Physiology of perivascular drainage of the brain and how it is affected by advancing age

Lead Research Organisation: University of Southampton
Department Name: Clinical and Experimental Sciences

Abstract

Fluid and soluble waste drain from all organs of the body to regional lymph glands. For organs such as the lung and liver, there are clearly defined channels along which the fluid and waste products drain. However, there are no such well-defined channels to drain fluid and waste products from the brain; instead, the drainage pathways are very narrow and confined to the walls of the arteries that supply the brain. This drainage pathway for waste products from the brain has received little attention in the past but its importance is becoming increasingly recognised because of its potential role in the decline of mental and psychological health. There are many unknown factors concerned with the perivascular drainage pathways from the normal brain and they need to be resolved before measures can be taken to maintain normal mental health in the elderly.

One of the important features of the ageing brain is the accumulation of an insoluble protein, amyloid-beta, within the brain substance. Our previous studies have shown that in normal young individuals, amyloid-beta drains out of the brain along the narrow pathways within the walls of brain arteries. However, as people become older, there is a failure of the drainage of amyloid-beta; it then accumulates in the brain and disturbs its normal biology. This is the reason why we are so interested in the drainage pathways.

Our project consists of three parts in which we investigate the normal drainage of waste products from the brain. With the information derived from the study, we can start to devise ways of improving the drainage of amyloid-beta from the brains of older people.

1] We will determine the force that drives fluid and waste products such as amyloid-beta out of the brain along the walls of arteries. In order to do this, we will inject a tracer substance that emits a fluorescent glow and then observe its progress along the walls of arteries in the brain using a two photon microscope. This microscope has been developed for viewing arteries in the brain and, by taking photographs at very short intervals, we will be able to test our hypothesis that the force driving fluid and waste products out of the brain is derived from the pulsations in the arteries. By using a drug, a beta-blocker, we will reduce the strength of the pulsations and measure the effect on the drainage of waste products from the brain. The main reason for using the beta-blocker is that it mimics the effects of ageing in brain arteries. As people get older, their arteries become stiffer and the strength of the pulsations reduces, preventing the elimination of amyloid-beta from the ageing brain.

2] The width of the drainage pathway by which fluid and waste products are eliminated in the brain along the walls of arteries is only 100-150nm thick (1nm equals one billionth of a meter). It is important, therefore, to know how large a protein molecule or particle can pass along the drainage pathway. If the proteins are too large, they may block the drainage pathway and this would prevent waste products from leaving the brain. We will inject particles up to 20nm in diameter into the brain to test the capacity of the drainage system. The results of this study will be combined with those in the previous study to determine how the capacity of the drainage system decreases with age in normal individuals.

3] We will study the composition of the drainage pathway itself. The drainage pathway is composed of a complex mixture of proteins. We know that the appearance of the layer under the microscope changes with age, so now we plan to determine the chemical changes that occur in this layer with age. By using innovative techniques of analysis (proteomics), the chemical structure of the normal pathway by which fluid and waste products drain from the brain will be revealed and furthermore, we will document the changes that occur with increasing age.

Technical Summary

Most organs in the body possess lymphatics by which proteins drain to lymph nodes for maintenance of immune competence and homoeostasis. The brain has no such traditional lymphatics but tracer studies in experimental animals have shown that interstitial fluid drains from the brain along the cerebrovascular basement membranes (BM) to regional lymph nodes in the neck. The same perivascular lymphatic drainage route is present in human brain as shown by the pattern of deposition of amyloid-beta (Abeta) in cerebral amyloid angiopathy (CAA) in the normal elderly brains.
The aims of this project are to:
1] Test the hypothesis that the motive force for perivascular drainage from the brain is derived from pulsations in cerebral arteries. We will directly observe the passage of tracers along BM in cerebral artery walls by Two Photon Microscopy. The effects of reduced vascular pulsations upon the efficiency of perivascular drainage will be observed following administration of beta-blockers. Data from this study are important because ageing cerebral arteries lose their elasticity and the amplitude of pulsations is reduced. Reduction in the motive force for drainage may result in deposition of Abeta in artery walls with age.
2] Define the size of molecules or particles that can pass along the perivascular drainage route. Preliminary studies suggest that particles of 20nm may deposit in BMs and temporarily obstruct the perivascular drainage route. So, nanoparticles 10-20nm will be injected to assess how efficiently they are drain along perivascular pathways. Data derived from the study will help to establish the capacity of the normal perivascular drainage pathway.
3] Test the hypothesis that changes in the morphology of BMs in cerebral artery walls with age reflect changes in their proteomics. The data will be essential for future planning of strategies to facilitate the drainage of solutes, from the ageing brain.

