Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : January 24, 2020
Accepted : May 17, 2020
Published online : June 30, 2020
doi:10.24659/gsr.7. 2_181

Review article

Perivascular drainage of amyloid beta (Aϐ) and waste products fromthe brain and its failure.
Nikola Barić
Private General Practice Office, Labin, Croatia

Abstract

The disorder of intracerebral accumulated amyloid beta (Aβ) drainage, as well as the drainage of various waste products
related to brain metabolism and permanent biochemical reactions, lead to brain homeostasis disorder, the breakdown ofimportant regulatory functions and to the occurrence of cerebral amyloid angiopathy and rapid development of geneticallyprogrammed Alzheimer's disease (AD). The brain does not have its own standard lymphatic networks and therefore thisimportant role is mostly taken over by the perivascular system which is located in the areas within the basement membranesof capillaries, arterioles and arteries. The driving force of this drainage is not formed by previously stated forces that areconditioned by heart functioning through the accompanying arterial pulsations, but by the newly discovered internal force ofarterial and arteriolar walls, the so-called vasomotion. Moving in the direction opposite from the direction of the blood stream
and pulse wave, these pulsating forces are based on rhythmic intracellular oscillations of Ca2+ ion concentration in vascularsmooth muscle cells. With the participation of a number of molecular components, these oscillations cause the vasomotion
phenomenon and their disturbance leads to perivascular drainage alterations and severe complications. This paper gives us athorough analysis of crucial events that lead to the perivascular system alterations.

KEY WORDS: Alzheimer's disease, cerebral amyloid angiopathy, perivascular drainage, hydroxyl radicals,

		oxidative stress

Introduction

Alteration of the perivascular drainage fromthe brain
A number of complex studies related to the etiologyof Alzheimer's disease (AD), the severe, chronic, and lethal
neurodegenerative phenomenon, increasingly indicate theenormous importance of the regular drainage from the brain.
This drainage refers to the Aβ peptide produced in the brain,
many metabolic waste products, as well as numerous waste
products resulting from the vitally important biochemical
processes. Not entering into the detailed pathophysiologyof AD, this disease, with its polygenetic etiology, is in fact
the result of the elevated neuronal production of amyloid
beta (Aβ) peptide, its enormous extracellular cerebral
accumulation, and altered drainage from the cerebral
parenchyma. Along with the mentioned problem connectedwith Aβ, it is also important to emphasize the elevatedintracerebral accumulation of transition metals in AD,
especially iron ions, the accompanying oxidative stress, aswell as their weakened drainage out from the brain (Fig. 1, 2).
Investigations into cerebral physiology, pathophysiology, andpathology, indicate a lot of important Aβ and superfluous
iron ions drainage pathways. The crucial factor which
determines the analysis of the perivascular lymphatic brain
drainage, connected with the basement membranes (BMs) of
the arterial, arteriolar, and capillary walls interior, is the
specificity of the brain, which does not have a standard
lymphatic drainage net like other body organs. The estimatedvalue of the drainage rate of the brain is in the range of 0.110.29 µL/min/g (data obtained from rat brain by combining
histological and mathematical analyses). Some considerably
investigated nonconventional brain lymphatic drainage
pathways are: drainage through the blood brain barrier(BBB), blood cerebrospinal fluid barrier (BCSFB), choroid
plexus, arachnoid granulations (AGs), paravascular drainage(connected with the cerebral vasculature surfaces), perineuraldrainage, lymphatic drainage through the orbits into the sinus
sagitalis superior, and nasal cavity (lymph inflow along theCorresponding to: Nikola Barić MD, PhD
Presika 153, 52220 Labin, Istra, Croatia

TEL: + 385-52-852- 008
e-mail: nikola.baric@pu.t-com.hr

Glycative Stress Research 2020; 7 ( 2) : 181-195(c) Society for Glycative Stress Research_ 181 _

Perivascular Drainage from the Brain

Tf

Capillary blood

Fee33++F

Tf R

Lum in al m e m brane

Reduction

Fe 2+

FPN1 (IRON-REGULATED TRANSPORTER 1)

Prote olysis of APP-typical for AD(E.C.S.)SIGNIFICANT RISE

(APP, integral membrane protein, speciallyconcentrated in the synapses of neurons)

NEUROPILFe 2+

MBD (His13-His14)
APP proteolysis

Great affinity
ATTRAC TIO N

α-secretaseγ-secretase

Aβ

APP

sAPPβ

Fe 3+

toxic

ELEVATIO N

Aβ

Fe 3+
"e"

sAPPβ
sAPPβ
AIC D

(I.C.S.)and β2 denote two different.Aβ (β1
strands of Aβ monomer)sAPPα

Me tS35 β2

.
Aβ

Aβ

(Located on the neighbouring cells)

CP

In LOAD α-secretase effect is minimal (proamyloidogenic pathway)
There is no the formation of the neuroprotective
fragment sAPPα (secreted ectodomain α)

Ne wcom e rβ
ATTRAC TIO N
β1 Aβse cre tase3+
Fixe d m onom e r Fe
Aggregation(E.C.S.) REDUC TIO N

Plasma membrane

APP

Oxidation

sAPPα
AIC D

Endosome

BBB

DMT 1

Abluminal membrane of endothelial cells
Aβ

Tf R

Fe rric re ductase

Cytosol of the capillary wall

Fe3+
Tf

In AD by effects of β and γ secretases
the APP is broken in the three separate units(Several Aβ monomers grouped together)

Fe 2+

ApoE

.

HDL

Fe3+ OXIDATIONAβAβREDUCTIONFe2+OXIDO /REDUCTIVE CYCLEREDUC TANT

MsrA

oxidation

Fe 2+ + H2O2 = Fe3+ + − OH + *OH
β1

Fenton reaction

Tissue destruction

Oxidative stress

Toxic, aggressive,
destructive

Methionine sulfoxide

2MetSOMet SO

2e −

- 2e−

O2*−

oxidation"e − h op" re duction

Met S35 (β2) + Aβ-Fe 3+(β1) = Met S35*+(β2) + Aβ-Fe 2+(β1)
methionine
Methionine sulfide radical

Fig. 1. Schematic presentation of events connected with Aβ and Fe 3 + elevated intracranial values.

Oxidation, loss of electrons; reduction, gain of electrons; hydroxyl radical (*OH), neutral form of the hydroxide ion (OH − ),highly reactive, aggressive and short lived; MsrA, peptide, methionine sulfoxide reductase, repair enzyme for proteins inactivatedby oxidation; MetS35, methionine, one of nine essential amino acids; Fe 3+, ferric iron; Fe 2+, ferrous iron; H 2 O 2 , oxidizer,
the simplest peroxide; CP, ceruloplasmin, a ferroxidase enzyme; Aβ, amyloid beta; APP, amyloid precursor protein; ApoE,
apolipoprotein E; Tf, transferrin; TfR, Tf receptor; DMT1, divalent metal ion transporter-1; MBD, metal binding domain; FPN1,
ferroportin 1, iron-regulated transporter 1; BBB, blood brain barrier; E.C.S., extracellular space; I.C.S., intracellular space; AD,
Alzheimer's disease ; LOAD, late onset AD; HDL, high-density lipoprotein; AICD, APP intracellular domain; sAPPα, secreted
ectodomain α: sAPPβ, secreted ectodomain β; CTFB, membrane bound fraction;.

