Fluid Flow and Mass Transport in Brain Tissue
Fluid Flow and Mass Transport in Brain Tissue
Fluid Flow and Mass Transport in Brain Tissue
Review
Fluid Flow and Mass Transport in Brain Tissue
Lori A. Ray and Jeffrey J. Heys *
Chemical and Biological Engineering, Montana State University, Bozeman, MT 59717, USA; [email protected]
* Correspondence: [email protected]
Received: 29 October 2019; Accepted: 22 November 2019; Published: 26 November 2019
Abstract: Despite its small size, the brain consumes 25% of the body’s energy, generating its own
weight in potentially toxic proteins and biological debris each year. The brain is also the only organ
lacking lymph vessels to assist in removal of interstitial waste. Over the past 50 years, a picture
has been developing of the brain’s unique waste removal system. Experimental observations show
cerebrospinal fluid, which surrounds the brain, enters the brain along discrete pathways, crosses a
barrier into the spaces between brain cells, and flushes the tissue, carrying wastes to routes exiting
the brain. Dysfunction of this cerebral waste clearance system has been demonstrated in Alzheimer’s
disease, traumatic brain injury, diabetes, and stroke. The activity of the system is observed to increase
during sleep. In addition to waste clearance, this circuit of flow may also deliver nutrients and
neurotransmitters. Here, we review the relevant literature with a focus on transport processes,
especially the potential role of diffusion and advective flows.
Keywords: biotransport; glymphatic system; perivascular circulation; interstitial flow; mass transport
1. Introduction
The brain is one of the last frontiers in physiology and medicine, and has been long under-explored
due to difficulties and concerns of breaching the cranial vault. Over recent decades, the combination of
new imaging technologies, transgenic (genetically-altered) animal models, and innovative surgical
procedures have shed great light on the workings of the brain, importantly the live brain. In addition,
non-invasive (or minimally-invasive) imaging technologies have enabled in vivo investigation of the
human brain. As more data become available for study, the opportunity and necessity for engineers to
contribute to the field of neuroscience is growing rapidly. A particular opportunity is integration of
data into comprehensive models based on fluid dynamics, material science, transport phenomena,
reaction kinetics and thermodynamic fundamentals.
The transport of molecules is an essential link in many physiological and pathological processes
of the brain. For example, transport is critical to delivery of nutrients, such as glucose [1], and other
materials needed for cell repair and renewal. Communication relies on molecular transport, where
transport rates determine the range-of-action for neurotransmitters [2,3] and transport effects cell-to-cell
communication [4]. The brain produces metabolic waste at a higher rate than any other organ, making
transport of molecules out of the brain (waste clearance) a critical cerebral process [5]. In addition,
fluid flow can initiate cell activity (mechano-transduction) such as the opening of cell receptors [6].
Finally, transport is a major determinant in delivery of therapeutics to the brain and the movement of
biomarkers to the peripheral body for detection [7].
Waste clearance has recently been the most studied role of molecular transport in the brain, as
its failure is a central feature of neurodegenerative disease [4] and injury, such a post traumatic brain
injury (TBI) [8,9]. Protein aggregates, mis-folded proteins that accumulate into insoluble clumps, are a
common feature in patients with amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s
disease and other neurodegenerative diseases, implying that reduced clearance from the brain could
be a shared phenomenon in neurodegeneration [10]. The removal of protein waste was traditionally
attributed to intra- and extra-cellular degradation [11], or for select proteins, by tailored protein channel
transport across vascular cell walls [12,13]. Contemporary researchers are elucidating an ordered
molecular transport pathway in the brain, which utilizes cerebrospinal fluid, brain-specific anatomy,
and state of consciousness (i.e., sleep), and may be a complimentary process for clearing potentially
toxic waste from brain tissue.
Due to the demonstrated role of this waste transport pathway in neurodegeneration, stroke [14],
diabetes [15], depression [16], multiple sclerosis [17], and others [18,19], the literature in this field has
become extensive. In this review, we seek to summarize what is known specifically with respect to
fluid and mass transfer mechanisms and the anatomy and physiology that directly affect transport.
Fluid dynamic and mass transport calculations and computational models in this field suffer from an
incomplete understanding of the physical situation, high variance in measured physical parameters,
and limited quantitative data for validation of theories put forward. An effort is made here to
thoroughly summarize the current understandings and key data as a reference for those interested
in transport in the brain. Transport theories and governing equations appropriate to the different
elements of brain transport are presented and discussed. The general focus is with respect to waste
clearance, but most concepts apply directly to the functions described in the preceding paragraphs.
Acronyms used throughout the text are summarized in Table 1.
Table 1. Acronyms.
2. Background
brain occurs via protein channels within the cell walls. Although many types of protein channels are
present, each is specific to a particular molecule or narrow group of molecules. In addition, lymph
vessels are absent from brain tissue (lymph vessels were recently identified in the meninges, which
surrounds the brain, but is outside the BBB. See Section 3.4.2). From this perspective, the brain appears
to lack a non-specific route for clearance, critical for molecules without a protein channel such as large
polar substances like sucrose [12] and protein aggregates implicated in neurodegenerative diseases.
Yet the brain’s high metabolic rate makes multiple routes of waste clearance imperative. It follows that
the brain must have a non-molecule-specific system of waste removal.
The brain sits within a thick-walled skull, surrounded by and “bathed in” cerebrospinal fluid
(CSF). CSF has long been thought to facilitate waste clearance, similar to lymphatics in the peripheral
body, however the exact processes were poorly understood [5,22]. As shown in Figure 1, CSF circulates
around the brain and spinal chord. It is produced mainly in the choroid plexus, a collection of special
endothelial cells that filter blood plasma to create CSF. CSF is about 99% water with several dissolved
ions, and has a much lower protein concentration than plasma (detailed compositions can be found
in [23]). The choroid plexus is located in the lateral and fourth cerebral ventricles near the center
of the brain. CSF flows through the ventricles to the cisterna magna at the lower back of the brain
and into the subarachnoid space (SAS) that surrounds the brain and spinal cord. The brain produces
approximately 500 mL of CSF daily. Only 100-150 mL fills the fluid spaces of the brain and spine at any
time, inferring constant circulation and reabsorption [5]. CSF efflux is via arachnoid granulations in
the SAS (Figure 1) and along nerve bundles exiting the brain and spine.
Unique to cerebral vasculature is an annular space surrounding each blood vessel as it penetrates
the brain tissue that is filled with fluid, called the perivascular space (PVS) [24]. As blood vessels
penetrate the tissue and branch into ever smaller vessels, the perivascular space follows at least to the
level of arterioles and venules, and possibly capillaries, reaching all parts of the brain. As illustrated in
Figure 2, the PVS is bounded on the inside by the vascular wall and on the outside by glial limitans,
which consist of layers of astrocyte endfeet. (Astrocytes are glial cells with several long cellular
processes terminating in endfeet. See reference [25] for descriptive illustrations of perivascular space
at different levels of the vasculature.) The inner wall is elastic, and transport of molecules is tightly
controlled by the BBB. The outer barrier, which we term the perivascular “wall” (PVW), is mechanically
more rigid and is permeable. Molecules can enter the interstitium through gaps between endfeet and
through specific protein channels in the endfeet walls, such as aquaporin-4 (AQP4) that transports
water. This perivascular space has been shown to be connected to the cerebrospinal fluid (CSF) of the
subarachnoid space [26,27], giving CSF thorough access to brain tissue.
Fluids 2019, 4, 196 4 of 33
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Figure 1.
Figure 1. Production
Production and
and Circulation
Circulation ofof Cerebrospinal
Cerebrospinal FluidFluid (CSF).
(CSF). Schematic
Schematic representation
representation of of the
the
pattern of intracranial CSF flow. (A) CSF is produced by the choroid plexus of the lateral
pattern of intracranial CSF flow. (A) CSF is produced by the choroid plexus of the lateral and fourth and fourth
ventricles. CSF
ventricles. CSFflows
flowsthrough
throughthe theventricles
ventriclestoto
thethe
cisterna
cisternamagna
magna at the lower
at the back
lower of the
back of brain and
the brain
into into
and the the
subarachnoid space
subarachnoid (SAS)
space that
(SAS) surrounds
that surrounds thethebrain
brainand
andspinal
spinalcord.
cord. CSF
CSF efflux
efflux is via
is via
arachnoid granulations
arachnoid granulations and
and along
along the
the olfactory
olfactory nerves
nerves through
through thethe cribriform
cribriform plate.
plate. (B)
(B) Schematic
Schematic
representation of
representation of CSF–interstitial
CSF–interstitial flow
flow from
from and
and toto the
the subarachnoid
subarachnoid space.
space. CSF can movemove in and out
of the brain parenchyma
parenchyma along
along the
the perivascular
perivascular space.
space. CSF can also exit through the meninges to the
lymphatics. Reprinted from Ref [28] Copyright 2016
dural lymphatics. 2016 with
with permission
permission fromfrom Elsevier.