Planned Impact

The main groups who will benefit from the results of the present proposal will be other researchers in the fields of neuroimmunology and biogerontology; finally, and probably the most important group, will be the elderly population for whom there is no fully developed and successful strategy for facilitating the removal of metabolic waste along the ageing cerebrovascular system.
Research into the neurophysiology of ageing moves forward on a broad front, ranging from genetics to therapy. As a basis for development in these fields, it is essential to have a firm understanding of the normal brain and its functions. Our research will help to clarify the normal physiology of drainage pathways for fluid and solutes from the brain and the effects that age has upon these pathways. It is increasingly recognised that the failure of elimination of amyloid-beta (Abeta) with increasing age is a major factor in the accumulation of Abeta in the brain and in the walls of cerebral blood vessels in ageing, contributing to cognitive decline. A number of mechanisms for the elimination of Abeta from the brain have been identified but the interrelationship between these mechanisms has not been firmly established. From previous animal experiments and observations in human brains, it appears that perivascular drainage of Abeta along the walls of cerebral arteries is a major route for the elimination of Abeta that fails with age. Establishing the normal physiology of perivascular drainage and the effects of age upon this pathway will form the foundation upon which future research can be based. For example: defining the molecular genetics of basement membranes in blood vessel walls may become an essential step in identifying populations who are most at risk of cognitive decline and for whom early therapy would be beneficial. Furthermore, the genes involved in the organisation and branching patterns of cerebral arteries during development may prove to be an important index to future failure of perivascular drainage of solutes, such as Abeta, from the ageing brain.
Little is currently known about the role of perivascular lymphatic drainage of the brain in neuroimmunological reactions. Establishing the normal physiology of perivascular drainage would form a basis for future studies on the drainage of antigens, be they brain antigens or proteins derived from infecting viruses, to regional lymph nodes. The picture of how immunological reactions develop in the brain is incomplete at the present time because the exact roles of lymphatic drainage of the brain and the regional lymph nodes has not been fully characterised. The results of our studies should form a firm basis upon which to build future research in neuroimmunology.
We hope that within the 3-5 years enough will be known about the physiology of lymphatic drainage of the brain to embark upon devising therapies that facilitate the perivascular elimination of Abeta from the brain for increasing the well-being and delaying the onset of cognitive decline in the elderly.

Publications

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Hawkes CA (2014) Failure of perivascular drainage of ß-amyloid in cerebral amyloid angiopathy. in Brain pathology (Zurich, Switzerland)

 
Description 1) We applied a novel proteomic technique on arteries from young and old normal brains, to identify the changes that occur with age in the walls of blood vessels. These changes are important to understand so we can maintain the health of the brain and the efficient elimination of waste from the ageing brain. We had the cooperation of Brain Banks in Edinburgh and Newcastle and we received arteries dissected from the surface of the brains of young and old human brains with short post-mortem delay followed by analysis. Our analysis is novel proteomic analysis that resulted in the identification of over 6000 proteins. We observed a gender difference between proteins and we were able to identify the proteins that change with age: for women- signalling between cells and proteins that make-up the back-ground tissue of the brain (extracellular matrix), proteins responsible for inflammation ; for men- the proteins changed were those of the extracellular matrix as well as proteins that are part of the cholesterol pathway. Our findings suggest that the proteins that change with age are those responsible for eliminating the metabolic waste of the brain and those responsible for inflammation- the first response to any injury. Clusterin was identified as a key protein also present as a biomarker in other studies. This was followed up in a subsequent study in collaboration with Mayo Clinic USA and led to a publication in PNAS in 2017.
2) In the absence of conventional lymphatics, drainage of interstitial fluid and solutes from the brain parenchyma to cervical lymph nodes is along basement membranes in the walls of cerebral capillaries and tunica media of arteries. Perivascular pathways are also involved in the entry of CSF into the brain by the convective influx/glymphatic system. The objective of this study is to differentiate the cerebral vascular basement membrane pathways by which fluid passes out of the brain from the pathway by which CSF enters the brain. Experiment 1: 0.5 µL of soluble biotinylated or fluorescent Aß or 1 µL 15 nm gold nanoparticles were injected into the mouse hippocampus and their distributions determined at 5 min by transmission electron microscopy (TEM). Aß was distributed within the extracellular spaces of the hippocampus and within basement membranes of capillaries and tunica media of arteries. Nanoparticles did not enter capillary basement membranes from the extracellular spaces. Experiment 2: 2 µL of 15 nm nanoparticles were injected into mouse CSF. Within 5 min. groups of nanoparticles were present in the pio-glial basement membrane on the outer aspect of cortical arteries between the investing layer of pia mater and the glia limitans. The results of this study and previous research suggest that cerebral vascular basement membranes form the pathways by which fluid passes into and out of the brain but that different basement membrane layers are involved.
Exploitation Route We anticipate that we can design methods in the future that rely on manipulating these proteins and pathways to maintain the health of the ageing brain.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.cararegroup.org
 