_ 18 2 _Glycative Stress Research

MBD (His13-His14)
Aβ peptide

Aβ

RAGE activation and expression
PKC activation
Phosphorylation and PKB (Akt) blockade,
PKB is GSK-3 blockader
GSK-3 (serine / threonine protein kinase) activation
tau protein hyperphosphorylation
stabilizer of microtubula in neurons (CNS),( PO3−)

tau protein

Formation of agregates of non bounded tau proteins
NFT, neurofibrillary tangles
Destabilisation and desintegration of microtubula
Alteration in the axonal transport,
favourable to the AD development and course

Two specific mutations in ADAM 10 gene with decline in α-secretase activity
Decline of cleavage Aβ fragment of APP protein
Complete Aβ peptide,after earlier APP cleavage by β and γ-secretases,
is released complete in synapse region Aβ
Accumulation of Aβ in synapse region with Aβ glycation and Aβ-AGE formation;
this is connected with the decline in formation of neuroprotective fragments
APPα induced by the normal ADAM 10 gene expression and α-secretase activity
LOAD development and its course acceleration

Fig. 2. Schematic presentation of events induced by RAGE activation and ADAM 10 gene mutations.

Aβ, amyloid beta; APP, amyloid precursor protein; AGEs, advanced glycation end products; RAGE, receptor for AGEs; glycation,non- enzymatic glycosylation, covalent attachment of sugar to protein; MBD, metal binding domain; PKC, protein kinase C,
phosphorylation, chemical addition of a phosphoryl group (PO 3− ) to an organic molecule; PKB (Akt), serine/threonine-specificprotein kinase; GSK-3, glycogen synthase kinase; NFT, neurofibrillary tangles; tau-protein, protein that stabilizes microtubulesin neurons; AD, Alzheimer's disease; LOAD, late onset AD; APPα, secreted ectodomain α; CNS, central nervous system.

_ 18 3 _Perivascular Drainage from the Brain

olfactory tract-tractus olfactorius). In addition, it is also
necessary to emphasize the processes of Aβ intracellular(proteasome and ubiquitin-proteasome route, lysosomal
cathepsin enzymes thiolmetalloendopeptidases), and Aβ
extracellular degradation (neprilysin, matrix-metalloproteinases
2, 3 and 9). However, this is not the theme of this study.
Perivascular drainage, connected with the walls of cerebral
vasculature, is increasingly the subject of intensified
investigations. This drainage, located in the BMs of the
leptomeningeal and cortical arteries, and in the BMs oftheir terminal branches, arterioles and capillaries, driven
by vasomotion forces, presents the crucial system of brain
homeostasis, and its failure leads to the occurrence of
cerebral amyloid angiopathy (CAA), earlier onset of AD, andits accelerated course (Fig. 2, 3 ) 1- 6).

Perivascular drainage pathway
Before the analysis related to the complex
pathophysiology of the development of perivasculardrainage alterations, the occurrence of CAA, and the onset
and acceleration of the course of AD, it is necessary topresent the general characteristics of this crucially important
pathway. The starting-point of this pathway is located on thewalls, or the BMs of cerebral capillaries. Molecules of Aβ
and other waste material first pass from the subarachnoid
space (SAS), where it has previously entered, and then acrossthe very permeable pia, into the paravascular space, locatedbetween the pia and the BM of the glia limitans. After thatthey pass across the BMs of glia limitans (passing throughthe astrocytic endfeet clefts and AQP4 channels), and finallyenter into the brain parenchyma, or travel further on distally
along the mentioned space up to the capillary level and here
enter into their BM.
One portion of Aβ and other waste products travel
further along the markedly narrow paravascular space, andat the parenchyma level pass through the astrocytic endfeet
clefts and AQP4 channels, and enter into the parenchyma,
while one part, moving distally, at the level of SAS, enters
across the permeable pia into its formerly abandoned space(SAS). In fact, the movement is circular. From SAS, thearriving Aβ and remaining waste can enter into the AGs andthrough them into the superior sagittal sinus (SSS), and further
on, by venous blood into the venous body system and heart.
A part of the Aβ and waste products enters the capillary wallinterior and its BMs, then into the cortical and leptomeningeal
artery media, where they move along their BMs between
vascular smooth muscle cells (VSMCs) in opposition to thedirection of blood flow. The driving force for this movement
is not dependent on arterial pulsations, or on pulse pressure,but exclusively on the vasomotion, i.e. on its vasomotion
waves. There are a number of written articles analyzing
the mechanism of this movement with the domination of
events linked with actin, myosin, and Ca 2+ oscillations in the
VSMCs. In order to understand the above presented facts, itis necessary to emphasize that Aβ is generated in the brain
parenchyma, by the proteolysis of the transmembrane protein
APP (amyloid precursor protein) located on neuronal cell
membranes. From there, Aβ goes on, passing through theBMs of glia limitans, the paravascular space, and pia, thenenters the SAS, and continues its circular pathway, as it has
been described earlier in the text (Figs. 3-5 ) 4 -9).

Basement membrane (BM) structure
The analysis of the BM by electron microscope showsthat it is composed of two layers (width about 50 µm) (Figs.
4, 5 ). The layer closer to the endothelium is the basal lamina(BL, basal membrane, tunica intima-width 20-100 nm),and the deeper layer connected with the previous one forms
the reticular connective tissue (lamina reticularis). Boththese two layers are connected by collagen VII molecularfibrils and fibrillin microfibrils. BL is composed of thesuperficially layer lamina lucida and the deeper layer, locatedalong the reticular layer of lamina reticularis, named lamina
densa. Lamina densa is 30-100 nm wide, and composed of
the collagen IV fibril net and the sulfate rich proteoglycan
perlecan (BM-specific heparan sulfate proteoglycan, whichcross links many extracellular matrix components- collagen,enzymes, glycoproteins). Lamina lucida is about 60 nm
wide and somewhat more luminous, and contains a lot of
laminins, integrins, entactins and dystroglycans. The main
components are laminins (high-molecular weight protein of
extracellular matrix, the major component of the BL). These
heterotrimeric glycoproteins (laminins) have connective
places for integrins, type IV collagen and heparan sulfate.
Their important function is the formation of the structural
net connected with the collagen IV net. Lamina reticularis isbuilt from the reticular fibers net composed of collagen type
III (homotrimer-protein composed of three long chains).
Fibers are synthetized by reticular cells. Due to the VSMCs
location (capillary BM does not have these cells) in thislayer, and due to their role in vasomotion wave’s generation,
it is evident that perivascular drainage takes place along thislayer.
The essential substance of the lamina reticularisis the poroelastic extracellular material and the fluid
component, i. e. interstitial fluid (ISF), which is primarily
water. Besides collagen, the other important componentsof the arterial wall are elastin molecules grouped in thinconcentric porous plates. Each of these laminas is connectedwith the concentric ring of VSMCs forming the flexible
lamellar unit of the arterial wall 10). BMs are becoming
increasingly important in biology 4). According to the mostrecent research, the BMs take part in blood filtration, muscle
homeostasis, the storing of growth factors, the cytokine
control of angiogenesis, and tumor growth. Their functions
are extensively presented by Moris AW et al. 11), Pozzi A etal. 12), and Hawkes CA et al. 13). The BL in the CNS is the
product of vascular endothelial cells secretion (continuous
endothelial lining). It is visible only by electron microscope.
Besides the supporting function to the neighboring cells, theBMs are also an important storage site for growth factors
and take part in physiological processes in these cells. It isespecially important to emphasize their role as the main
components of the perivascular drainage pathway (IPAD,
intra-mural-peri-arterial drainage) (Figs. 4, 5 ).

