypothesis that: (1) tracers from the CSF enter the cerebral cortex along pial-glial basement membranes as there are no perivascular “spaces” around cortical arteries, (2) tracers leave the brain along smooth muscle cell basement membranes that form the Intramural Peri-Arterial Drainage (IPAD) pathways for the elimination of interstitial fluid and solutes from the brain. 2 μL of 100 μM soluble, fluorescent fixable amyloid β (Aβ) were injected into the CSF of the cisterna magna of 6–10 and 24–30 month-old male mice and their brains were examined 5 and 30 min later. At 5 min, immunocytochemistry and confocal microscopy revealed Aβ on the outer aspects of cortical arteries colocalized with α-2 laminin in the pial-glial basement membranes. At 30 min, Aβ was colocalised with collagen IV in smooth muscle cell basement membranes in the walls of cortical arteries corresponding to the IPAD pathways. No evidence for drainage along the walls of veins was found. Measurements of the depth of penetration of tracer were taken from 11 regions of the brain. Maximum depths of penetration of tracer into the brain were achieved in the pons and caudoputamen. Conclusions drawn from the present study are that tracers injected into the CSF enter and leave the brain along separate periarterial basement membrane pathways. The exit route is along IPAD pathways in which Aβ accumulates in cerebral amyloid angiopathy (CAA) in Alzheimer’s disease. Results from this study suggest that CSF may be a suitable route for delivery of therapies for neurological diseases, including CAA.

Aβ, amyloid beta, CAA, cerebral amyloid angiopathy, CT, computed tomographic scanning, IPAD, intramural periarterial drainage, ISF, interstitial fluid, MRI, magnetic resonance imaging, PVS, perivascular spaces, WMH, white matter hyperintensities.Cerebral white matter hyperintensities (WMH) observed on magnetic resonance imaging (MRI), or low attenuation on computed tomographic scanning (CT), are the most frequent brain imaging finding in patients with small vessel disease or dementia. It has been assumed that WMH are due to arteriosclerosis or blood-brain barrier breakdown, though recently it was demonstrated that WMH have distinct molecular signatures in Alzheimer's disease (AD) where markers of Wallerian degeneration are present, compared to normal ageing [1]. This article is protected by copyright. All rights reserved.

Meninges that surround the CNS consist of an outer fibrous sheet of dura mater (pachymeninx) that is also the inner periosteum of the skull. Underlying the dura are the arachnoid and pia mater (leptomeninges) that form the boundaries of the subarachnoid space. In this review we (1) examine the development of leptomeninges and their role as barriers and facilitators in the foetal CNS. There are two separate CSF systems during early foetal life, inner CSF in the ventricles and outer CSF in the subarachnoid space. As the foramina of Magendi and Luschka develop, one continuous CSF system evolves. Due to the lack of arachnoid granulations during foetal life, it is most likely that CSF is eliminated by lymphatic drainage pathways passing through the cribriform plate and nasal submucosa. (2) We then review the fine structure of the adult human and rodent leptomeninges to establish their roles as barriers and facilitators for the movement of fluid, cells and pathogens. Leptomeningeal cells line CSF spaces, including arachnoid granulations and lymphatic drainage pathways, and separate elements of extracellular matrix from the CSF. The leptomeningeal lining facilitates the traffic of inflammatory cells within CSF but also allows attachment of bacteria such as Neisseria meningitidis and of tumour cells as CSF metastases. Single layers of leptomeningeal cells extend into the brain closely associated with the walls of arteries so that there are no perivascular spaces around arteries in the cerebral cortex. Perivascular spaces surrounding arteries in the white matter and basal ganglia relate to their two encompassing layers of leptomeninges. (3) Finally we examine the roles of ligands expressed by leptomeningeal cells for the attachment of inflammatory cells, bacteria and tumour cells as understanding these roles may aid the design of therapeutic strategies to manage developmental, autoimmune, infectious and neoplastic diseases relating to the CSF, the leptomeninges and the associated CNS.

Alzheimer's Disease (AD) is the most common form of dementia and to date there is no cure or efficient prophylaxis. The cognitive decline correlates with the accumulation of amyloid-β (Aβ) in the walls of capillaries and arteries. Our group has demonstrated that interstitial fluid and Aβ are eliminated from the brain along the basement membranes of capillaries and arteries, the intramural periarterial drainage (IPAD) pathway. With advancing age and arteriosclerosis, the stiffness of arterial walls, this pathway fails in its function and Aβ accumulates in the walls of arteries. In this study we tested the hypothesis that arterial pulsations drive IPAD and that a valve mechanism ensures the net drainage in a direction opposite to that of the blood flow. This hypothesis was tested using a mathematical model of the drainage mechanism. We demonstrate firstly that arterial pulsations are not strong enough to produce drainage velocities comparable to experimental observations. Secondly, we demonstrate that a valve mechanism such as directional permeability of the IPAD pathway is necessary to achieve a net reverse flow. The mathematical simulation results are confirmed by assessing the pattern of IPAD in mice using pulse modulators, showing no significant alteration of IPAD. Our results indicate that forces other than the cardiac pulsations are responsible for efficient IPAD.