Elsevier.
In the
the current
current literature,
literature, thethe terms
terms ‘perivascular’
‘perivascular’ and and ‘paravascular’
‘paravascular’ are each each used,
used, sometimes
sometimes
interchangeably, to to refer
refer toto the
the annular
annular spacespace described
described above.
above. Some authors use both terms to to
differentiate two hypothesized compartments
two hypothesized compartments surrounding surrounding the cerebral vasculature:
cerebral vasculature: perivascular,
referring to the the space
space within
within thethe basement
basement membrane
membrane of the the smooth
smooth muscle
muscle cells adjacent
adjacent to the the
vascular
vascular wall, and paravascular, referring referring to the the space
space adjacent
adjacent to the
the astrocytes
astrocytes (the reason
reason for thisthis
hypothesized
hypothesizedseparation
separationisisdiscussed
discussed inin
Section
Section3.1). “Virchow–Robin
3.1). “Virchow–Robin space” is also
space” a term
is also used used
a term to referto
to theto
refer paravascular space.space.
the paravascular No consensus
No consensus currently existsexists
currently within the field
within on this
the field onterminology,
this terminology,in partin
because there there
part because is not is
yetnot
a clear
yet aanatomical understanding
clear anatomical of the nature
understanding of theofnature
these spaces
of theseand whether
spaces and
they differthey
whether from eachfrom
differ other.each
For other.
simplicity, we use the we
For simplicity, termuse‘perivascular’ to indicate the
the term ‘perivascular’ fluid-filled,
to indicate the
annular spaceannular
fluid-filled, surrounding
spacecerebral
surrounding vasculature, andvasculature,
cerebral do not distinguish
and do separate compartments.
not distinguish separate
Brain tissue, also called the parenchyma, is made up of neural and glial cells, as well as the
compartments.
fluid-filled spacesalso
Brain tissue, between
calledthethecells, called theisextracellular
parenchyma, or interstitial
made up of neural and glial space.
cells,The interstitial
as well fluid
as the fluid-
(ISF)
filledthat fills between
spaces the extracellular
the cells,space
called (ECS)
the is believed to or
extracellular have a composition
interstitial space.similar to CSF [29],
The interstitial around
fluid (ISF)
99%
that water.
fills theWithin the interstitial
extracellular space (ECS)fluid,ision concentrations
believed to have are regulated tosimilar
a composition optimize signaling,
to CSF and at
[29], around
the
99%same
water.time wastethe
Within must be promptly
interstitial fluid,removed to minimizeare
ion concentrations neurotoxicity
regulated to[29]. optimize signaling, and
at theThe
sameECStime
andwaste
possiblymustthebePVS contain removed
promptly a networktoofminimize
proteins called the extracellular
neurotoxicity [29]. matrix (ECM).
ECMThe mayECS impedeandfluid and mass
possibly the PVStransport
containin the ECS andof
a network PVS. Cerebral
proteins ECM
called is primarily
the extracellularcomposed
matrix
of brain-specific
(ECM). ECM may glycosaminoglycans
impede fluid and (GAGs) [5]. GAGs
mass transport in give the ECM
the ECS characteristics
and PVS. Cerebral ECM of excellent shock
is primarily
absorption and high polarity, attracting water molecules and acting as
composed of brain-specific glycosaminoglycans (GAGs) [5]. GAGs give the ECM characteristics ofa lubricant.
excellent shock absorption and high polarity, attracting water molecules and acting as a lubricant.
Fluids 2019, 4, 196 5 of 33
Fluids 2019, 3, x 5 of 33
2.2.2.2. Relevant
Relevant Transport
Transport
ForForthethe application
application ofofwaste
wasteclearance,
clearance,molecules
moleculesto to be
be transported
transported out out of
ofthethebrain
brainreside
resideininthe
the
extracellular space, which is filled with interstitial fluid. The ECS is a tortuous and narrow spacespace
extracellular space, which is filled with interstitial fluid. The ECS is a tortuous and narrow where
thewhere
widththe width of channels
of channels measuresmeasures
40–60 nm40–60 nm [30] 2(Figure
[30] (Figure insert).2 Molecules
insert). Molecules
move by move by diffusion
diffusion down a
down a concentration gradient, colliding with each other and the cell
concentration gradient, colliding with each other and the cell walls. Some may interact with the walls. Some may interact with
cell
the cell walls or ECM, slowing their progress. Diffusion may be
walls or ECM, slowing their progress. Diffusion may be augmented by advection, or interstitial flow. augmented by advection, or
interstitial flow. If flow exists in the interstitial spaces of the brain, it may be at most similar in
If flow exists in the interstitial spaces of the brain, it may be at most similar in magnitude to interstitial
magnitude to interstitial flow in other tissues of the body, measured at 10–100 μm/min [20,21]. The
flow in other tissues of the body, measured at 10–100 µm/min [20,21]. The interstitial fluid of the brain
interstitial fluid of the brain is believed to be similar to the CSF, mostly water and a Newtonian fluid
is believed to be similar to the CSF, mostly water and a Newtonian fluid [31]. The Reynolds number
[31]. The Reynolds number (Re) of a potential flow in the interstitial spaces is−7therefore around Re =
(Re) of a potential flow in the interstitial spaces is therefore around Re = 10 to 10−6 , indicating a
10−7 to 10−6, indicating a creeping (Stokes) flow where viscous forces dominate. For large molecules,
creeping (Stokes) flow where viscous forces dominate. For large molecules, diffusive and advective
diffusive and advective rates are similar, and the coupling of fluid and mass transport leads to more
rates are similar, and the coupling of fluid and mass transport leads to more efficient waste removal.
efficient waste removal.
In In
thetheperipheral
peripheralbody body(outside
(outsidethe thebrain),
brain), aa molecule
molecule moves through the
moves through the ECS
ECStotothethenearest
nearest
capillary or lymph vessel, an average of 20 µm away. The molecule permeates
capillary or lymph vessel, an average of 20 μm away. The molecule permeates the wall of the vessel the wall of the vessel
andandenters a “pipe” with significant −1
enters a “pipe” with significantflow.flow.The
Theaverage
averagevelocity
velocityofoflymph
lymph is is about
about 11 mmmm ss−1 [32]
[32](1000
(1000
times faster than interstitial flow). Re for lymphatic vessel flow is about 0.1,
times faster than interstitial flow). Re for lymphatic vessel flow is about 0.1, indicating laminar flow indicating laminar flow
where
where inertial forces also govern fluid dynamics. In brain tissue, due to the BBB and the absence ofof
inertial forces also govern fluid dynamics. In brain tissue, due to the BBB and the absence
lymph
lymph vessels,
vessels,molecules
molecules must
musttransport
transporteither
eitherall allthe
theway
wayto to the
the CSF surrounding
surroundingthe thebrain
brainorortotothe
the
nearest perivascular space. The path from ECS
nearest perivascular space. The path from ECS to perivascular to perivascular space is approximately 60 µm
approximately 60 μm and the and the
path from
path fromECSECS to to
CSFCSFis is
a distance
a distanceofofmillimeters.
millimeters.ItIt waswas traditionally thought
thoughtthat thattransport
transportthrough
through
brain tissue
brain was
tissue was bybydiffusion
diffusion only
onlytotothe CSF.
the CSF.If Ifperivascular
perivascularadvection
advectionoccursoccurswith
withvelocities
velocitiesatat even
even a
a fraction
fraction of lymphatic
of lymphatic flow,flow,
waste waste clearance
clearance rates
rates areare significantly
significantly faster.
faster.
Periarterial Perivenous
influx 100 μm efflux
���
����
���&
Diffusion
?
Figure 2. Illustration
Figure 2. Illustration ofof
details
detailswithin
withinBrain
BrainTissue.