Perivascular drainage alterations in AD
The analysis of the alterations of the perivascular Aβ
drainage of iron ions, as well as the harmful waste products
resulting from a number of biochemical processes in thebrain, crucial for the onset and course of AD, primarily
requires an insight into the complex morphological and_ 18 4 _Glycative Stress Research

Subdural cavity
O uter basement membrane of VSMC s
S UPERIO R SAGITAL SINUS
Endoth e lium

Inner basement membrane of SMC

Dura mater

Entrance into the sinus

AG

Dura mater

SS S

Outer

To the he artm e diaHDL

e n dothe liu m

Tf-Fe3+Arterial blood

media

SS S
arachnoidea
VS MC

Aβ

endothelium
pia

Paravascular drainage Aβ
Blood from the heart
Perivascular drainage

Aβnutrient

CSF

Pial fun ne l

Vein

Aβ

pia

endothelium
Virch ow-Robin spaceCAβarachnoidea

AβAβPe Perivascular drainage
Aβ

ArteryAβAβAβ

Ve sse l pia

O2

VRS

BM

C6H12O6

Cortical pia
CSF ISF(E.C.S.)AβParavascular drainage
Basement membraneof glia limitansAβe ndoth e lium
Astrocytic endfeet

ApoE

Vein wall
Blood to the
heart

Aβ

Aβ

capillaryAβS

Astrocyte

Vein wall

CO2
WASTE

Aβ

HDL

Venous blood

BBB

Arterioles

CSF

pia SAS

endothelium

AβVenuleDirection of bloodendotheliumBM
AβAstrocytic
endfeetO
2
BRAIN PARENCHIMA
AβAβCO2 , waste
GlucoseIS F
Brain parenchimanutrient
Paravenous
space
Aβ
Leptomeningeal artery
Leptomeningeal vein
Paravascular drainage
Perivascular drainage
NEUROPILAβ , IS F

Capillary

AQP4

Fig. 3. Schematic presentation of the Aβ, iron, and waste products drainage from the brain.

BBB, blood brain barrier; this figure represents the additional survey of Figure 3 in Reference 5), On the left side of the figurea rectangle limited by small red lines is visible-it in fact presents Fig. 5. The other rectangle including capillaries presents Fig.
4. Basement membrane (BM) at the capillary level is the result of the proximal integration of the outer and inner BM of VSMCs.
VSMC, vascular smooth muscle cell; SSS, superior sagital sinus; HDL, high-density lipoprotein; CSF, cerebrospinal fluid;
E.C.S., extracellular space; Aβ, amyloid beta; VRS, Virchow-Robin space; O 2, oxygen; C 6 H 12 O 6 , glucose; CO 2, carbon dioxide;
ISF, interstitial fluid; SMC, smooth muscle cell; AQP4, aquaporin 4, channel located on astrocytic endfeet, important for Aβ
drainage; SAS, subarachnoid space; AG, arachnoid granulation; Tf -Fe 3+, transferrin-ferric ion complex; ApoE, apolipoprotein E.

_ 18 5 _Perivascular Drainage from the Brain

O2, glucose, nutrients
ApoE

Angiopep-2

CO2,waste

Tf-Fe 3+

BBB - Start of the perivascular drainage
HDL

Aβ

LDL

LDLR

C ovalently conjugated drug

RAGE

Aβ

ABCB1

LRP1

SR-B1

O2
Glucosenutrients

nucleusFe2+

Lysosom ee ndocytosis

Aβ FPN1

lipidation

Luminal side
Recycling

Fe2+

Fe rric redu ctaseFe 3+
re duction 2+ DMT1
Fe

endosome

endosome

ABC B1

ABCA1

Tf R

LRP1

Tf R

BM

Tf R LRP1

Basal lamina≈ 50 µm

ApoE

Aβ Tf Fe3+

Lamina reticularis

Aβ

.

Aβ

Aβ

Aβ

3+
TfFe

Tf Fe3+AβAβpericyte

Tf Fe3+
Starting point of
the perivasculardrainage pathway

Very permeable Aβ

TfFe3+Aβ AβNeuropilnutrients Neuropil

Aβ

Tf Fe3+

Intercellular astrocytic
endfeet cleft (relatively large)

glucose.

Aβ
pericyte
NeuropilAstrocytic endfeet
2+3+

Fe + H2O2 = Fe
HDL

Apo E

fibrillin
Elastic fibers
Collagenous fibers

In the capillary region, the BM is
a complex resulting fromthe
proximal fusion of the outer andinner BMs of VSMCs.

AQP4

O2

bl.

collagenVIIAβAβVery permeable

Endothelial cell

FPN1 LRP1 Abluminal sidebinding

Perivasculardrainage

1-2µm thick~135µm length

Fe2+

LDL

HDL

nucleus40-60 nm30-100 nm

Lamina densa

Tf R

TfR-Tf-Fe 3+

Tight
junction

Fe3+

Lamina lucida
Hp oxidation
Fe3+
Tf

LUMEN RAGE

TfR
Tf

TfR
endocytosis
Recycling AβAβtranscytosis

Aβ

Fe 3+ CO2
Waste
Tf
Aβ

. Blood flow (cerebral capillary) Aβ+ − OH + *OH(E.C.S)
Astrocyte

Basement membrane of glia limitans

Paravascular drainage pathway
Pia disappears proximally

CO2

APP
2+

Fe

waste

3+

→ Fe
CP

Paravascuar space and drainage

Fig. 4. Structure of the cerebral capillary basement membrane: effects of aging and AD (longitudinal cross-section).

In the upper part of the figure two endothelial cells connected by a tight junction are visible. Above the luminal side of both cells, the redarrow denotes the capillary lumen, anterograde blood flow direction, some receptors (TfR, RAGE) and Aβ peptide molecules. Below thecells the basement membrane is visible which is composed of two layers, the basal lamina (bl.) and lamina reticularis. The basal lamina iscomposed of the lamina lucida (below the endothelium) and lamina densa; Hp, hephaestin, a transmembrane copper-dependent ferroxidase,functionally closely bound with the FPN1 receptor, responsible for the oxidation and transport of Fe2+ from the cell interior across its
abluminal cell membrane. The obtained Fe3+ binds promptly with the locally present Tf ; Angiopep2, conjugated drug; AD, Alzheimer's
disease; Aβ, amyloid beta; APP, amyloid precursor protein; CP, ceruloplasmin; E.C.S., extracellular space; AGEs, advanced glycation
end products; RAGE, receptor for AGEs; LDL, low-density lipoprotein; HDL, high-density lipoprotein; LDLR, LDL receptor; ApoE,
apolipoprotein E; BBB, blood brain barrier; Tf, transferrin; TfR, Tf receptor; ABCA1, ATP-binding cassette transporter 1; ABCB1, ATPbinding cassette sub-family B member 1; FPN1, ferroportin 1; LRP1, low density lipoprotein receptor-related protein 1; DMT1, divalent
metal ion transporter-1; VSMCs, vascular smooth muscle cells; BM, basement membrane; AQP4, aquaporin 4.

_ 18 6 _Glycative Stress Research

Inner basement membrane of VSMc

Aβ.

. .waste
Aβ

Tf

Tf-Fe

From the heart

.

Aβ
RAGE

LDLR

3.0 nm thick
12 nm length

.

SR-B1

Aβ
HDL
recycling

LDL

. .

Tf

Fe3+
DMT1

. Fe endosome
Endothelial cell
. FeFPN1 .Aβ
Tf R Aβ
Tf R

re duction2+

lysosome2+Tight
jun ction

FPN1

Hpoxidation

Lamina lucida

Fe3+

Hp

BM

Lamina densa Fe3+oxidation

Lamina reticularis

Very flexible

LRP1

. Aβ

Fe2+

LRP1

Abluminal side

Tf R

TfRTf-Fe3+
ApoEε4
Lipid
Tf R
Ferric reductase
recycling
SR-B1
LDL
HDL
Fe3+
reduction
nucleusFe2+
endosome
Fe2+
Aβ TfR
Lum in al side

TfRTf-Fe 3+
re cicling

nucleus1-2 µm thick~135 µm length

LDLR
Aβ RAGETf R

transcytosis.Aβ

Blood flow

3+

. Aβ

O2
CO2, glucose
waste nutrients

Longitudinal cross section Fe3+
Tf
lipid

bl.

.

.

sinking.
pressure

Aβ

LR

.

.

