Vascular dementia: failure of fluid drainage from cerebral white matter:

Cerebral small vessel disease (SVD) is key feature of vascular dementia, radiologically defined by the presence of white matter hyperintensities, lacunar infarcts, microbleeds and perivascular spaces.  Cerebral arteriolosclerosis resulting in loss of elasticity and segmental disorganisation of the arterial wall leads to damage of the deep white matter.  The primary functions of penetrating and perforating cerebral arteries supplying blood and drainage of fluid and solutes from the parenchyma along intramural perivascular pathways along the arterial basement membranes are impaired.  In this project, using animal models and post-mortem brain tissue from stroke survivors with SVD (CogFAST study) along with light sheet 3D microscopy and post-mortem MRI, we will assess the immunocytochemical pattern of distribution of AQP4, α-dystrobrevin and β-dystroglycan.  We will then test the hypotheses that 1) disruption in the anchoring system of the basement membranes such as that observed in α-dystrobrevin knock-out mice and 2) disruption of gliovascular end feet tracked by aquaporin 4 (AQP4) knock-out mice there is failure of perivascular clearance of fluid from the deep gray matter and the corpus callosum.  Our aim is to demonstrate that failure of perivascular drainage of fluid from the brain is a mechanism underlying SVD and this could be targeted therapeutically.

Matthew MacGregor Sharp

The selective permeability of leptomeninges:

The meninges are the interface between cerebrospinal fluid and interstitial fluid, with controversy regarding their permeability. Through a partnership with molecular microbiologists (Prof Myron Christodoulides) we are investigating the exact properties of the leptomeninges with particular emphasis on their selective permeability. This is relevant for a) the transfer of Aβ from the parenchyma to the CSF where it is measured as a biomarker for disease progression and b) the pathology of complications of subarachnoid haemorrhage.

In collaboration with Professor Myron Christodoulides

In vivo MRI imaging of the motive force driving intramural perivascular clearance:

Based on results from mathematical modelling we now know that the motive force for perivascular clearance is provided by spontaneous vasomotion resulting from the intrinsic contractions of pericytes and cerebrovascular smooth muscle cells and not by the pulsations derived from cardiac cycle (Dr Alexandra Diem, Prof Neil Bressloff). Collaborations are in place with senior neuroradiologists in University College London and Leiden, The Netherlands to demonstrate in vivo in real time using MRI, the features of the motive force for efficient intramural perivascular clearance and correlate this with findings in different stages of Alzheimer’s disease and mild cognitive impairment. This could be the first marker for impaired clearance of cerebral interstitial fluid in humans.

Roxana Aldea, PhD student

Maternal Low Protein Diet leads to morphological and biochemical modifications of cerebrovascular basement membranes:

In this project we test the hypothesis that a maternal diet low in fat leads to modifications of the proteoglycans and glycoproteins that make up the cerebrovascular basement membranes, resulting in a failure of perivascular clearance of fluid from the brain.

Ben Burwood, Joanna Gould. In collaboration with Dr Sandrine Willaime-Morawek, Prof Tom Fleming

Markers of lymphatic vessels in the brain parenchyma:

In this project, using human tissue from the Newcastle Brain Tissue Resource, we assess the pattern of immunostaining of LYVE-1, a marker of lymphatic endothelia, in the brain.

Tom Wand, Masters in Medical Sciences

Does maternal high fat diet lead to dementia?

In this project we test the hypothesis that exposure to a high fat diet during development and early life, leads to the remodelling of the neurovascular unit and reduces the efficiency of Aβ clearance from the brain, leading to increased CAA severity. A mouse model of pre- and postnatal high fat diet exposure will be established by feeding female mice (C57Bl/6), either a standard (21% kcal fat) or high fat (45% kcal fat) diet for 4 weeks before conception and during gestation and lactation. At weaning, male and female offspring will be fed either a normal or high fat to generate 4 groups of experimental mice. We will create a separate group of female pregnant mice that will be treated with Metformin to assess whether this therapeutic agent is effective in halting the pathological process. We aim to demonstrate that simple measures like improving the diet of pregnant mothers, managing hypercholesterolaemia, diabetes will prevent or delay the onset of dementia.