Tissue.Arterioles
Arterioles and and venules penetratethe
venules penetrate thetissue,
tissue,each
each
surrounded
surrounded by by
perivascular
perivascular space (PVS).
space TheThe
(PVS). blood-vessel
blood-vesselwall forms
wall the the
forms inner barrier
inner of the
barrier PVS,PVS,
of the note
thenote
tight junctions between cells. Astrocytic endfeet form the outer barrier of the
the tight junctions between cells. Astrocytic endfeet form the outer barrier of the PVS, with looser PVS, with looser
gaps between
gaps between cells allowing
cells allowingtransport
transportinto
intothe
theinterstitium.
interstitium. The interstitium
interstitium isis crowded
crowdedwith withcells
cells
where both fluid and solutes move along a tortuous path in a restricted extracellular
where both fluid and solutes move along a tortuous path in a restricted extracellular liquid volume liquid volume that
comprises approximately
that comprises 20% of the
approximately 20%total
of volume
the total(see insert).(see
volume Green arrows
insert). Greenindicate
arrows fluid transport,
indicate fluidby
advection
transport,andby diffusion.
advection In the
and glymphatic
diffusion.hypothesis, fluid moves
In the glymphatic along the periarterial
hypothesis, fluid movesspace intothe
along the
interstitium
periarterialand outinto
space along
the the perivenous
interstitium andspace. Purple
out along indicates interstitial
the perivenous space. Purplesolutes; solutes
indicates exit the
interstitial
interstitial
solutes; space
solutesthrough
exit thegaps in the astrocytic
interstitial endfeet
space through gapsto the perivenous
in the astrocyticspace,
endfeetwhere theyperivenous
to the are cleared
space, where
to primary they are drainage
peri-venous cleared topathways
primary peri-venous
or the CSF. drainage pathways or the CSF.
Fluids 2019, 4, 196 6 of 33
the field of brain transport, convection is the combination of both diffusion and advection). Diffusion
2.2.1. Diffusion and Advection
occurs via the random motion of molecules from high to low concentration. Diffusion is always
occurring,Movement
and its rate of depends
moleculesupon
in a fluid occurs
the size of by
thetwo possibleAdvection
molecule. mechanisms: diffusion
is the and advection
transport of a substance
(in flow.
by bulk the field
Bulkofflow
brainrequires
transport, convection
an external is the force,
driving combination of both
a common one diffusion and advection).
being a pressure gradient. In
Diffusion occurs via the random motion of molecules from high to low concentration. Diffusion is
a free medium, advection is molecular-size independent; all solute molecules move in the direction
always occurring, and its rate depends upon the size of the molecule. Advection is the transport of a
and with the velocity of the bulk flow. The characteristic time scale (Figure 3), useful in comparing the
substance by bulk flow. Bulk flow requires an external driving force, a common one being a pressure
rates gradient.
of different processes, for each transport mechanism is
In a free medium, advection is molecular-size independent; all solute molecules move in
the direction and with the velocity of the bulk flow. The characteristic time scale (Figure 3), useful in
L2
comparing the rates of different processes,τfor each
di f f usion transport
= , mechanism is (1)
D
𝜏 = 𝐿 , (1)
τadvection = L/v,𝐷 (2)
where
L = relevant length scale,
𝐿 = relevant length scale,
v = velocity, and
𝑣 = velocity, and
D =𝐷 diffusivity.
= diffusivity.
Figure
Figure 3. Characteristic
3. Characteristic transport
transport time
time in in
thethe interstitialspace
interstitial spaceofoftissues
tissuesas
asaafunction
function of
of molecular
molecular size.
size. For small molecules, diffusion is much faster than advection. However, as
For small molecules, diffusion is much faster than advection. However, as molecular size increases, molecular size even
increases, even the slow advective velocities present in interstitial tissue have a significant impact on
the slow advective velocities present in interstitial tissue have a significant impact on transport rate.
transport rate.
Often, both mechanisms occur simultaneously and the Peclet (Pe) number is utilized to understand
Often, both mechanisms occur simultaneously and the Peclet ( 𝑃𝑒 ) number is utilized to
their understand
relative impact on overall rate of transport. Pe is the rate of advection over the rate of diffusion
their relative impact on overall rate of transport. 𝑃𝑒 is the rate of advection over the rate
(or characteristic diffusion timediffusion
of diffusion (or characteristic divided time
by the characteristic
divided advectionadvection
by the characteristic time). For Pe
time). For1,𝑃𝑒
transport
≪ 1, is
predominantly
transport is predominantly Pe 1 transport
diffusion, anddiffusion, and 𝑃𝑒 ≫ 1is transport
primarily by advection.
is primarily by advection.
𝑟𝑎𝑡𝑒 𝑜𝑓 𝑎𝑑𝑣𝑒𝑐𝑡𝑖𝑜𝑛 𝐿𝑣
𝑃𝑒𝑐𝑙𝑒𝑡 𝑁𝑢𝑚𝑏𝑒𝑟 (𝑃𝑒) =rate o f advection =Lv (3)
Peclet Number (Pe ) = 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑑𝑖𝑓𝑓𝑢𝑠𝑖𝑜𝑛= 𝐷 (3)
rate o f di f f usion D
2.2.2.2.2.2. Osmotics
Osmotics
SinceSince
bodybody fluids
fluids contain
contain manysolutes
many solutes(CSF
(CSF osmolality
osmolality ==289 Osm/L),
289 Osm/L),osmosis
osmosismustmust
always be be
always
considered. If large solute gradients exist that are not rapidly adjusted by diffusion, they result in an
considered. If large solute gradients exist that are not rapidly adjusted by diffusion, they result in an
osmotic potential, or pressure. Osmotic pressure can drive water flow across cell walls to balance the
osmotic potential, or pressure. Osmotic pressure can drive water flow across cell walls to balance the
overall solute concentration, via dilution. Within the brain interstitium, most of this flow occurs
overall solute concentration, via dilution. Within the brain interstitium, most of this flow occurs locally
locally and is managed locally. Aquaporin channels (AQP), protein channels specific to water, in the
and is managed locally. Aquaporin channels (AQP), protein channels specific to water, in the vascular
Fluids 2019, 4, 196 7 of 33
walls are constantly transporting water between the interstitial fluid (ISF) and the blood to balance
osmotic differentials associated with local solute variations.
∇·v = 0, (5)
where
The mass transfer equation (Equation (4)) includes a diffusion term, a source term, a reaction or
adsorption term, and an advection term. It is adapted for porous media by including void volume and
tortuosity. Void volume is the fraction of the ECS volume to the total volume. Tortuosity represents the
degree to which molecular transport is slowed by interaction with the porous medium; it is a property
of both the medium and the molecule. Tortuosity incorporates (1) the additional distance a molecule
must travel to move around obstacles in the medium, including dead spaces (“dead-end” pores), and
(2) how its progress is slowed by interaction with the walls and extracellular matrix, or exclusion from
pathways due to molecular size. The continuity equation (Equation (5)) is a statement of conservation
of mass, shown here for a fluid of constant density.
Darcy’s law for flow in a porous media (Equation (6)) relates superficial velocity to hydraulic
conductivity and pressure gradient. Superficial velocity is a non-physical variable peculiar to porous
media flow; it is a hypothetical flow velocity calculated as if the mobile (liquid) phase were the only
phase present in a given cross-sectional area. Superficial velocity leads to ease of calculations for
volumetric flow and other variables. Intrinsic velocity is the actual liquid velocity within the ECS at
a specific location. Average intrinsic velocity (vi ) is simply superficial velocity (v) divided by void
volume, vi = v/α. Hydraulic conductivity is a parameter of the tissue, characterizing the ease with
which a fluid moves through a porous media. The hydraulic conductivity of brain ECS has been shown
to vary with void volume fluctuations [33].
Fluids 2019, 4, 196 8 of 33
Darcy’s law assumes an incompressible fluid and a flow dominated by viscous forces (Re << 1,
Stokes Flow). These assumptions hold true in brain tissue due to the very small dimensions of the
interstitial space and the small pressure differences possible within the brain (see Section 3.6.2.), leading
to small velocities. More complex models exist for flow in porous media [34], but Darcy’s law is the
most appropriate for the very low Reynolds numbers encountered in interstitial flow. “Darcy’s law has
been utilized successfully in several biomedical applications” [34] (see Swartz et al. [21] for a more
detailed discussion of interstitial flow and mass transport in biological tissues).
∂u µ αg
∂t
+ (u·∇)u − 2
ρ∇ u = − ρ1 ∇P − k0 u, (7)
inertial viscous pressure porous media
∇·u = 0, (8)
where
u = intrinsic velocity;
µ = µ λ/α = effective viscosity;
ρ = density;
P = pressure;
α = void volume;
g = gravitational acceleration, and
k0 = hydraulic conductivity
Perivascular flow may be driven by a pressure difference, ISF or CSF production, or by peristaltic
motion due to pulsation of the vascular wall (discussed further in Section 3.6.1.). In the case of
peristaltic flow, the inner boundary deforms transiently with the passage of arterial pulse waves,
adding significant complexity to the solution of Equations (7) and (8).