LRP1

endocytosis

nucleus
VSMC
Vasomotion wave

..
. ..
...
.. .
ApoEε4 .
.P
.
Aβ .
.
.
.
.
. .
.
waste
.
squeeze
Lipid
.
Ca
VSMC
MEDIA P
. ... pressure .
.
Vasomotion wave
. . . . . . .
.
.
.
.
.
Aβ
. ..
.
nucleus
IPAD
exit
P
..
Ca
.
.
. Ca
VSMC
Aβ
VSMC relaxation VSMCs sink into the lam. retic.
.
IPAD

HDL

To the heartAβelastin

LRP1

BM poreFrom the capillary

2+

HDL

collage n

ISF

ApoE

2+

Lamina reticularis

Aβ

E.C.S.

LRP1

2+

Vessel pia

Direction of vasomotion contraction

Ca2+
pressure
MEDIA

Direction of Aβ and waste particles moving
Outer basement membrane of VSMCs

.

.

.

. .
. Aβ Pia is very peameable
Poroelastic structure dilated. wasteParavascular drainage
Cortical pia. . .. . . . .
.
. .
.. ..
.
Aβ
AQP4 .
.
. Pericytes
Aβ. . waste
Astrocytic endfeet
NEUROPIL E.C.S.
..
.
SAS

Aβ

LR

VSMC contraction.
Virchow-Robin space

Aβ

Fig. 5. Schematic presentation of intra-mural peri-arterial drainage (IPAD)

Poroelastic structure compressed.

Pia disappears distally
Aβ

. ..
.

waste Aβ

ISF
CSF

The LRP1/Aβ endocytosis = IPAD pathway is visible; VSMC, vascular smooth muscle cell; AQP4, aquaporin 4; Tf, transferrin;
TfR, Tf receptor; Hp, hephaestin; AGEs, advanced glycation end products; RAGE, receptor for AGEs; CSF, cerebrospinal fluid;
ISF, interstitial fluid; BBB, blood brain barrier; Aβ, amyloid beta; APP, amyloid precursor protein; LDL, low- density lipoprotein;
HDL, high-density lipoprotein; LDLR, LDL receptor; ApoE, apolipoprotein E; VSMC, vascular smooth muscle cell; LRP1, lowdensity lipoprotein receptor- related protein 1; SR-B1, scavenger receptor class B member 1; FPN1, ferroportin 1; DMT1, divalent
metal ion transporter-1; SAS, subarachnoid space; E.C.S., extracellular space;BM, basement membrane; bl., Basal lamina; LR,
lipoprotein receptor.

_ 18 7 _Perivascular Drainage from the Brainfunctional changes of the cerebral vasculature connectedwith the aging process, but without the presence of active
AD. Perivascular drainage alterations during normal aging(also with the presence of AD), manifests itself with Aβ
aggregation in the middle layer of leptomeningeal artery
walls (media), i.e. in the BMs which envelop VSMCs, aswell as in the BMs of their final elements, arterioles andcapillaries. The occurrence of this aggregation is definedas CAA. It is important to emphasize that it occurs in 30 %of aged people without any sign of AD, and in 90 % with
AD. This study does not deal with the complex etiology ofAD, nor with the analysis of the whole complex of genes
important for the onset and course of this severe, chronic,
and lethal neurodegenerative disease; it is primarily involvedin the problem of perivascular drainage, connected with
the BMs of the mentioned vessels, whose alteration has a
crucial role in AD pathophysiology, and consequently inthe development and course of the clinical picture and thefinal lethal result of the disease. Before further analysis, it
is important to emphasize that AD occurs in two essential
forms: the early form or EOAD (early onset AD) before 65
years, about 5% of all cases, and the late form or LOAD (lateonset AD), 65 years and more, about 95% of all cases. The
clinical picture of both forms is practically the same. The
crucial signs of the disease are progressive loss of memory,loss of speech, and failure of cognitive functions. The courseof the disease is marked with increased weakness of thepatient, who at the end is completely disabled and dependent
on someone else's help and nursing 5-7).
Now it is necessary to concentrate our attention to thearteries and their terminal units (arterioles and capillaries)
in the aging process without the presence of AD. The
investigation of the perivascular drainage starting point
structure, i.e. capillary BMs of aged human brains, showstheir vacuolization, reduplication, and thickening. The
investigation of the capillary BM width in mice aged 23
months, by transmission electron microscopy, has shown asignificant increase in the width (thickness in nm) of theircortex BMs, hippocampus, and thalamus, as compared tomice aged 2 and 7 months (2 months: 65.79, 62.57, 70.11
nm; 23 months: 97.49, 109. 50, 108.43 nm). In mice aged
23 months, in cortical BMs, there is a significant drop in
the amount of collagen IV, laminin, and nidogen 2. At thesame time, in these mice, there is a significant increase of
N-terminal fibronectin fragment levels, as well as an increaseof perlecan levels. Collagen IV, laminins, and nidogens
are all markedly defined inhibitors of Aβ aggregation anddestabilizers of previously formed Aβ fibrils. Heparansulfate proteoglycans (HSPGs), i. e. agrin and perlecan,
accelerate the mentioned aggregation. The presented data
show changes in the cerebral vasculature BMs connectedwith aging, which are favorable for the development of theproamyloidogenic environment. Again, it is necessary toemphasize that thickening, cleavage, and duplication, as wellas the occurrence of abnormal inclusions of cerebrovascularBMs, is a frequent phenomenon in the brain of aged humans
and animals, and that this phenomenon is much more visible
in the brains affected by AD ( Table 1 ) 10, 13-21).
Further in the discussion, it is necessary to point out
that in the presented figures, close to receptors, up anddown directed arrows are visible. Their direction indicatesthe receptors activity, i. e. their expression, and is related

to the decline in magnitude of perivascular drainage. The
drainage decline is closely linked with increasing age. It isvery instructive to present the example of LRP1 receptor(low density lipoprotein receptor-related protein1, receptor
mediated endocytosis of Aβ and its effective drainage fromthe brain) located at the abluminal side of the endothelial
cells (BBB). Experiments with laboratory mice show asignificant difference in LRP1 expression (significant drop in
its expression and effectiveness) between 34 month old miceand 3 month old mice (Fig. 4 ) 22).

Vascular alteration with aging
In their earlier work, Hawkes CA et al. 13) present findings
related to the Aβ deposition in the walls of leptomeningeal
and cortical arteries in form of CAA in the normal aging
process, and its accelerated rate in AD. They declare that
the Aβ deposits in the cerebral vasculatory can be observedin nearly 30-40% of aged individuals without any sign ofdementia. This percent is much higher in demented persons.
The Aβ deposition is accompanied by capillary thickening,
vessel tortuosity, vasoconstriction, angiogenesis inhibition,and pericyte, endothelial cell, and VSMC death. During theaging process, the enzymatic degradation, Aβ absorption in
the blood, as well as its influx into the microglia, are also
diminished or disturbed. The already mentioned compounds,
the integral components of the vasculature, laminin, collagenIV, and nidogen by direct effect on Aβ, obstruct its aggregationand favor the dissolution of already formed fibrils. On theother hand, agrin and perlecan ( both are HSPGs) induce
Aβ fibrillization and the stabilization of already formed
fibrils. All presented changes, in fact linked with the courseof normal aging, lead to the dysfunction of the perivasculardrainage and induce Aβ deposition. The authors emphasize
their own experimental results related to dextran moleculedrainage by hippocampal microvasculature. Mice aged 22
months have an evidently weaker drainage compared to mice
aged 3 and 7 months. They have found evident differencesin the capillary structure thickness and in the protein
composition of their walls. The subsequent paper by Hawkes
CA et al. 19) published two years later, confirms these findings.
Keable A et al. 23), show the disturbance of solubleAβ perivascular drainage from the brain induced by age.
By the use of Image J (a Java-based image processingNational Institutes of Health and Laboratory for Opticaland computational Instrumentation, LOCI, Universityof Wisconsin) and confocal microscopy (optical imaging
technique for increasing optical resolution and contrast ofa micrograph), the authors analyze the immunostaining
paraffin sections of post-mortem human occipital cortex,
and find a marked elevation of nidogen 2 (enactin) parallelwith aging. In CAA, its level is markedly reduced. By
aging, arteries become increasingly stiffer and rigid andlose their innervation. The authors continually emphasize
the biochemical changes of the cerebrovascular BMs which
go along with the aging process. The Aβ aggregation in thevascular walls of aged persons without symptoms and signsof dementia, strongly indicates the aging induced disturbanceof Aβ elimination from the brain. Through time, this leads tothe occurrence of CAA, as is in the case of AD.
ZekonyteJ et al. 24) in their study related to the dynamics
of perivascular drainage, emphasize the great importance_ 18 8 _Glycative Stress Research

Table 1.
Structural changes of basement membranes related to "normal" aging without any signs of Alzheimer's disease (AD).