Matthew MacGregor Sharp and Antigoni Manousopoulou. In collaboration with Dr Felino Cagampang

Innervation of cerebral arteries is key to maintenance of efficient clearance and flow:

This project, funded by Alzheimer’s Research UK in collaboration with Dr Cheryl Hawkes (Open University), tests the hypothesis that loss of perivascular innervation by cholinergic neurons leads to dysfunctional regulation of vascular tone, thereby reducing the motive force for perivascular drainage of Aβ leading to a worsening of cerebral amyloid angiopathy. Researcher: Mrs Maureen Gatherer

Maureen Gatherer. In collaboration with Dr Cheryl Hawkes

Regional differences in the ultrastructure of cerebral blood vessels. Significance for cerebral small vessel disease and vascular cognitive impairment:

Cerebral small vessel disease (SVD) is characterised by neuroimaging features including dilated perivascular spaces (DPVS) and white matter hyperintensities (WMH) which are associated with cognitive impairment. DPVS exist within the white matter (WM) but are absent from cortical grey matter (GM). Differences in the arrangement of leptomeningeal cells around blood vessels may underlie this regional specificity. We hypothesised that one leptomeningeal layer exists around cortical GM arterioles and two layers around WM vessels, providing the anatomical basis for DPVS.

One cause of WMH is periventricular venous collagenosis (PVC) which describes the build-up of concentric layers of collagen around post-capillary venules in the periventricular white matter (PVWM). PVC is a compensatory response to shear stress on venule walls caused by abnormal extension of the arterial pulse-wave into the venous system. The exclusivity of PVC to the PVWM may be explained by ultrastructural differences in the expression of intramural cells (pericytes, smooth muscle cells) which constitute the limited pulse-wave-attenuating properties of venules. We hypothesised that intramural cell expression is lower in PVWM venules compared to superficial WM venules, increasing their susceptibility to PVC.

Using transmission electron microscopy, we are analysing the ultrastructure of blood vessels in the cortical GM, subcortical WM and basal ganglia of an intracardially perfused 12-year old Beagle brain (supplied by Invicro, USA).

Theodore Criswell, Master of Medical Sciences Student,
Matthew MacGregor Sharp

Subarachnoid haemorrhage leads to accumulation of Ab in the cortical arterioles:

Our new and yet unpublished data demonstrate that the motive force for efficient clearance of solutes along the walls of blood vessels is provided by vasomotion, which consists of the spontaneous series of contractions of vascular smooth muscle cells, propagated from the arterioles towards the leptomeningeal arteries. As such, the disturbance in the tone and the vasoconstriction induced by an overlying subarachnoid haemorrhage would lead to impaired clearance of solutes in the cortical arteries that are branches of the vessels where the haemorrhage occurs. This study tests the hypothesis that a subarachnoid haemorrhge results in the deposition of Ab in the cortical arteries underlying the haemorrhage.

Matthew Myers, MMedSc. In collaboration with Ian Galea (neurology) and Diederik Bulters (neurosurgery)

Hyperhomocystaeinaemia leads to a failure of perivascular clearance of Ab:

Hyperhomocystaeinaemia (HHCy, induced mainly by low vitamin B levels) is a risk factor for Alzheimer’s disease. A novel model of HHCy developed by our collaborator Prof Donna Wilcock in the University of Kentucky USA displays all features of small vessel disease. When HHCy is induced in a mouse model of Alzheimer’s disease, there is a significant deposition of amyloid in the walls of blood vessels. We are testing the hypothesis that HHCy results in modifications of the pathways for drainage of fluid from the walls of blood vessels and a failure of perivascular drainage of fluid from the brain.

Karis Hodgson, BMedSc In collaboration with Prof Donna Wilcock, University of Kentucky, USA

Development of an in vitro perivascular clearance system:

Using a novel Quasi Vivo in vitro system developed by Kirkstall Ltd and mouse astrocytes that express humanised ApoE, (collaboration with David Holtzman, Washington University, USA), we aim to test the hypothesis that flow of Aβ over astrocytes expressing different forms of ApoE results in morphological alterations to the astrocytes expressing ApoE4, compared to those expressing ApoE2 or ApoE3.