Literature values for the key parameters describing transport in the brain are given in Table 2 and
discussed further in Section 3.
Table 2. Key Parameters describing Transport in the Brain and their Values.
outward, advective transport of tracers from the parenchyma along the basement membranes of arterial
walls, confirming this idea [55].
A mechanism was sought for this unexpected flow within the highly-resistive basement membrane
and in opposition to blood flow—“retrograde” flow [52,56,58,59]. Weller et al. introduced the idea of a
Fluids 2019, 3, x 10 of 33
drainage pathway along the basement membrane of the smooth muscle cells, which make the outer
wall of capillaries
make the outer and
wallarteries, directly
of capillaries andtoarteries,
cervicaldirectly
lymphtonodes and
cervical separate
lymph nodesfrom
andCSF drainage
separate from [57].
This direct route from
CSF drainage the parenchyma
[57]. This direct route from to the
the lymphatic
parenchymasystemto the provided a significant
lymphatic system provided pressure
a
differential for driving
significant pressure advective
differential flow (further
for driving discussed
advective flowin (further
Sectiondiscussed
3.6.4.). However,
in Section it3.6.4.).
required
However,
a physical it required
separation a physical
of the separation
periarterial spaceof into
the periarterial space into two
two compartments: (1) acompartments: (1) a
basement membrane
basement
drainage route membrane drainage route
and (2) a perivascular andconnecting
route (2) a perivascular
SAS CSF route connecting
to the parenchyma SAS CSF to the
(Figure 4). The
parenchyma (Figure 4). The basement membrane route was termed
basement membrane route was termed the “perivascular” space by Weller and has been called the “perivascular” space by the
Weller and has been called the intramural periarterial space by others [12,29]; intermural periarterial
intramural periarterial space by others [12,29]; intermural periarterial is used throughout this text. The
is used throughout this text. The route connecting the SAS to the parenchyma was termed the
route connecting the SAS to the parenchyma was termed the “paravascular” space by Weller and the
“paravascular” space by Weller and the extramural periarterial space by others [12,29], extramural
extramural periarterial
periarterial space by others
is used throughout this text.[12,29],
A layer extramural periarterial
of pia-arachnoid tissue was isproposed
used throughout
to separatethis
the text.
A layer of pia-arachnoid
two spaces. tissue was proposed to separate the two spaces.
Figure
Figure 4. Hypothetical
4. Hypothetical perivascular
perivascular (called
(called intramural
intramural periarterial
periarterial in the
in the text)
text) andand paravascular
paravascular (called
(called periarterial
extramural extramural periarterial
in the text) in thepathways
flow text) flow in
pathways in Paravascular
an artery. an artery. Paravascular flow
flow moves movesfrom
inward
inward
the SAS from
to the thetissue
brain SAS tobetween
the brainastrocyte
tissue between astrocyte
end feet and piaend feet and
mater. pia mater. Perivascular
Perivascular flow moves flow
outward
moves outward from the brain tissue in basement membranes between
from the brain tissue in basement membranes between smooth muscle cells, ultimately smooth muscletocells,
cervical
ultimately to cervical lymph vessels. Reprinted from Ref [60]. Copyright 2018 Biomedical Central.
lymph vessels. Reprinted from Ref [60]. Copyright 2018 Biomedical Central.
In 2012, an imaging study enabling observation of tracer movement in live subjects shed further
In 2012, an imaging study enabling observation of tracer movement in live subjects shed further
light upon this idea of preferential transport routes. Using in vivo two-photon laser scanning
light upon this idea of preferential transport routes. Using in vivo two-photon laser scanning
microscopy, Iliff et al. (the Nedergaard group) showed movement of tracers from subarachnoid CSF
microscopy,
along the Iliff et al. (thespace
periarterial Nedergaard group)
into the brain [27].showed movement
Fluorescent ovalbumin of tracers
(43 kDa)from was subarachnoid
shown to enter CSF
alongthethebrain
periarterial space into the brain [27]. Fluorescent ovalbumin (43 kDa)
specifically within the periarterial space, between the smooth muscle (of the outer was shown to enter
vascular
the brain specifically within the periarterial space, between the smooth muscle (of the
wall/inner perivascular wall) and the astrocytic endfeet (of the outer perivascular wall). Three tracers outer vascular
wall/inner perivascular
of varying molecularwall) and
size (3, 40,the
andastrocytic
2000 kDa)endfeet (of the to
were observed outer
moveperivascular
from the SAS wall). Three
rapidly and tracers
at
similarmolecular
of varying rates along size
the periarterial
(3, 40, and space
2000into the brain,
kDa) which was to
were observed interpreted
move from as advective
the SASflow. The and
rapidly
two smaller
at similar rates alongtracers
thecrossed the “periarterial
periarterial space into thewall”, moving
brain, which into
wastheinterpreted
interstitium.asThe rate of flow.
advective
fluorescence increase in the interstitium was slower for the larger molecule (see Section 3.6.2. for the
The two smaller tracers crossed the “periarterial wall”, moving into the interstitium. The rate of
effect of molecular size on transport rates). Iliff et al. used ex vivo microscopy to observe the tracers
fluorescence increase in the interstitium was slower for the larger molecule (see Section 3.6.2. for the
leaving the interstitium along large venous structures to primary perivenous drainage pathways.
effect of molecular size on transport rates). Iliff et al. used ex vivo microscopy to observe the tracers
Investigating tracer transport from the brain interstitium outward, Iliff and colleagues observed
leaving
the clearance rate of along
the interstitium large venous
interstitially deliveredstructures
dextran-10to(10
primary
kDa) was perivenous
identical to drainage
mannitolpathways.
(380 Da)
Investigating tracer transport from the brain interstitium outward,
[27], confirming the findings of Cserr. Having observed transport from the CSF Iliff and colleagues observed
into the brain
the clearance
interstitiumratetoofbeinterstitially
molecular-size delivered
dependentdextran-10 (10 kDa)
with exclusion was
of the identical
largest tracerto mannitol
from (380 Da) [27],
the interstitium,
and transport from the interstitium out to be molecular-size independent; Iliff et al. hypothesized a
molecular-size dependent resistance at the periarterial wall [27].
Fluids 2019, 4, 196 11 of 33
confirming the findings of Cserr. Having observed transport from the CSF into the brain interstitium
to be molecular-size dependent with exclusion of the largest tracer from the interstitium, and transport
from the interstitium out to be molecular-size independent; Iliff et al. hypothesized a molecular-size
dependent resistance at the periarterial wall [27].
Seeking to understand the biological mechanisms at the perivascular wall, Iliff et al. conducted
experiments comparing clearance from Aqp4 knockout (KO) mice, mice lacking AQP4 channels, to
wild-type (WT) mice [27]. AQP4 is a water channel that is preferentially expressed on the astrocytic
endfeet (covering approximately 50% of their surface [61]) comprising the perivascular wall. A 70%
reduction in mannitol clearance was observed in KO mice compared to the WT mice. Periarterial influx
was observed to be unaffected, while the 3 kDa tracer movement into the interstitium was abolished
in the KO mice. The authors hypothesized that astroglial AQP4 channels might be a low-resistance
pathway for water movement through the interstitium, maintaining the advective flow circuit from the
periarterial to perivenous space.
1. CSF from the subarachnoid space moves along periarterial spaces into the brain (in the same
direction as blood flow, termed antegrade) by advective transport;
2. From the periarterial space the fluid moves into the brain interstitium, facilitated by AQP4
channels on astrocytic endfeet comprising the perivascular wall;
3. The fluid flows across the interstitium, dissolving or entraining waste molecules, and
4. Carries them to the perivenous space, where they are transported out of the brain via primary
perivenous drainage pathways.
This theory was named the glymphatic system due to its dependence on glial cells (astrocytes)
and its lymphatic-like purpose—a circulatory system outside the cardiovascular system that clears
extracellular waste. The glymphatic system thus proposed both perivascular and interstitial advection,
adding perivascular velocity, perivascular wall permeability, and interstitial hydraulic conductivity to
the list of key parameters describing transport in the brain (Table 2).
Several researchers have confirmed the antegrade perivascular flow observed by Iliff et al.—solutes
moving from the SAS into the parenchyma along periarterial space and out of the parenchyma via
perivenous space [14,25,50,62–65]. In addition, new findings offer different interpretations of previous
observations supporting intramural periarterial flow. New theories of beta-amyloid plaque formation,
e.g., shear-induced amyloid formation [66], have developed that are independent of the direction of
perivascular flow. Bedussi et al. demonstrated a single periarterial compartment, rather than physically
separated compartments that might allow flow in opposite directions [65]. Fixation artifacts [39] may
explain Carare’s observation of intramural periarterial transport. However, multiple observations,
including in vivo, of periarterial efflux [55,67–69] have not yet been explained.