Different parameters
Cappilary basement membranes:
Vacuolization
Reduplication
Thickening
Cleavage
Collagen IV – drop

→ Amyloid beta (Aβ) aggregation inhibition and destabilization

Laminin→ of early formed Aβ fibrils;– dropNidogen 2 (enactin) elevation (in the case of present CAA clear drop)
N-terminal fibronectin fragment – elevation→

accelerate AβAggregation and HSPG Agrin →its fibrilization

Perlecan – elevation

Aβ sedimentation in the form of CAA – elevation
30-40 % elderly subjects without AD have CAA;
Besides capillary thickening, cerebral arteries show tortuosity, vasoconstriction, inhibition ofangiogenesis, death of pericytes, endothelial cells, and VSMCs;
It is also visible enzymatic Aβ degradation, drop of the Aβ absorption
and drop of its influx

into the blood,

into microglia;

Arteries with aging loss their elasticity, become stiffer and loss inervation.
The data on the table are related to the essential events connected with the aging of brain structures and functions; the data are not influenced by AD;
to obtain this data, among different methods, transmission electron microscopy was primarily used; collagen IV, a type of collagen primarily locatedin the basal lamina, structural protein; laminin, high- molecular weight protein located in the extracellular matrix of basal lamina, it is biologicallyvery active; nidogen 2, enactin, protein located in basal lamina, biologically very active; N- terminal fibronectin fragment, some different functiions
are mentioned, effects in the mononuclear phagocyte function, inhibition of fibronectin to fibroblasts, inhibition of platelet aggregation; perlecan,
heparin sulfate proteoglycan 2, included in binding and cross-linking of many extracellular matrix components and cell surface molecules, primarily
are located in the extracellular matrix of basement membranes; HSPG agrin, heparin sulfate proteoglycan, important for the structure and function of
the neuromuscular junction, it binds with many other proteins; VSMCs, vascular smooth muscle cells; Aβ, amyloid beta; AD, Alzheimer's disease;
CAA, cerebral amyloid angiopathy.

of continuous and effective Aβ elimination from the brain.
The accumulation of this peptide, regardless of the presenceof AD, permanently occurs in the brain parenchyma and its
vasculature, especially in the carriers of APOEε4 allele. The
authors relate the disturbance of this drainage to the reductionof pulse waves linked with the aging process of arteries. As
distinguished from a number of recent researchers, among
them, especially Aldea R et al. 4), who declare that pulse
waves, do not have any effect on the perivascular, but only
on the paravascular drainage, Zekonyte J et al. 24) give great
importance to the, until now, generally current attitudeemphasizing the crucial role of pulse waves, especiallyreflexion waves. According to them, the reduced strengthof linking ApoEε4/Aβ complex to the BM structures of thevascular wall (laminin), as related to the evidently stronger
linking of the mentioned complex with the other two APOE

alleles (ε2 and ε3), makes possible its oscillations during
the passing of pulse waves and in this way, the decline ofthe drainage intensity. However, according to a number ofrecent researches, the drainage process still depends on acompletely different mechanism (see Aldea R et al. 4) ). The
most recent experiments indicate the crucial importance ofthe system of cerebral arteries and arterioles BMs and among
them, the embeded VSMCs.

The role of ApoE
The APOEε4 gene codes the ApoEε4 protein. In thecentral nervous system (CSN), ApoEε4 protein synthesis
is established in astrocytes and microglia (astrocytes aspart of the BBB surround cerebral capillaries and secrete
ApoE proteins into the narrow paravascular space). In_ 18 9 _Perivascular Drainage from the Brain

relation to the other two alleles, ε2 and ε3, ApoEε4 is astrong risk factor for the occurrence of AD. It is establishedthat ApoEε4 is an amphipathic peptide whose N-terminus
carries two hydrophilic groups of residues (res. 55-79 andres. 14 0-158), and C- terminus which carries hydrophobic
residues 261- 287. In the perivascular space, this allele issignificantly less effective in the events related to Aβ
drainage. The drainage process is otherwise induced by theaction of the ABCA1 (ATP-binding cassette transporter1,cholesterol efflux regulatory protein, CERP), located on
the abluminal side of the cerebral endothelial cells. It leads
to the lipidation ( joining with the lipid component) of the
ApoE protein with the formation of lipoprotein particles.
The investigations show that the lipidation of ApoEε4lipoprotein is significantly weaker as related to ApoEε2 andApoEε3. ApoEε4-HDL (high-density lipoprotein) particles
are markedly smaller than the other two alleles. Otherwise,
through aging, ABCA1 expression and activity declines.
ApoE when linked with a lipid conditions its interaction with
Aβ, with the prompt transport of the whole complex by theeffect of LRP1 and ABCB1 receptor. ABCB1 (ATP-binding
cassette sub-family B member 1) is located on the luminal
and abluminal side of the endothelial cells’ membranes. It isan active mediator for the Aβ peptide transport out from thebrain. ApoEε4 protein (lipidated by ABCA1), in relation tothe other two alleles, has an evidently weaker linking with
Aβ, and the generated complex transport is also weaker.
The consequent result is stronger Aβ accumulation in the
structures of the perivascular space (PVS), as well as its
stronger accumulation in the brain parenchyma. On bothlevels, its aggregation is stronger. The LRP1 expression dropin the aged has been emphasized previously (Figs. 4-6 ) 25).
Tokuda T et al. 26), at the beginning of their study,emphasize that apolipoprotein E (ApoEε4) is the dominantrisk factor for sporadic AD (LOAD, 95% of all cases) andfamilial AD (EOAD, 5% of all cases). ApoE isoforms
bind directly with Aβ in vivo and in vitro. By analyzing
this binding, using the lipidated and delipidated ApoE3
and ApoE4 isoforms, Tokuda T et al. 26) have found that
lipidated ApoE3 isoforms have an evidently higher rate ofbinding with Aβ than ApoE4 isoforms. This is related toboth Aβ1- 40 and Aβ1-42. Delipidation of both isoforms
leads to the drastic 5-10 fold drop of their affinity towards
Aβ, with the loss of difference between them. The injectingof all the isoforms into the special mixture of lipoprotein
particles leads to the return of all isoform binding rates totheir usual state, especially ApoE3. The strength of isomer
binding to Aβ is inversely proportional to the risk for LOAD
occurrence. The authors examine the possible use of ApoE3
in the cleaning of the perivascular pathways.
It is important to emphasize the findings by Safieh M
et al. 27) obtained by analyzing the human CSF, in which isfound a markedly lower lipidation of ApoEε4 with HDL,
than in the case of ε2 and ε3 isoforms.