Description We have completed the project: 1. validated a novel proteomics technique and have applied the technique to human arteries dissected freshly from the surface of the brain, with excellent published results; 2. validated the technique of intracerebral injections and assessing clearance using two photon microscopy; 3. synthesised nanoparticles in-house and we have fully described the pattern of clearance of nanoparticles in a publication in Acta Neuropathologica
First Year Of Impact 2017
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Biogen - PhD studentship
Amount £75,000 (GBP)
Organisation Biogen 
Sector Public
Country United Kingdom
Start 09/2014 
End 10/2017
 
Description INVICRO
Amount £100,000 (GBP)
Organisation Invicro 
Sector Private
Country United States
Start 04/2015 
End 06/2017
 
Title Quantitative assessment of cerebrovascular basement membranes 
Description We have developed a system of assessing reliably the thickness of the cerebrovascular basement membranes as they are observed by electron microscopy. 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2013 
Provided To Others? Yes  
Impact PMID: 23413811 
 
Description Ageing of the arterial wall 
Organisation British Neuropathological Society
Country United Kingdom 
Sector Learned Society 
PI Contribution A medical student who is undergoing the Masters in Medical Sciences is assessing the changes that occur in the vascular walls as the brain ages. It appears that the structure of arteries in the anterior part of the brain is different to that in the posterior part of the brain.
Collaborator Contribution Bursary to a medical student
Impact Not yet. Presentation at BNS 2018 in preparation.
Start Year 2016
 
Description Clusterin and perivascular clearance 
Organisation Mayo Clinic
Country United States 
Sector Charity/Non Profit 
PI Contribution Dr John Fryer has provided the model for the study of perivascular clearance in the absence of clusterin
Collaborator Contribution Knock-out mice provided by Dr Fryer in Mayo clinic
Impact Pilot data suitable for a publication
Start Year 2014
 
Description Novel molecular analysis of brains, for detection of new biomarkers 
Organisation Protea Biosciences Group, Inc.
Country United States 
Sector Private 
PI Contribution I have secured a partnership with Protea Biosciences who will apply the LAESI technology for the first time to brains, at their cost and with £10,000 towards our research costs.LAESI® (Laser Ablation Electrospray Ionization), is used with mass spectrometry to detect the presence of up to, and over, one thousand distinct molecules from a single analysis of samples.
Collaborator Contribution Protea will cover the costs for analysis of mouse brains and have pledged £10,000 towards our research costs
Impact Press release
Start Year 2014
 
Description Oligodendrocyte and white matter changes in Alzheimer's disease 
Organisation University of Portsmouth
Department School of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution I provide the knowledge of the pathogenesis of Alzheiemr's disease and associated white matter changes and expertise in human immunocytochemistry, as well as tissue.
Collaborator Contribution Prof Arthur Butt and his team have a long track record in the changes that oligodendrocytes and oligodendrocyte precursor cells undergo in normal and pathological conditions. We plan to apply this to Alzheimer's disease.
Impact Summer placement for PhD student, aiming to complete data for a publication.
Start Year 2013
 
Description Role of dystrobrevin in perivascular clearance 
Organisation University of Portsmouth
Country United Kingdom 
Sector Academic/University 
PI Contribution Using alpha-dystrobrevin knock-out mice provided by our collaborator Prof Darek Gorecki in the University of Portsmouth, we are studying the perivascular clearance of amyloid-beta in this model.
Collaborator Contribution Prof Gorecki provided the mice
Impact PIlot data included in Stroke Association grant proposal
Start Year 2015
 
Description Routes of communication between the extracellular spaces of the brain and cerebrospinal fluid 
Organisation Biogen Idec
Country United States 
Sector Private 
PI Contribution Through PhD funding as well as direct research funding we perform experimental work and analysis of the distribution of tracers injected into the brain parenchyma and CSF, aiming to clarify the connections between the brain parenchyma and CSF.
Collaborator Contribution Financial, expertise and tissue.
Impact Results that will be presented in part at the Alzheimer's Association International Conference
Start Year 2014
 
Description Townswomen Guild 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Two cheques for over £500 were collected

Funds were donated to ARUK. I have recently been invited to give another talk by Townswomen, in Chandlers Ford
Year(s) Of Engagement Activity 2013,2014