Using coated coverslips, the astrocytes are plated and left to adhere for 24 hours before being loaded into the QV500 chamber or 24 well plate for static experiments. A solution of astrocyte growth medium supplemented with 100nM Aβ 1-40 is circulated around the system for 24 hours. The coverslip is removed and fixed in 4% PFA, immunostained and examined by confocal microscopy.

We have already optimised the system for testing the activity of Quasi Vivo and concluded that the Quasi Vivo system has no significant toxic effect on the growth or viability of the cells when cells grown under flow were compared with static controls. There appears to be a decrease in cell number when Aβ is applied to ApoE4 astrocytes under flow. We have also shown that there are morphological changes to ApoE4 astrocytes when Aβ is applied that are further enhanced when combined with flow. These changes are not seen in ApoE2 or ApoE3 astrocytes. We also observed that Aβ appears to be concentrated where there are clusters of cells, though this has only been seen in ApoE3 astrocytes. This work suggests that the dynamics of interactions between  Aβ and astrocytes are dependent on their APOE genotype and this is likely to contribute to the reduced clearance of Aβ in APOE4 individuals

Abby Keable PhD student

2017 - 2020

Vascular dementia: failure of fluid drainage from cerebral white matter:

Cerebral small vessel disease (SVD) is key feature of vascular dementia, radiologically defined by the presence of white matter hyperintensities, lacunar infarcts, microbleeds and perivascular spaces.  Cerebral arteriolosclerosis resulting in loss of elasticity and segmental disorganisation of the arterial wall leads to damage of the deep white matter.  The primary functions of penetrating and perforating cerebral arteries supplying blood and drainage of fluid and solutes from the parenchyma along intramural perivascular pathways along the arterial basement membranes are impaired.  In this project, using animal models and post-mortem brain tissue from stroke survivors with SVD (CogFAST study) along with light sheet 3D microscopy and post-mortem MRI, we will assess the immunocytochemical pattern of distribution of AQP4, α-dystrobrevin and β-dystroglycan.  We will then test the hypotheses that 1) disruption in the anchoring system of the basement membranes such as that observed in α-dystrobrevin knock-out mice and 2) disruption of gliovascular end feet tracked by aquaporin 4 (AQP4) knock-out mice there is failure of perivascular clearance of fluid from the deep gray matter and the corpus callosum.  Our aim is to demonstrate that failure of perivascular drainage of fluid from the brain is a mechanism underlying SVD and this could be targeted therapeutically.

Matthew MacGregor Sharp

2017 - 2018

The selective permeability of leptomeninges:

The meninges are the interface between cerebrospinal fluid and interstitial fluid, with controversy regarding their permeability. Through a partnership with molecular microbiologists (Prof Myron Christodoulides) we are investigating the exact properties of the leptomeninges with particular emphasis on their selective permeability. This is relevant for a) the transfer of Aβ from the parenchyma to the CSF where it is measured as a biomarker for disease progression and b) the pathology of complications of subarachnoid haemorrhage. In collaboration with Professor Myron Christodoulides

2017 - 2018

In vivo MRI imaging of the motive force driving intramural perivascular clearance:

Based on results from mathematical modelling we now know that the motive force for perivascular clearance is provided by spontaneous vasomotion resulting from the intrinsic contractions of pericytes and cerebrovascular smooth muscle cells and not by the pulsations derived from cardiac cycle (Dr Alexandra Diem, Prof Neil Bressloff). Collaborations are in place with senior neuroradiologists in University College London and Leiden, The Netherlands to demonstrate in vivo in real time using MRI, the features of the motive force for efficient intramural perivascular clearance and correlate this with findings in different stages of Alzheimer’s disease and mild cognitive impairment. This could be the first marker for impaired clearance of cerebral interstitial fluid in humans. Roxana Aldea, PhD student

2017 - 2019

Does maternal high fat diet lead to dementia?