The disturbance in Aβ perivascular transport
The passing of Aβ molecules along the BMs whichsurround VSMCs is characterized by their fairly chaotic
movement with mutual approaching and detaching. This isespecially expressive in lower velocities and in the higher
resistance to the flow. The approach of the two moving

neighboring Aβ monomers and their binding is induced
by intermolecular hydrophobic interactions, electrostaticinteractions, H-bonding, and concurrent cross-linking throughadvanced glycation endproduct (AGE) compounds from theenvironment, or formed from precursors in situ, primarily
GOLD (glyoxal-lysine dimer) and MOLD (methylglyoxal
lysine dimer). In the course of this binding, there is a great
possibility for the interaction of MetS35 on one monomer
and the MBD (metal binding domain with Fe 3+ ) on theother. The result of this event is a great possibility of anelectron "hop" from the S of MetS35 onto the mentioned
MBD (Fe 3+ ). This is an ideal situation for the reduction of
Fe 3+ to Fe 2+, for the occurrence of the dangerous Fenton
reaction, and the generation of aggressive and toxic hydroxyl
radicals (*OH).
By means of oxidative attack on the neighboring
VSMCs cell membranes and on the protein chains ofcollagen, *OH steals electrons (e¯ ), and this leads to serious
damage of these structures. This attack with further Aβaggregation diminishes the strength of vasomotion waves
and disturbs the perivascular transport. With the drop of
the drainage velocity, conditions are achieved for the failureof arterial walls with changes typical for atherogenesis andthe CAA phenomenon 28, 29). For a better understanding of
the mentioned problem, it is necessary to emphasize thatthe length of Aβ monomer is about 12 nm and the width isabout 3.0 nm. The Aβ monomer actually has an amphiphilic
nature. Hydrophobic regions lie on the residues 29-40,
and the hydrophilic N-terminal domain covers residues
1-28. The diameter of a water molecule is approximately
0.275 nm. The lipidation induced by ABCA1 significantly
elevates the hydrophobicity of the central part of HDL and
LDL (low-density lipoprotein) and hydrophilicity of theirsurface. It can be assumed that during the development of
the vasomotion wave pressing force, the markedly present
hydrophilic complexes on their surface (well soluble inwater, i.e. in the ICF, with water as a dissolvent), induce
an excellent solubility of these particles, with an evidently
faster movement along the perivascular drainage pathway.
HDL particles diameter, measured by the NMR procedure,in persons with low cardiovascular risk is 9. 4-14.0 nm,
while in persons with high risk is 7. 3- 8.2 nm (this is ingood correlation with the earlier analysis of ApoEє4/
HDL particles). Lamina reticularis of the BMs is otherwise
sufficiently wide to enable the drainage of HDL particles,
which as well as phospholipids and cholesterol, carry ApoE
and Aβ proteins 28, 30).

The driving forces for drainage
VSMC contractions (vasomotion) along the perivasculardrainage pathway lead to the intermittent pressure gradientin the arterial media, which drives Aβ molecules dissolvedin the ISF and linked with lipids and ApoE protein. The
direction of drainage along the BM is contrary to the bloodflow in the lumen of arteries. According to the authors of
this study, if the surface hydrophilicity of HDL particles,or their polarity, is greater, the resistance to the flow (infact solubility) is reduced as related to the main element of
matrix, i.e. water (ECS, ECF). Stronger binding of Aβ withthe particles of complex HDL/ApoE by ε2 and ε3 APOE
alleles, leads to the faster cleaning of Aβ from ECS/ECF_ 19 0 _Glycative Stress Research

ABCA1
ABCA1
Cholesterol
effluxregulatory
protein,in humans encoded by the
Cholesterol efflux regulatory protein, in humans encoded by the ABCA1 gene,locatedontheabluminal
side
of
the
cerebralcapillary endothelia
located on the abluminal side of the cerebral capillary endothelial cells.
Lipidation
ApoE onfromblood binds to LDLR locatedLipidation inductor. ApoE from the bloodbinds toinductor.
LDLR locatedthethecellsurfaceofcapillaryendothelial
cells.
surface of capillary endothelial cells. The generated complex is transported The generated complex isthroughmembrane,into
andtheApoE
continues its translocation int
through the membrane, and ApoE continues
itsthetranslocation
matrixofcapillarybasement
membrane.
ApoE
of capillary basement membrane. ApoE is produced by liver and in the CNS is produced by liver andby macrophages.
APOE
gene Chr.19q13.32. ABCA1 gene Chr
by macrophages. APOE gene Chr.19q13.32.
ABCA1 gene
Chr.9q31.1.

ApoE pApoE protein
ApoE
producedbyastrocytes,
Lipidation(ApoE+ lipid)
ApoE produced by astrocytes, Lipidation (ApoE + lipid)
lipid-HDLinthecytoplasm
of
the
EC.
in the cytoplasm of the EC.

/ lipid complex– interaction
ApoE / lipid complex – interaction withApoE
Aβ peptide,mutually binding
and with Aβ peptide, mutua
ApoE
/
lipid
/
Aβ
complexformation.
ApoEε4 isomer-has a v
ApoE / lipid / Aβ complex formation. ApoEε4 isomer-has a very poorbindinginrelationtothe
ApoEε2
isomer,and especially tobinding in relation to the ApoEε2 isomer, and especially to the ApoEε3.
There
is
also
the
identical
relationtothetransport
and draThere is also the identical relation to the transport and drainage
induced
by
the
LRP1
and
ABCB1
receptors.
Transport
thr
induced by the LRP1 and ABCB1 receptors. Transport through theperivascular
space
has
a
retrograde
directioninrelationtoperivascular space has a retrograde direction in relation to thedirection
of
the
blood
flow.
A
PO
Eε4
is
the
most
prevalentrdirection of the blood flow. APOEε4 is the most prevalent risk factor for AD.
Aβlipid - HDL
ApoE / lipid / Aβ complex

Aβ

ApoE

lipid - HDL
ApoE / lipid / Aβ

ApoEFig. 6. Schematic presentation of ApoE protein lipidation and ApoE/lipid/Aβ complex formation.

The APOE gene codes APOEε2, APOEε3 and APOEε4 alleles. The worldwide frequency of alleles is 8.4% for ε2, 77.9 %for ε3, and 13.7% for ε4. In the CNS, microglia and astroglia produce ApoE. ApoE is a transporter for lipids, fat-soluble
vitamins and cholesterol. Chr.19.q13. 3 codes all three alleles. It is composed from 299 AAs. ApoE in the brain is themain cholesterol transporter. ApoEε4 is less lipidated by ABCA1 than ApoEε3. Lipidation, the covalent binding of a lipid
group to a peptide chain. Astrocyte, star- shaped glial cell of the CNS. Perivascular space, the virtual space located in thecapillary basement membranes, and in basement membranes in the media of brain arteries and arterioles. Perivascularspace is responsible for drainage of Aβ and waste products out from the brain. LDL, low-density lipoprotein; HDL, highdensity lipoprotein; LDLR, LDL receptor, cell surface receptor, composed of 839 AAs, transmembrane glycoprotein,
mediates the endocytosis of cholesterol-rich LDL; ABCA1, ATP-binding cassette transporter 1; ABCB1, ATP-binding
cassette sub-family B member 1; EC, endothelial cell; ApoE, apolipoprotein E; LRP1, low density lipoprotein receptorrelated protein 1. Aβ, amyloid beta; AD, Alzheimer's disease; CNS, central nervous system; AA, amino acid.