Smith et al. (the Verkman group) repeated Iliff et al.’s experiments in 2017, corroborating transport
along the periarterial space faster than diffusion, but finding diffusion alone was adequate to describe
transport in the interstitium and also observing no differences in clearance for Aqp4 KO vs. WT
mice [62]. To our knowledge, Smith’s work is the only attempt at a direct reproduction of Iliff’s AQP4
experiments. However, several groups investigating the impact of the glymphatic system on different
pathologies have corroborated Iliff et al.’s findings. A recently published meta-analysis cites decreased
ISF tracer clearance in Aqp4 KO mice and rats in five of six studies (the one outlier being Smith et al.).
and demonstrates a clear positive correlation between AQP4 and ISF tracer clearance [70]. Further,
Smith et al. used different injection methods and a different strain of Aqp4 KO mice. “The meta-analysis
identified the anesthesia protocol, wide range of experimental animals, and injection paradigm were
potential causes for the conflicting data reported by Smith” [70].
Fluids 2019, 4, 196 12 of 33
Arachnoid granulations are small protrusions of arachnoid matter into the dural sinuses (Figure 1B).
They are the traditional outflow route for CSF from the SAS into the venous system. Bradbury et al.
hypothesized that most of the large solutes in ISF flowed out of the parenchyma into a portion of
CSF that subsequently left the brain via the cribiform plate to the nasal mucosa [69]. Perineural CSF
surrounding olfactory nerve sheets carries fluid to nasal lymph vessels that cross the cribiform plate
(Figure 1A), which separates the cranium from the nasal mucousa [79]. Nasal lymphatic vessels are
possibly the most important route for CSF outflow to extracranial lymph vessels [80–82]. Meningeal
lymph vessels are described below (Section 3.4.2.). Looking at Figure 1, the above list offers exit routes
surrounding the brain, meaning a local CSF efflux route would be available to any perivascular exit
from the parenchyma.
The exact proportion of clearance along each CSF efflux route is not yet known and may vary
adaptively in response to other efflux routes [63,79,83]. For example, mice born without meningeal
lymphatic vessels demonstrate no change in intracranial pressure (ICP) or brain water content [63],
indicating another pathway has increased its CSF efflux to balance CSF production. With multiple
efflux routes identified, it follows that fluid and solutes may simply leave the cranial vault by the path
of least resistance.
The second hypothesis, efflux through a route that does not mix with the CSF, has been supported
and investigated by Carare [55], Weller [57], and others [58,84]. They propose an intramural periarterial
route that passes through the subarachnoid space, but remains physically separated from the CSF.
“Interstitial fluid and solutes drain from the brain parenchyma into the basement membranes of
capillaries (and then along) the basement membranes between smooth muscle cells of arteries. The
ISF and solutes follow (a similar route along surface) arteries and continue through the base of the
skull along the carotid artery to cervical lymph nodes. A layer of pia-arachnoid separates (the route in
Fluids 2019, 4, 196 14 of 33
surface) arteries from the CSF in the SAS” [57]. This intramural periarterial efflux route correlates with
observations of beta-amyloid deposition and periarterial efflux, and provides a pressure difference
for periarterial efflux [55,68,84]. Using molecular characterization, Hannocks et al. recently found no
evidence of a pial barrier physically separating surface vessels (both arteries and veins) from the SAS
CSF [26], which was in contrast to previous electron micrograph morphological studies [85].
Table 3. Estimated interstitial velocity for awake state based on measured changes in void volume
from Xie et al. and proposed interstitial velocity in the anesthetized state for mice.
State Void Volume Hydraulic Conductivity (cm2 mmHg−1 s−1 ) Pavg (mmHg) Velocity (µm min−1 )
Asleep 0.23 [37] 2× 10−6 [43,44] 0.5 15 [46]
Awake 0.14 [37] 2 × 10−7 0.5 <1 [46]
Fluids 2019, 4, 196 15 of 33
Fluids 2019, 3, x 15 of 33
1.8
Simulation
Experiment
1.6
Awake
1.4
0.8
Asleep
0.6
0.4
0.2
0
0 25 50 75 100 125 150 175 200
Time (Sec)
Figure 5.
5. Comparison
Comparison ofof
RTI experimental
RTI experimentaldatadata
[37][37]
(orange) and finite-element
(orange) simulation
and finite-element [46] (blue)
simulation [46]
using parameters
(blue) from Table
using parameters from 3Table
for asleep and awake
3 for asleep states. states.
and awake CurvesCurves
report tracer
reportconcentration versus
tracer concentration
time. Simulations
versus agree well
time. Simulations agreewith
well experimental data,data,
with experimental supporting the hypothesis
supporting of an
the hypothesis increase
of an in
increase
hydraulic
in hydraulicconductivity
conductivityand
andbulk
bulkflow
flowduring
during sleep.
sleep. Reprinted
Reprinted from
from Ref
Ref [46]. Copyright
Copyright 2019
Biomedical Central.
Additional evidence suggests changes in ECS structure and glymphatic activity with state of
consciousness. The The effect of anesthesia type and protocol on tracer penetration and clearance in the
brain was
was investigatedusing
investigated usingDCE DCEMRI, MRI,with withdiffering
differingresults
results [5,72].
[5,72].Apparent
Apparent diffusion
diffusion coefficient of
coefficient
water
of water(ADC w ) measurement
(ADC w) measurement by diffusion tensor-weighted
by diffusion imagingimaging
tensor-weighted (DTI) MRI has also
(DTI) MRIbeen has developed
also been
as a non-invasive
developed method of tracking
as a non-invasive method changes in ECSchanges
of tracking structure, inespecially between
ECS structure, wakefulness
especially and
between
anesthesia [72,90]. Vorisek and Sykova concluded ADC is less exact than
wakefulness and anesthesia [72,90]. Vorisek and Sykovaw concluded ADCw is less exact than RTI, but RTI, but can be correlated
with
can be changes in void
correlated withvolume
changes [90].inThe
void most significant
volume [90]. Thesupport
mostissignificant
a recent fMRI studyisina humans
support that
recent fMRI
demonstrates
study in humans enhanced fluid flow in enhanced
that demonstrates the brain during
fluid flowsleepin[91]
the (see
brain Section
during3.6.1 for[91]
sleep further
(seedetails).
Section
3.6.1 Xie
for et al.’s experiments
further details). are unique thus far, having trained an animal to sleep in the experimental
apparatus. Sleep
Xie et al.’s has a complex
experiments are unique biochemistry
thus far, havingand trained
physiology, making
an animal it difficult
to sleep to simulate
in the experimental
pharmacologically.
apparatus. Sleep hasThe recovery biochemistry
a complex from anesthesia andtophysiology,
a “normal” making awake state is also to
it difficult difficult
simulateto
objectify. The work The
pharmacologically. referenced
recovery in this
fromsection, combined
anesthesia with Mestre
to a “normal” et al.state
awake [70] highlights a strong
is also difficult to
correlation
objectify. Theof glymphatic systeminfunction
work referenced with both
this section, anesthesia
combined withand alertness.
Mestre et al. Until these relationships
[70] highlights a strong
are better understood,
correlation of glymphatic studies shouldfunction
system be interpreted
with bothwith care. (The typical
anesthesia anesthesiaUntil
and alertness. for inthese
vivo
microscopy
relationships andareRTI experiments
better understood,in this field isshould
studies ketamine/xyaline
be interpreted administered
with care.by injection.
(The typicalThe typical
anesthesia
anesthesia
for in vivoformicroscopy
DCE MRI experiments in this fieldin
and RTI experiments is isoflurane
this field administered by inhalation.)
is ketamine/xyaline administered by
injection. The typical anesthesia for DCE MRI experiments in this field is isoflurane administered by
3.6. Unanswered Questions
inhalation.)
The glymphatic hypothesis (see Figure 2, and Section 3.2) leaves a few questions unanswered:
3.6.what
(1) Unanswered Questions flow in the periarterial space, (2) what provides the pressure differential
drives advective
for flow
The across the interstitium,
glymphatic hypothesis (3) (seehow do AQP4
Figure 2, and water
Sectionchannels
3.2) leaves (preferentially
a few questions expressed on the
unanswered:
astrocytic endfeet of the perivascular wall) facilitate transport, and (4) what
(1) what drives advective flow in the periarterial space, (2) what provides the pressure differentialis the route and mechanism
of
forperivenous
flow acrossefflux? The following
the interstitium, sections
(3) how discusswater
do AQP4 the physics,
channels theories, and dataexpressed
(preferentially regardingon these
the
unanswered questions.
astrocytic endfeet Notperivascular
of the the marked difference in results
wall) facilitate from computational
transport, and (4) what modelling due to and
is the route use
of different parameter
mechanism of perivenousvaluesefflux?
and assumptions.