In this chapter we describe in detail the tissue processing techniques we employ for the study of cerebral tissue by transmission electron microscopy (TEM). In particular, we explain a technique that enables quantification of changes in cerebral basement membranes at the ultrastructural level. This is significant, as age related pathological conditions affecting the brain are often accompanied by ultrastructural changes in the cerebral vasculature.Briefly, experimental mice are fixed by perfusion and their brains removed. Brains are then vibratomed into 100 μm slices with regions of interest microdissected and processed for TEM following a protocol optimized for the preservation of cerebral tissue. Changes in the thickness of cerebral basement membranes are then quantified using novel software. Some prior knowledge of general TEM specimen preparation and sectioning will be useful when performing this protocol.

Although there are no conventional lymphatic vessels in the brain, fluid and solutes drain along basement membranes (BMs) of cerebral capillaries and arteries towards the subarachnoid space and cervical lymph nodes. Convective influx/glymphatic entry of the cerebrospinal fluid (CSF) into the brain parenchyma occurs along the pial-glial BMs of arteries. This project tested the hypotheses that pial-glial BM of arteries are thicker in the midbrain, allowing more glymphatic entry of CSF. The in vivo MRI and PET images were obtained from a 4.2-year-old dog, whereas the post-mortem electron microscopy was performed in a 12-year-old dog. We demonstrated a significant increase in the thickness of the pial-glial BM in the midbrain compared with the same BM in different regions of the brain and an increase in the convective influx of fluid from the subarachnoid space. These results are highly significant for the intrathecal drug delivery into the brain, indicating that the midbrain is better equipped for convective influx/glymphatic entry of the CSF.

In this chapter we describe in detail the surgical and imaging techniques employed for the study of the anatomical routes of drainage of cerebrospinal fluid (CSF) and interstitial fluid (ISF) from the brain. The types of tracers, sites of injection, and volumes injected are crucial. For example, when testing the drainage of ISF from the parenchyma, volumes larger than 0.5 μL result in spillage of ISF into the ventricular CSF.

Dilatation of periarteriolar spaces in MRI of the ageing human brains occurs in white matter (WM), basal ganglia and midbrain but not in cerebral cortex. Perivenous collagenous occurs in periventricular but not in subcortical WM.Here we test the hypotheses that (a) the capacity for dilatation of periarteriolar spaces correlates with the anatomical distribution of leptomeningeal cells coating intracerebral arteries and (b) the regional development of perivenous collagenous in the WM correlates with the population of intramural cells in the walls of veins.The anatomical distribution of leptomeningeal and intramural cells related to cerebral blood vessels is best documented by electron microscopy, requiring perfusion-fixed tissue not available in human material. We therefore analysed perfusion-fixed brain from a 12-year-old Beagle dog as the canine brain represents the anatomical arrangement in the human brain. Results showed regional variation in the arrangement of leptomeningeal cells around blood vessels. Arterioles are enveloped by one complete layer of leptomeninges often with a second incomplete layer in the WM. Venules showed incomplete layers of leptomeningeal cells. Intramural cell expression was higher in the post-capillary venules of the subcortical WM when compared with periventricular WM, suggesting that periventricular collagenosis around venules may be due to a lower resistance in the venular walls. It appears that the regional variation in the capacity for dilatation of arteriolar perivascular spaces in the white WM may be related to the number of perivascular leptomeningeal cells surrounding vessels in different areas of the brain.

Non-amyloid cerebral small vessel disease (CSVD) and cerebral amyloid angiopathy (CAA) may be interrelated through the damaged basement membranes (BMs) and extracellular matrix changes of small vessels, resulting in a failure of β-amyloid (Aβ) transport and degradation. We analyzed BM changes and the pattern of deposition of Aβ in the walls of blood vessels in spontaneously hypertensive stroke-prone rats (SHRSP), a non-transgenic CSVD model. In 45 SHRSP and 38 Wistar rats aged 18 to 32 weeks: (i) the percentage area immunostained for vascular collagen IV and laminin was quantified; (ii) the capillary BM thickness as well as endothelial and pericyte pathological changes were analysed using transmission electron microscopy (TEM); and (iii) the presence of vascular Aβ was assessed. Compared with controls, SHRSP exhibited a significantly higher percentage area immunostained with collagen IV in the striatum and thalamus. SHRSP also revealed an age-dependent increase of the capillary BM thickness and of endothelial vacuoles (caveolae) within subcortical regions. Endogenous Aβ deposits in the walls of small blood vessels were observed in the cortex (with the highest incidence found within fronto-parietal areas), striatum, thalamus and hippocampus. Vascular β-amyloid accumulations were frequently detected at sites of small vessel wall damage. Our data demonstrate changes in the expression of collagen IV and of the ultrastructure of BMs in the small vessels of SHRSP. Alterations are accompanied by vascular deposits of endogenous Aβ. Impaired β-amyloid clearance along perivascular and endothelial pathways and failure of extracellular Aβ degradation may be the key mechanisms connecting non-amyloid CSVD and CAA.