(BM interstitium), and its weaker aggregation. ApoEε4lipidation is weaker (binding with HDL and LDL particles)
as related to the other two alleles, which all results in thegreater possibility of agglomeration and aggregation of Aβ,
in the drop of its drainage, and the development of CAA 4).
The perivascular drainage pathway is actually atridimensional poroelastic net along which pass collagenand elastic fibers. Lipidated Aβ molecules, located on the
Aβ/HDL/ApoE complex, are placed in the way that theirhydrophilic parts are congruent with the hydrophilic regionsof the other two components. The complete system has anevident hydrophilic surface and is well soluble in ECF,
whose major component is water. Lower lipidated Aβ,
due to weaker ApoE4 lipidation, and weaker Aβ binding,inclines to its aggregation with the formation of increasingly
higher aggregates that are very poorly soluble in water.
The restoration of the pressure gradient (vasomotion) leads
to the movement of fluid and its present particles. The

smaller, lipidated particles can more easily move along thetridimensional net of the pathway, while the greater Aβaggregates meet a higher resistance, and their movement issignificantly retarded. This retardation additionally increases
Aβ aggregation. This general retardation of flow raises
the local concentration of moving iron ions (Fe3+ boundto transferrin). Aβ has a strong affinity towards Fe3+. Aβ
aggregation is favorable for the contacts of monomers,
electron " hops" from the MetS35 of one monomer to the
MBD ( Fe3+ ) of the other, and the Fe3+ onset. The generated
Fe3+ reduction induces the Fenton reaction with the
production of the toxic and aggressive *OH. This is followed
by further oxidative damages of the local molecular structures(formation of cross-links between neighboring collagenchains-dityrosine and disulfide bridges). The drainage
increasingly slows down. For better explanation of these
events, it is necessary to present some facts about the iron
efflux out from the endothellial cells. FPN1 (ferroportin 1:_ 191 _

Perivascular Drainage from the Braintransmembrane protein, only known iron exporter) is the iron
exporter from cells. But, for this function it is necessary themutual action of neighbouring protein hephaestin. Hephaestin
is a transmembrane copper-dependent ferroxidase, functionallyclosely bound with the FPN1 receptor, responsible for theoxidation (electron loss) and transport of ferrous iron ( Fe2+ )from the cell interior across its abluminal cell membrane.
The obtained Fe3+ binds promptly with the locally present
transferrin. Its deficiency results in a serious defect of thementioned transport 31).
Albargothy NJ et al. 3), in their study related to BM,
present an excellent pictorial review of the CSF influx
pathways in the brain and the drainage of the CSF/ISF
mixture along the capillary and periarterial BMs out fromthe brain. They especially emphasize the entrance of CSF
into the brain parenchyma and its mixing in the ECS(extracellular space) with the ISF. The CSF/ISF mixture
exits from the ECS and after passing through the narrowparavascular space around capillaries, enters into their BMs,
and by vasomotion waves travells to the arteriolar and arterial
BMs proximally to the lymph nodes around the carotid. The
authors emphasize that the paravascular space is locatedbetween the pia mater and astrocytic glia limitans and it ischaracterized by the presence of α2-laminin. This spacepractically disappears in the direction towards capillaries.
It is important to emphasize that the mixture of fluids fromECS enters the mentioned space through the AQP4 channels
and astrocytic endfeet clefts. The authors also point out thatthe inner and outer BMs of VSMCs do not participate inperiarterial drainage. This drainage is exclusively related to
the BMs in the media. The authors have not found signs ofdrainage along the venous walls. Perivascular drainage isextremely important for brain homeostasis, but during theaging process it progressively declines. In the carotid walls,
regardless of the subject’s age, only a small amount of Aβmolecules have been found.
Weller RO et al. 2), present the results of investigations
related to the accumulation of insoluble Aβ deposits in thebrain parenchyma and cerebral vasculature walls (CCA).
In these walls, Aβ accumulation takes place in the arterialand capillary BMs, which are the substrate of perivasculardrainage, i.e. cerebral lymphatic drainage. According to theauthors, the driving forces for this drainage are the arterial
pulsations. During the progression of arterial stiffness linked
with the aging process, the pulsations decline, resulting inAβ aggregation in the BMs, a decline in drainage, and theoccurrence of CAA. The CAA complications may result in
vascular wall ruptures with intracerebral hemorrhages.

The formation of amyloid fibrils
Castillo GM et al. 20) investigate the binding of perlecan(specific heparin sulfate proteoglycan) with Aβ (1- 4 0) andAβ (1- 42). The binding capacity closely correlates with theintensity of Aβ fibril formation. Perlecan has also a strong
effect on the stability of already formed fibrils.
Bronfman FC et al. 17) emphasize that the formation ofamyloid fibrils is a nucleation-dependent polymerization
process induced by different factors essential for thedevelopment, prevention, and treatment of amyloidosis.
They present the great capacity of laminin as the inhibitorof Aβ (1- 40) fibril formation. They also point out its evident

effect to maintain already formed fibrils.
Kiuchi Y et al. 18) present data about fibrilogenesis
induced by Aβ protein as the cause of AD onset and its
progression. According to their investigation, type IV
collagen is a strong inhibitor of Aβ fibril formation.
Cotman SL et al. 21) emphasize that agrin, extracellularheparan sulfate proteoglycan, according to a number ofinvestigations, has an important role in AD. Its possibility toaccelerate Aβ fibril formation as well as to obstruct the Aβ
proteolysis has been proven. Agrin is found in senile plaquesand cerebrovascular deposits. Cerebral capillaries containing
agrin indicate agrin as the cause of pathological alterationsin AD.

The role of VSMCs in perivascular drainage
Rannikmäe K et al. 32), in their study, investigate thegenetic background of CAA. A frequent connection of
CAA is established with brain aging, dementia, and lobar
intracerebral hemorrhage. A meta-analysis of 24 studies(3,520 examinees) has shown the link of ApoEε4 with CAA.
A statistically significant link with ApoEε2 has not beenfound. However, a specific link is found with the transforming
growth factor β1 gene, translocase of outer mitochondrial
membrane 40 gene, and complement component receptor
gene 1.
Biffi A et al. 33), present their findings which show thatthe carriers of APOEε2 and ε4 are exposed to a greater riskof intracerebral hemorrhage (ICH), probably due to thegreater risk of CAA.
Aldea R et al. 4), in their detailed study, present theexperimentally and theoretically confirmed completely new
standpoint concerning the role of VSMCs in the generation of
the driving force for perivascular drainage. This standpoint isbased on the new comprehension that arterial pulsations are
not strong enough to be the driving force for the perivasculardrainage. The drainage is in fact driven by the innate
mechanism of the arterial wall, i.e. VSMC contractionsand vasomotion phenomenon (V-IPAD, vasomotion driven
IPAD). By means of Darcy's hydrodynamic law, the authors
have performed a detailed analysis of the fluid flow (here
ISF) along the porous reticular lamina of the cerebral
arteries media. By this analysis they have obtained a strong
confirmation of the VSMCs as generators for the driving
force of perivascular drainage (IPAD). The authors do not
go into a complex explanation of this hydrodynamic law,
adapted for the fluid motion through the brain vasculature.
They present components of the essential Darcy's law
equation:Q = k*A* (Pb-Pa)/µ*L.
The components of the equation are: Q, total discharge,
flow rate (cm3/sec); A, cross-sectional area to flow (cm 2 );k, intrinsic permeability of the medium, coefficient of
permeability (cm/sec); Δp, (Pb-Pa), total pressure drop (atm,pascals)); µ, cp, dynamic viscosity; L, length over which thepressure drop is taking place (cm); from the equation it isvisible that the Q value is directly proportional to the valueof k, A, and Δp, and inversely proportional to µ and L. It
should be noted that arterial elongation (L) develops withage. Aβ, by the elevation of its concentration, induces toxic
damages in VSMCs and the adequate drop of Pb.