The following sections discuss the physics, theories, and data
regarding these unanswered questions. Not the marked difference in results from computational
3.6.1. Perivascular Flow: Influx
modelling due to use of different parameter values and assumptions.
Potential drivers of perivascular influx are static pressure differential, arterial wall pulsation,
3.6.1.CSF
and Perivascular Flow:
production. Influx
The simplest driving force for perivascular flow would be a static pressure
gradient. However, in the glymphatic hypothesis, flow is from the SAS (via the periarterial space, then
Potential drivers of perivascular influx are static pressure differential, arterial wall pulsation,
and CSF production. The simplest driving force for perivascular flow would be a static pressure
gradient. However, in the glymphatic hypothesis, flow is from the SAS (via the periarterial space,
Fluids 2019, 4, 196 16 of 33
interstitium, then perivenous space) back to the SAS. Local variations in SAS CSF pressure are no more
than 0.03 mmHg [60,92], insufficient to drive the hypothesized flows [60].
Iliff et.al. proposed that arterial pulsation might drive periarterial flow [27]. In subsequent work,
Iliff used in vivo and ex vivo analysis to test this hypothesis. Increasing the pulsatility of cerebral
arteries with an adrenergic agonist accelerated CSF influx into brain tissue, while decreasing pulsatility
through carotid artery ligation (a commonly used experimental model of cerebral hypo-perfusion)
reduced perivascular CSF influx [93]. In earlier studies, Rennels et al. observed “rapid paravascular
influx of (tracer) could be prevented by stopping or diminishing the pulsations of the cerebral arteries
by aortic occlusion or by partial ligation of the brachiocephalic artery” [50].
Pulsation-driven flow in the periarterial space is hypothesized to occur by peristalsis. During
propagation of the arterial pressure wave, the periarterial space is occluded, forcing fluid to move
forward in the direction of blood flow. In the case of a circular tube, it has been shown that for
Newtonian fluids there is always a net forward flow of fluid in the same direction as the travelling
wave, even against a modest opposing pressure gradient [94]. Annular flow in the direction of the
traveling wave was confirmed by computational model for the dimensions of perivascular space
and an arterial pulse wave [95], predicting a high periarterial velocity of 105 µm s−1 with Re = 30.
However, this study used a pulse wavelength of 100 µm, while the actual wavelength is believed to be
on the order of meters [96]. Asgari et al. built a similar computational model incorporating porous
media dampening (Equation (7)) and using instead a wavelength of 0.1 m, and concluded there was
essentially no net flow [41]. Asgari et al. further showed how dispersion caused by wall pulsations
could increase transport rate over diffusion alone (dispersion occurs when non-ideal flow patterns,
such as within a pulsating vessel, accelerate transport of a molecule relative to diffusion and without
net forward advective motion). Table 4 presents a comparison of periarterial computational modeling
approach, assumptions, and simulation results to experimental measurements.
In 2018, both Bedussi et al. and Mestre et al. measured perivascular flow around a surface artery
that was pulsatile, correlated with the cardiac cycle, and had net antegrade flow [39,40]. Measurements
were made by tracking the movement of fluorescent microspheres. Mestre et al. observed an average
Fluids 2019, 4, 196 17 of 33
perivascular velocity of 18.7 µm s−1 and Bedussi et al. 17–19 µm s−1 , which give characteristic transport
times around 1000 times faster than diffusion-only transport. The speed of the arterial wall matched
that of the CSF, “suggesting arterial wall motion is the principal driving mechanism” of perivascular
flow [39]. Interestingly, images of the perivascular space show a cross-sectional shape that is elliptical
with a high eccentricity and comparative in area to the vascular cross section [39]. Mestre et al. observed
a parabolic flow profile. Parabolic flow results from laminar flow through “empty space”, as opposed
to porous space that would give a flattened profile. This result suggests the ECM in the PVS offers
little resistance to flow.
Bedussi et al. also performed ex vivo confocal microscopy to determine whether microspheres
continued from surface arteries into penetrating vessels. Some penetration into the brain parenchyma
was observed along paravascular spaces at the ventral (bottom) side of the brain, but none at the lateral
(side) and dorsal (top) parts of the brain [40]. This distribution agrees with DCE MRI results, where
tracer progresses from the cisterna magna at the back of the brain, forward along the ventral surface of
the brain at early times, progressing laterally at intermediate times and to the ventral surface at much
later times (within the brain of mice and rats, arteries are predominant on the ventral surface and
veins on the dorsal surface). It is unstated how much time passed in Bedussi’s experiments between
injection of microspheres and sacrifice of the animal, perhaps not enough for tracer to reach the lateral
and ventral surfaces of the brain. Bedussi et al. propose that either (1) the 1 µm microspheres were
excluded from the PVS of penetrating arteries, or (2) CSF flows along PVS on the brain surface, and
instead of penetrating the brain tissue, simply moves toward the front of the brain and exits the skull.
Mestre et al. found fixation, required for ex vivo studies, to collapse the perivascular space and
disrupt perivascular tracer distribution [39]. Upon chemical fixation, the PVS cross-sectional area was
shown to decrease by a factor of 10, with tracer moving vigorously towards the cisterna magna, in the
retrograde direction, and into the basement membrane. This result calls into question ex vivo studies of
perivascular flow, and provides a potential explanation for intramural periarterial flow observations.
A recently published study on the human brain suggests changes in cerebral blood volume
coordinated with changes in CSF production may be a driver of fluid flow through brain tissue. Fultz
et al. demonstrated the following coordinated sequence [91]
1. slow neural waves that occur during non-rapid eye movement (NREM) sleep;
2. followed by a decrease in cerebral blood flow;
3. followed by an increase in CSF production.
The low-frequency oscillatory dynamics associated with NREM are an indicator of oscillating
suppression of neural activity. The decrease in neural activity results in a decrease in blood demand.
The decrease in blood flow decreases cerebral blood volume in the fixed cranial volume. CSF flow
increases to fill the space previously occupied by blood, expanding the CSF and ISF-filled volumes
of brain tissue. This coordinated sequence was observed to oscillate with a period of approximately
20 s, and therefore is not coordinated with cardiac cycle or respiration. The results are in agreement
with observed increases in extracellular volume during sleep [37]. As this was a study of whole-brain
dynamics, it leaves open several questions about the microscopic details. Is periarterial and/or
extracellular volume increased? What are the routes of perivascular influx and efflux? How is pressure
affected in the CSF and throughout the brain tissue? However, it provides evidence of an additional
driver of perivascular flow—CSF production and cerebral blood flow dynamics.
In summary, periarterial flow has been measured and independently verified in surface arteries at
a velocity of 17–19 µm/sec [39,40], about 1000 times faster than diffusion. Although it would be ideal to
measure periarterial flow in penetrating arteries, a significant body of data supports faster-than-diffusion
movement of tracers into the brain along periarterial routes [5,25,27,37,62,65,72,77]. Antegrade flow
has also been well demonstrated, although the few observations of retrograde periarterial flow have
not yet been explained. Work remains to understand the driving force of perivascular flow, although
there is good support for arterial wall pulsation. Recent results suggest coordinated changes in cerebral
Fluids 2019, 4, 196 18 of 33
blood volume and CSF production, especially during sleep, may also contribute to fluid flow through
the brain.
Table 5. Summary of Computational Models of interstitial Flow and Mass Transport. No measurements
have been reported to date for interstitial flow.
Ray et al. used published RTI experimental data from three research groups to verify a
3-dimensional porous-media finite-element model of interstitial transport, based on Equations (4)–(6).
Using the full range of hydraulic conductivities reported in the literature and the range of pressure
differences described above, Ray et al. calculated average superficial velocities of 0.05–250 µm min−1
(Table 6). Quantitative comparison of tracer transport simulation results to RTI experimental data
for anesthetized subjects gave best agreement at a superficial velocity of order v = 10 µm min−1 [46],
comparable to interstitial velocity measure in other tissues (Figure 3) [20,21].
Table 6. Interstitial superficial-velocity simulation results for various authors. A range of hydraulic
conductivity values and a range of periarterial wall pressures were used (further described in the text).