In this project we test the hypothesis that exposure to a high fat diet during development and early life, leads to the remodelling of the neurovascular unit and reduces the efficiency of Aβ clearance from the brain, leading to increased CAA severity. A mouse model of pre- and postnatal high fat diet exposure will be established by feeding female mice (C57Bl/6), either a standard (21% kcal fat) or high fat (45% kcal fat) diet for 4 weeks before conception and during gestation and lactation. At weaning, male and female offspring will be fed either a normal or high fat to generate 4 groups of experimental mice. We will create a separate group of female pregnant mice that will be treated with Metformin to assess whether this therapeutic agent is effective in halting the pathological process. We aim to demonstrate that simple measures like improving the diet of pregnant mothers, managing hypercholesterolaemia, diabetes will prevent or delay the onset of dementia. Regan Doherty, Matthew MacGregor Sharp and Antigoni Manousopoulou. In collaboration with Dr Felino Cagampang

2016 - 2018

Innervation of cerebral arteries is key to maintenance of efficient clearance and flow:

This project, funded by Alzheimer’s Research UK in collaboration with Dr Cheryl Hawkes (Open University), tests the hypothesis that loss of perivascular innervation by cholinergic neurons leads to dysfunctional regulation of vascular tone, thereby reducing the motive force for perivascular drainage of Aβ leading to a worsening of cerebral amyloid angiopathy. Researcher: Mrs Maureen Gatherer Maureen Gatherer. In collaboration with Dr Cheryl Hawkes

2016 - 2017

Subarachnoid haemorrhage leads to accumulation of Ab in the cortical arterioles:

Our new and yet unpublished data demonstrate that the motive force for efficient clearance of solutes along the walls of blood vessels is provided by vasomotion, which consists of the spontaneous series of contractions of vascular smooth muscle cells, propagated from the arterioles towards the leptomeningeal arteries. As such, the disturbance in the tone and the vasoconstriction induced by an overlying subarachnoid haemorrhage would lead to impaired clearance of solutes in the cortical arteries that are branches of the vessels where the haemorrhage occurs. This study tests the hypothesis that a subarachnoid haemorrhge results in the deposition of Ab in the cortical arteries underlying the haemorrhage. Matthew Myers, MMedSc. In collaboration with Ian Galea (neurology) and Diederik Bulters (neurosurgery)

2016 - 2017

Hyperhomocystaeinaemia leads to a failure of perivascular clearance of Ab:

Hyperhomocystaeinaemia (HHCy, induced mainly by low vitamin B levels) is a risk factor for Alzheimer’s disease. A novel model of HHCy developed by our collaborator Prof Donna Wilcock in the University of Kentucky USA displays all features of small vessel disease. When HHCy is induced in a mouse model of Alzheimer’s disease, there is a significant deposition of amyloid in the walls of blood vessels. We are testing the hypothesis that HHCy results in modifications of the pathways for drainage of fluid from the walls of blood vessels and a failure of perivascular drainage of fluid from the brain. Karis Hodgson, BMedSc In collaboration with Prof Donna Wilcock, University of Kentucky, USA

2015 - 2019

Development of an in vitro perivascular clearance system:

Using a novel Quasi Vivo in vitro system developed by Kirkstall Ltd and mouse astrocytes that express humanised ApoE, (collaboration with David Holtzman, Washington University, USA), we aim to test the hypothesis that flow of Aβ over astrocytes expressing different forms of ApoE results in morphological alterations to the astrocytes expressing ApoE4, compared to those expressing ApoE2 or ApoE3. Using coated coverslips, the astrocytes are plated and left to adhere for 24 hours before being loaded into the QV500 chamber or 24 well plate for static experiments. A solution of astrocyte growth medium supplemented with 100nM Aβ 1-40 is circulated around the system for 24 hours. The coverslip is removed and fixed in 4% PFA, immunostained and examined by confocal microscopy. We have already optimised the system for testing the activity of Quasi Vivo and concluded that the Quasi Vivo system has no significant toxic effect on the growth or viability of the cells when cells grown under flow were compared with static controls. There appears to be a decrease in cell number when Aβ is applied to ApoE4 astrocytes under flow. We have also shown that there are morphological changes to ApoE4 astrocytes when Aβ is applied that are further enhanced when combined with flow. These changes are not seen in ApoE2 or ApoE3 astrocytes. We also observed that Aβ appears to be concentrated where there are clusters of cells, though this has only been seen in ApoE3 astrocytes. This work suggests that the dynamics of interactions between  Aβ and astrocytes are dependent on their APOE genotype and this is likely to contribute to the reduced clearance of Aβ in APOE4 individuals Abby Keable PhD student