_ 19 2 _Glycative Stress Research

The decline of vasomotion
The perivascular drainage starts with the entrance ofAβ and other waste materials into the structure of capillaryBMs, and takes its course along the BMs in the media of
cerebral arterioles and arteries.
BMs are poroelastic membranes, in fact special softplates internally filled with a poroelastic medium, whose
pores (diameter ~7-17 µm) open and close depending on the
VSMCs relaxation or contraction. The VSMCs contraction,induced by vasomotion waves, forces the fluid filled with
dissolved Aβ monomers and other molecules out fromthe open pores. The fluid flows further in the directionof the vasomotion wave. All these events are completely
independent from heartbeat, innervation, and respiration.
The vasomotion wave has a length of about 2,000 µm, itsperiod is about 10 sec, average wave speed is 200 µm/sec,
and VSMCs contraction strength (S) is about 100 kilopascals(kPa). In the relaxed state, this strength is 0 kPa. This
contraction declines during the aging process. The VSMCs
contractions drop, induced by increased arterial stiffness
and toxic damage caused by the presence of Aβ, generating
the decline of the vasomotion induced pressure in the wall(Pb) and the consequent drop of the hydrostatic pressure
gradient (Δp, Pb-Pa). The result is the decline of the fluid
flow velocity and the increased tendency for stronger Aβaggregation ( Fig. 5 ) 4, 13, 19).
As previously mentioned, vasomotion is a vascular
phenomenon, defined as spontaneous oscillations of thevascular musculature tension, independent from heartbeat,pulse wave moving, innervation, and respiration. Their
origin lies in the rhythmic intracellular oscillations of Ca 2+
ions concentration in the VSMCs. The starting point of thevasomotion wave is related to small arterioles, metarterioles,
and precapillary sphincters. According to Raffaello A et al. 8),
the mentioned Ca 2+ concentration oscillations are the resultof its periodical release from stores located in the VSMCs
endoplasmic reticulum (ER). Gaspers LD et al. 9), in theirstudy also present the complete mechanisms underlying thevasomotion phenomenon. Baric N 6), in his last paper, has alsopresented a comprehensive review related to this problem.
Kalaria RN et al. 34) present the changes in cerebral
capillaries connected with aging. During the process of
aging, in the cerebral capillary endothelium (BBB) the lossof cytoplasm, drop of mitochondria number, loss of strong
connections between endothelial cells (tight junctions) andthe thickening of the BMs linked with collagen accumulation
occur. Astrocytic endfeet retract and the alteration of
pericytes occurs. Linked with these changes, there are often
lacunar infarcts, cortical and subcortical microinfarcts,
microhemorrhages and diffuse damage of white matter linked
with the loss of myelin and alterations in axon structure.
Thomsen MS et al. 35) emphasize the importance of theBMs which contribute to the integrity of BBB composedfrom the capillary endothelial cells. The BMs width is about
20-200 nm. In fact they are a tridimensional network, mainly
formed with proteins which belong to the big glycoproteingroups: laminins, collagen IV isoforms, nidogens, and HSPGs.
Apart from them, in these groups there are also some otherproteins: insoluble fibronectin, fibulin 1 and 2, collagen type
XVIII, thrombospondins 1 and SPARC (secreted protein
acidic and rich in cysteine). The present integrins anddystroglycan contribute to a better interrelated connectionof the mentioned proteins. The changes in the structure andcomposition of BMs in AD include the sedimentation of Aβ,
their thickening, and changes in protein structure. Laminins,
collagen IV, and nidogen, break the Aβ1-40 and Aβ1-42
fibril formation. In AD, the concentration of perlecan andfibronectin in capillaries is elevated.

The lymphatic drainage pathways
Bakker ENT et al. 36) emphasize that the lymphaticdrainage pathways in the brain are completely different ascompared to the other parts of the body. They are not only
important for the regulation of ISF and CSF volume, but also
for the drainage of waste products from the brain, among
them especially Aβ. The cleaning of this waste predominantly
takes place while sleeping. The authors emphasize theestablished communication between ISF and CSF, as well as
the connection between CAA and disorders of the perivasculardrainage. Astrocytic endfeet form a complete circle aroundcapillaries. Between astrocytic endfeet and capillary
endothelium there is a space 40-100 nm wide filled withthe capillary BM. In the capillary region, this membrane
is in fact composed of two joined layers, outer and inner
BM of VSMCs. Paravascular space on the capillary level isvery narrow and it is located between the astrocytic endfeet
and the outer surface of the joint membranes. Between
the BM and ECS there is good communication by AQP4
channels and intercellular astrocytic endfeet clefts (Figs.
3-5 ). In this region, the pia is practically absent. Contractile
pericytes are attached close to the endfeet. The width of
the basal lamina (bl.) is on an average about 100 nm. It is
produced by endothelial cells and VSMCs. After entering
into the BM, ISF drains further on by bulk flow along its
interior. According to the authors, the BMs are dominantly
composed of collagen type IV, laminin, fibronectin andheparan sulfate proteoglycans. They possess the capability of
autoremodeling. Confocal microscopy has shown that tracers
co-localize with laminin in the capillary BMs and arterial
tunica media.
Marques F et al. 37) present a detailed analysis of aging
alterations in the cellular elements of neurovascular units,
and in the choroidal plexus (CP) endothelium. They emphasize
the endothelial necrosis, accumulation of the extracellularmatrix components in the VSMCs, the drop of mitochondria
in the cells, the occurrence of pinocytotic vesicles, loss oftight junctions, retreat of astrocytic endfeet, stiffening ofvessel walls and loss of elasticity. Regional blood flow andmetabolism decline, as well as the oxidative processes, andthe whole drainage process.

Prevention and therapy for AD
After this presentation, it is clear that presently aneffective prevention and therapy for AD and CAA still does
not exist. The cleaning of the perivascular drainage pathway
will also be a great problem in the future. The actual therapy
for AD with cholinesterase blockers (donepezil, rivastigmine,galantamine) is not satisfactory, and a number of other current
therapeutics have a problematic effect. Currently, there areattempts with immunotherapy, therapy with free radical
scavengers and chelators (for example curcumin). Cross-_ 193 _

Perivascular Drainage from the Brainlinker breakers is also widely used in therapy. The correctionof fasting glucose level (nonenzymatic glycosylation of Aβ
retardation) has an important preventive role. Generally
speaking, AD therapy presently includes antioxidants, AGE
breakers, RAGE (Receptor for AGEs) blockers and antiglycation compounds.
Recently, researchers are directed to the effectsof alagebrium (ALT- 711), aminoguanidine, DPTC,
tiamine, benfotiamine, and piridoxamine. Related to theextracellular degradation of Aβ, the effects of neprilysin(metalloendopeptidase) and matrix-metalloproteinases 2, 3
and 9 inhibitors, are intensively investigated, as well asfunctions of proteasomes and the ubiquitin-proteasome route.
It is also necessary to mention vitamin C, vitamin E,
superoxide dismutase (SOD), Ginkgo biloba, and greentea (among the polyphenols found in green tea the mostimportant is epigallocatechin-EGCG) 5, 6, 38 - 41).
It is evident that during the aging process, especiallywith the presence of AD, there is a breakdown of many
cerebral functions. The recognition and comprehension ofthis breakdown, as well as its prevention and therapy, allrequire an intensive approach; otherwise we can expect
serious disturbances of social relations in human society
with unforeseeable consequences.

disorientation, and general drop of cognitive functions. The
costs for its prevention and therapy exponentially grow. Dueto the great importance of Aβ accumulation and aggregationin the AD brain, it is evident and logical that a numberof recent investigators and their institutions increasingly
take part in the research of the etiology, genetics, andpathophysiology of this disease. These investigations areespecially related to the failures of perivascular drainage andmodes of its improvement. The presented study is obviously
also included in this research.

Acknowledgements
Part of this research was presented at the 29th Summer
Stroke School "Healthy Lifestyle and Prevention of Stroke"on June 7, 2019, in Dubrovnik, Croatia.

Conflict of interest
The authors claim no conflict of interest in this study.

Conclusion
Due to the increasing rise of AD incidence andprevalence, this serious, chronic, and at the end lethal disease,is becoming one of the crucial problems of modern society.
The world population is increasingly older, absolutely andrelatively. AD is closely connected with age and aging. This
neurodegenerative disease with a chronic and progressive
course, is marked with a progressive decline of memory,

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