Velocity reference is Ray et al. [46] unless otherwise noted.
starts to become dominant and less molecular-size dependence is expected. Further, the characteristic
diffusion time for Dex 40 kDa is about 200 min, while the characteristic advection time is around
20 min. Slow flows that are not significant to the transport of small molecules can be hugely impactful
for large, slow-to-diffuse molecules.
Abbott argues that interstitial advection would lead to disruption of the delicate chemical
micro-environment required for brain function, and suggests the possibility of perivascular flow to the
capillary level as an alternative to interstitial flow [29] (the glymphatic hypothesis states perivascular
flow to the arteriole level, where the PVS terminates, truncating flow). This idea follows from the
work of Hannock et al. who used cellular markers to identify the perivascular space and observed
two molecularly distinct basement membranes through to the level of capillaries, hence a potential
complete perivascular route from arterioles through capillaries to venules [26]. Capillaries are separated
by approximately 10–20 µm, resulting in a short diffusional path through the interstitium. If this
pericapillary theory were correct, given current experimental resolutions, observations would be
similar to those for interstitial flow.
In summary, it remains unclear if transport in the interstitium is diffusion-only transport or a
combination of advective and diffusive transport. Molecular-size dependent transport does not exclude
slow interstitial flow, which could have a significant impact on transport of large, slow-to-diffuse
molecules. By conservation of mass, the periarterial influx must go somewhere. A method for
direct measurement of interstitial flow would be very useful. In the absence of direct measurement,
quantitative transport analysis and modeling, inclusive of anatomical details, using existing tracer
data can provide useful insight and supplement currently available experimental approaches.
This theory is silent on how the solutes present in the ISF cross the perivascular wall, as AQP4 is
incapable of transporting most ISF solutes, including macromolecules [29]. However, most solutes are
able to pass through the gaps between astrocytic endfeet. AQP4 is known to play critical roles in the
transport of water between compartments [29]. Perhaps the role of AQP4 in the glymphatic system is
less direct. AQP4 channels move water between local PVS and the astrocyte cell/endfoot, swelling or
shrinking the endfoot and adjusting the gap size and therefore promote or restricting flow. Asgari et al.
tested solute transport for 20 nm and 14 nm astrocytic endfeet gaps, with the smaller gap leading to a
significant reduction in transport [41].
Improved understanding of the elements affecting transport of water and solutes across the
“perivascular wall” is an area of great opportunity. EM data of the wall structure are variable;
in vivo observation would likely improve knowledge. Experimental observations of tracer movement
demonstrate a marked decrease in PVW transport in the absence of AQP4 [27]. AQP4 presence is
strongly correlated with glymphatic function, but its mechanism is not understood.
1. Perivenous efflux to the CSF, but subsequent exit through a proximal route (glymphatic
hypothesis); or
2. Efflux via an intramural periarterial route that passes through the subarachnoid space, but remains
physically separated from the CSF, and ultimately connects to the cervical lymph.
Below, we discuss static pressure and arterial pulsation driving forces for each theory. Refer to
Section 3.6.1. for the discussion of flow driven by CSF production and cerebral blood flow dynamics.
No pulsation occurs in the veins, therefore, for perivenous efflux pressure driving force may come
from static pressure or pressure generated by periarterial pulsation. Faghih et al. calculated a very low
required pressure difference for hypothesized flow through the perivenous (called paravenous in their
paper) tree of 0.00023 mmHg [60]. Although, as discussed in Section 3.6.1, static pressure variations in
the CSF are not sufficient to drive the entire glymphatic flow circuit, arterial pulsation will result in
intermittent elevated pressure at the periarterial wall [95]. Such pressure may provide the difference
required to drive fluid across the interstitial space and still deliver the pressure difference (0.00023
mmHg) needed to propel fluid along the perivenous tree to the SAS.
Intramural periarterial efflux offers a substantial pressure differential for driving fluid efflux,
which was a major motivation for the theory. Lymph vessels are at very low or even negative pressure.
Therefore, a significantly higher pressure differential exists between the parenchyma and the lymph,
than the parenchyma and the CSF (CSF pressure is 7–15 mmHg; pressure inside a lymph vessel is
typically 1 mmHg). In addition, reflection waves from arterial pulsation on the periarterial walls have
been hypothesized to also drive periarterial efflux [59]. Calculations by Asgari et al. [105] and Faghih
et al. [60] show the resistance to flow of the basement membrane is far too great to allow the estimated
physiological flows.
In summary, intramural periarterial efflux is unlikely due to the high resistance of the basement
membrane. Perivenous efflux (following the glymphatic hypothesis) would require a small pressure
difference (0.00023 mmHg) that may be generated by periarterial pulsation driven flow and translated
across the porous interstitial space to the perivenous space.
Fluids 2019, 4, 196 22 of 33
of cell bodies and non-myelinated cell processes. These cell components are spatially disorganized,
and therefore grey matter behaves in an isotropic way. White matter is found in the deeper regions of
the brain, with grey matter making up the majority of the outer brain, or cortex.
On the subject of sleep and glymphatic function, increases in CSF beta amyloid concentrations,
measured via PET, were reported after one night of total sleep deprivation [75,109] and following the
disruption of slow wave sleep [110]. DTI MRI studies have reported changes in ADCw as a function of
sleep or arousal indicative of dynamic changes in void volume [111]. A recently published study on the
human brain shows slow-wave oscillations in neural activity during NREM sleep induce changes in
cerebral blood volume coordinated with changes in CSF production that may be a driver of increased
fluid flow through brain tissue during sleep [91].
4. Discussion
Two calculations, grounded in the observations and data reported above, are discussed below.
These calculations are preliminary, requiring assumptions to complete, but none-the-less provide
perspective about transport processes occurring in the brain, especially with respect to how transport
is affected by molecular size.
Figure 6.
Figure Illustrations for
6. Illustrations for characteristic
characteristic time
time and
and mass
mass balance
balance calculations.
calculations. (a)
(a) A
A cerebral
cerebral penetrating
penetrating
artery and
artery and vein
vein with
with perivascular space. Fluid
perivascular space. Fluid (green
(green arrows)
arrows) moves
moves into
into the
the brain
brain along
along periarterial
periarterial
space, branching into arteriole PVS. Characteristic length for periarterial transport is the
space, branching into arteriole PVS. Characteristic length for periarterial transport is the entireentire path from
path
penetrating
from penetrating artery to terminating arteriole (drawing not to scale). From periarterial space,enters
artery to terminating arteriole (drawing not to scale). From periarterial space, fluid fluid
the interstitium
enters and moves
the interstitium to perivenous
and moves space.space.
to perivenous In an In
optimized system,
an optimized τPV =𝜏 τIS .=(b)
system, 𝜏 Whole-brain
. (b) Whole-
mass balance. Flow in through periarterial space, measured at the dotted line for
brain mass balance. Flow in through periarterial space, measured at the dotted line for branchesbranches of major
of
cerebral arteries, equals flow out across the interstitium, represented by dotted line between arterioles
major cerebral arteries, equals flow out across the interstitium, represented by dotted line between
and venules. (c) Control volume: one venule surrounded by six arterioles [44]. Fluid flow is from
arterioles and venules. (c) Control volume: one venule surrounded by six arterioles [44]. Fluid flow is
periarteriole to perivenule space (green arrows). Cross-sectional area (XCIS ) is a cylinder surrounding
from periarteriole to perivenule space (green arrows). Cross-sectional area (XCIS) is a cylinder
the venule at the midpoint between the arterioles and the venule.
surrounding the venule at the midpoint between the arterioles and the venule.
Using the equations for characteristic time (Equations (1) and (2)) and results from the literature,
Using the equations for characteristic time (Equations (1) and (2)) and results from the literature,
characteristic transport times are given in Table 7. The perivascular space is like the highway, where
characteristic transport times are given in Table 7. The perivascular space is like the highway, where
molecules move a long distance at a fast rate. Since periarterial advection is quite fast, diffusion is
molecules move a long distance at a fast rate. Since periarterial advection is quite fast, diffusion is
insignificant to overall transport within the PVS. The characteristic time for periarterial transport is
insignificant to overall transport within the PVS. The characteristic time for periarterial transport is
about 9 min. The interstitial spaces between cells are like the neighborhood streets, where molecules
about 9 min. The interstitial spaces between cells are like the neighborhood streets, where molecules
move slowly over a short distance. Since interstitial flow is likely slow, characteristic interstitial
move slowly over a short distance. Since interstitial flow is likely slow, characteristic interstitial
transport time will be a combination of diffusion and advection, where the characteristic time for
transport time will be a combination of diffusion and advection, where the characteristic time for
diffusion varies with molecular size.
diffusion varies with molecular size.
The dominant mechanism of interstitial transport depends on molecular size. For small molecules,
The dominant mechanism of interstitial transport depends on molecular size. For small
interstitial diffusion alone has a similar characteristic time to periarterial transport. Interstitial advective
molecules, interstitial diffusion alone has a similar characteristic time to periarterial transport.
flow is not needed to optimize the system. However, for larger molecules, diffusion across the interstitial
Interstitial advective flow is not needed to optimize the system. However, for larger molecules,
space is slower than periarterial transport, and interstitial advection likely plays an important (or
diffusion across the interstitial space is slower than periarterial transport, and interstitial advection
dominant) role in augmenting diffusion to achieve an optimal transport time. For Dextran 3 kDa, of
likely plays an important (or dominant) role in augmenting diffusion to achieve an optimal transport
similar size to beta amyloid, the characteristic times for interstitial advection and diffusion are nearly
time. For Dextran 3 kDa, of similar size to beta amyloid, the characteristic times for interstitial
equal—both processes work together to achieve an optimal interstitial transport time of around 7
advection and diffusion are nearly equal—both processes work together to achieve an optimal
min. A very large molecule, like IgG (150 kDa), has a characteristic diffusion time of over 500 min
interstitial transport time of around 7 min. A very large molecule, like IgG (150 kDa), has a
in interstitial space. Without interstitial advection, its transport would be extremely slow. Slow
characteristic diffusion time of over 500 min in interstitial space. Without interstitial advection, its
interstitial advection may be a significant factor for transport of large molecules, and particularly
transport would be extremely slow. Slow interstitial advection may be a significant factor for
protein aggregates implicated in neurodegenerative diseases.
transport of large molecules, and particularly protein aggregates implicated in neurodegenerative
This same idea applies to concerns over interstitial flow “upsetting” the chemical microenvironment
diseases.
required for proper neural function. For small molecules, like the ions active in neural processes,
This same idea applies to concerns over interstitial flow “upsetting” the chemical
diffusive transport is happening so much faster than potential advection that small molecules will
microenvironment required for proper neural function. For small molecules, like the ions active in
quickly diffuse to maintain concentrations. For example, the potassium ion (K+ ) has a characteristic
neural processes, diffusive transport is happening so much faster than potential advection that small
diffusion time of about 1 min in interstitial space, while characteristic advection time is around 19 min.
molecules will quickly diffuse to maintain concentrations. For example, the potassium ion (K+) has a
By comparison, neural activities occur on a time scale of msec.
characteristic diffusion time of about 1 min in interstitial space, while characteristic advection time is
around 19 min. By comparison, neural activities occur on a time scale of msec.
Fluids 2019, 4, 196 25 of 33
Table 7. Characteristic Transport Times in the Mouse Brain. In an optimized continuous system,
characteristic times for each step will be nearly equivalent. Periarterial transport is dominated
by advection, therefore its overall transport time is equal to the characteristic time for advection.
Interstitial transport is by diffusion and advection, with the relative importance of each depending on
molecular size. For the overall interstitial characteristic time, diffusion is augmented by advection,
with 1/τoverall = 1/τdi f f usion + 1/τadvection .
1. Flow rates for transport in tissues are most frequently reported as either velocity or volumetric
flow rate per gram of tissue, known as rate of perfusion, and
2. Fluids in the brain are incompressible and at nearly constant temperature, making mass and
volume equivalent.
For overall transport in the brain, we equate total flow into the brain along perivascular routes
of penetrating cerebral arteries with total flow across the interstitium (see dotted lines in Figure 6b).
Equating these two flow streams makes an inherent assumption that any sources or sinks of fluid in
the interstitium are small compared to the overall flow. (Metabolic production of water by cells is a
small percentage of CSF production (0.03/0.3 µL g−1 min−1 = 10% [12]), and we assume a net zero flow
of fluid across the BBB). Volumetric flow rate (Q) is given by
Q = XC ∗ v, (9)
where XC is cross-sectional area. Proposed values for perivascular and interstitial velocity are available
from experimental measurements and simulations. The key assumptions are made in estimating the
cross-sectional area to calculate a volumetric flow rate. Results are reported in Table 8.
Fluids 2019, 4, 196 26 of 33
Table 8. Volumetric flow balance comparisons for periarterial and interstitial flow in the mouse brain,
compared with published clearance rates estimating overall clearance by a perivascular route.
Interstitial—
Description Periarterial Interstitial— Awake
Anesthetized/Asleep
Total Cross-Sectional Area (mm2 ) 0.2 200–500 200–500
Velocity (um/sec) 18 [39,40] 0.2 [46] 0.01 [46]
Total Volumetric Flow Rate (µL min−1 ) 0.2 2–6 0.2–0.4
1
Calculated Perfusion Rate (µL g−1 min−1 ) 0.5 5–15 0.4–1
0.3–5.5 [37] 2.8–5.5 [37] 0.3–1.5 [37]
Literature Perfusion Rate1 (µL g−1 min−1 )
0.2–1.2 [116] 0.8–1.2 [116] 0.2–0.5 [116]
1 Based on clearance of normally occurring molecules lacking a BBB transport route from the mouse or rate brain
For periarterial flow, the calculation is made for the perivascular space of all penetrating arteries
entering the brain tissue (Figure 6b). Detailed knowledge of mouse anatomy [117] and the shape
of a periarterial cross section [39] were used to estimate a total periarterial cross-sectional area of
about 0.2 mm2 . Assuming a constant average periarterial velocity of 18 µm/sec [39,40], gives a total
periarterial volumetric flow rate of about 0.2 µL min−1 , or 0.5 µL g−1 min−1 for a 0.4 g mouse brain.
Possible sources of error include (1) exclusion of transport across the pial surface (outer surface of
the brain) and perineural routes that would add to periarterial flow and may result in a higher total
inflow, and (2) every branch of the major arteries may not have been included, resulting in a low value
for periarterial flow. The estimated periarterial perfusion rate agrees with the lower end of reported
ranges based on clearance rate of endogenous substances from the brains of mice and rats [37,116].
For interstitial flow, a representative cross-sectional area for all the interstitial space is needed,
“perpendicular” to the superficial interstitial flow. First let us consider an appropriate “control volume”
for the smallest representative vascular arrangement. The control volume proposed by Jin et al. [44]
(Figure 6c) and the range of vascular separation reported in Section 3.6.2 are used to calculate a
cross-sectional area per vascular volume. Having an ISF cross-sectional area per vascular volume, we
multiply by the vessel volume fraction in the mouse brain (0.6–0.9% of the total volume [117]) to get
a total cross-sectional area of approximately 200–500 mm2 , 1000 times greater than the periarterial
cross-sectional area. Multiplying by an average interstitial velocity of 10 µm/min [46], gives a
total interstitial volumetric flow rate of 5–15 µL g−1 min−1 . Interstitial cross-sectional area may be
overestimated due to possible overlap of the idealized cross sections in real tissue, resulting in a high
value for total interstitial flow.
Recall that interstitial advection is believed to increase with sleep, and the interstitial perfusion
range calculated above is for an anesthetized state shown to be similar to sleep. Xie et al. measured
a clearance rate for inulin, eliminated by a perivascular route, in sleeping and anesthetized mice of
2.8–5.5 µL g−1 min−1 [37], which agrees with the low end of the range calculated here. Based on the
decrease in void volume from sleep to wakefulness measured by Xie et al., Ray et al. estimated a
decrease in interstitial flow of 93% [46]. Estimated interstitial perfusion range adjusted for wakefulness
would be 0.4–1 µL g−1 min−1 . Groothius et al. and Xie et al. report waking clearance rates of
0.2–0.5 [116] and 0.3–1.5 [37] µL g−1 min−1 , respectively.
periarterial perfusion rates agree with reported ranges for the awake state. A mass balance also shows
estimated anesthetized interstitial flow exceeds periarterial flow. Errors inherent to the calculations
may account for this difference.
size change with state of alertness or other factors? By what mechanism does AQP4 affect transport
from periarterial to interstitial space? Such research is outside our breadth of knowledge, but many
neuroscientists are actively seeking this answer.
Fundamental knowledge of fluid flow and mass transfer in brain tissue described here and
continuing to be discovered can ultimately be applied to the development and delivery of therapeutic
solutions for the numerous disease states affected by glymphatic dysfunction.
Author Contributions: L.A.R. wrote the article. J.J.H. reviewed and edited the article.
Funding: This research was partially funded by the National Science Foundation, grant number DMS-1361240.
Acknowledgments: Roderick J. Ray provided valuable editorial input, and improved ease of reading.
Conflicts of Interest: The authors declare no conflict of interest.
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