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1

Weihong Pan, M.D., Ph.D.

Pennington Biomedical Research Center

Baton Rouge, LA

[email protected]

The Blood-Brain and Blood-CSF Barriers

Principles o! Pharmacolog", #ulane Med $%%&


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Course objectives

#he lood'C() and lood'rain arriers as

d"namic regulator" inter!aces

#he morphological*cellular asis andsustrates o! the arriers

Anal"tical methods

)unctional implications in normal andpathological conditions

Pharmacological manipulations


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1+%% Lewandows-" Prussian

lue

1+%$ hrlich /ital staining

with man" d"es1+%+ 0oldmann tr"pan lue

i

1+12 0oldmann tr"pan lue

into C()

1+3% #schirgi d"e'proteincomple4

1+5%6s peptides cross the

BBB

$%%%6s lood'rain regulator"

inter!ace

Short history


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Cerebrospinal fluid (CSF)

Production !loor o! the lateralentricle and third entricle, "choroid ple4us

Circulation L./. '7 888./. '7 8/./.'7 e4it entricular s"stem intoarious asal cisterns and thento suarachroid space

Drain ac- to lood iaarachnoid granulation to superiorsagittal sinus, or ia spinal nereroots, or ia ol!actor" tracts

)rom 9ohanson C


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5

CSF volume in human based on

NMR imaging (Kohn 1991)

#otal 1&:.3 ; :


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Function of CSF

Maintenance o! a constant e4ternal

enironment !or neurons and glia

Mechanical cushion to protect the rain anduo"ant to the hea" rain =1:%% g>

(ere as a l"mphatic s"stem and a conduit

!or neuropeptides p? o! C() regulates pulmonar" entilation

and CB)


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Cerebral Edema

/asogenic edema A state o! increased

e4tracellular !luid olume Brain tumor, ascess, in!arct,hemorrhage Glucocorticoids, hypertonic solutions

C"toto4ic edema #he swelling o! cellular

elements ?"po4ia*asph"4ia, water into4ication, meningitis,encephalitis, Re"e6s s"ndrome

8nterstitial edema Attriuted to increased waterand sodium in perientricular white matterstructie h"drocephalus Surgical shunting, acetazolamide


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Hydrocephalus

structie h"drocephalus Congenital mal!ormations A!ter in!lammation or hemorrhage Mass lesions

ormal pressure h"drocephalus Communicating h"drocephalus

erproduction o! C() De!ectie asorption o! C() /enous drainage insu!!icienc"

?"drocepalus e4 acuo =rain atroph">


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CSF sampling

Contraindiation or !reaution in" #$C%

Pressure !rom lumar puncture &3'1+3 mm C() =?$>, or

3 13 mm?g

Cells % " in!lammation, tumor, or other cereraldamage

Protein E 23 mg*dl in loc-age or increased BBB

permeailit". M( shows higher F'gloulin and oligoclonal

ands 0lucose 7 :% mg*dl G in meningeal tumor, !ungal or #B

in!ection, sarcoidosis

Appearance not cloud" or 4anthochromia


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Blood'C() Barrier


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)luid compartments o! the lood'rain'C() s"stem


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Blood-Brain Barrier (BBB) and Blood-

Spinal Cord Barrier (BSCB) Barrier and regulator" unit etween Hcapillaries

and postcapillar" enulesI J H4tracellular

compartment o! rain and spinal cordI Composed o! ascular endothelia, their

asement memrane, peric"tes, and glial

periascular !eet =the end!eet>

#ight Kunctions in'etween endothelial cells


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13

)rom Dais #P


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CD21 immunostaining showing lood essels

and prealence o! the B(CB


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Put B(CB into the conte4t


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olarity

Distinctie apical and asolateral sur!aces

Di!!erent distriution o! transporters

Directional transport

Resolution in in'itro models 2'D or :'D

cultures


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ermeability of a membrane

and !inetic analysis 8ssues related to mechanisms o! penetration

simple di!!usion, carrier'mediated transport,

and actie transport

(tead"'state distriutions

!!ects o! lipid soluilit" and !low

Multiple compartments and regionaldi!!erences


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"inetic aspects of the BBB

Permeability the characteristics o! a

memrane, or a group o! memranes,

separating two !luids

Permeability coefficient how rapidl" a giensustance will permeate the memrane

dM*dt %A=Cpl CBr=W>>

Transfer coefficient PA*/wW P(*/wstimated !raction o! rain weight /w %.13 !ore4tracellular !luid onl" and %.


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#athematical treatment

Crone6s single'pass techniOue =1+&%6s> A'/ di!!erenceo! test and re!erence sustances

Brain upta-e inde4 =ldendor!, 1+ re!erence

sustance =2?$, or 1:C'utanol with P(*) o! 1. hasrapid, !low'dependent permeation to compensate !orCB) ariations

nidirectional !lu4 in rapidl" eOuilirating space Patla-'

Blaserg')enstermacher treatment =1+5%6s> (lowl" penetrating compounds that are stale in

circulation Ban-s'astin multiple'time regressionanal"sis =1+5%6s>


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Cerebral blood flo$

)ic- principle dCi )=Ca'C>

8ndicator !ractionation techniOue with 1:C'

utanol or 1:C'iodoantip"rine Quantitatie autoradiograph" !or regional

di!!erences

Cereral lood olume measured " 31Cr'RBC or 11C inhalation

and positron emission tomograph" =P#>

4tracellular space inulin space


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%isruption of the BBB

#rauma mechanical, irradiation, chemical

8n!lammation

8schemia *hemorrhage #umor

Dem"elinating disease

eurodegeneration Certain metaolic and congenital disorders

pileps"


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EBA SF Overlay

Left

hemisphere

Right

hemisphere25 m

Demonstration o! BBB disruption " an6s lue alumin

and sodium !luorescein sample !rom mouse rain


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&smotic opening of the BBB

A therapeutic approach

What are the osmotic agents

' mannitol, RMP'< =rad"-inin receptor agonist>, etc. ?as een used clinicall" in C( tumor chemotherap"

Question:

What are other ways to deliver agents to the CNS?- viral vectors fusion !roteins carrier antibodies

immunoli!osomes


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8ssue o! circumentricular organs =C/>


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'*-eptide

#ermination

o! actions

#ransc"tosis 'transport

8nteractionswith othere!!ectormolecules

!!ects oncells

Bindinginteractions

Receptordistriution8ntracellular

degradation

Degradation

4tracellular

matri4on'speci!icassociation

/olume o!

distriution

In blooIn bloo At the barrierAt the barrier InsieInsie!"S!"S


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+ransport of substances across the

BBB "lood-to-CNS

influ#

"rain-to-blood

efflu#

8ons

Amino Acids and organic acids

0lucose and other caroh"drates Biogenic amines

ucleotide precursors

Peptides, proteins and lipoproteins

(teroid and th"roid hormones /itamins, trace metals

Chemotherap" agents, antiiotics


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, specific eample. C/S chemotherapy
http://www.jci.org/cgi/content/full/116/3/642/F1


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Chemotheraphy to the C/S

("stemic #4 not routine since most agents hae

limited penetration despite partiall" disrupted BBB

8ntrathecal therap" is inasie and con!ined to onl"

three agents methotre4ate, c"tosine arainoside,

thiotepa

niOue side'e!!ects aseptic meningitis =:2S>,

chemotherap"'related leu-oencephalopath"

ewer approaches include gene therap" =with iral

ectors>, osmotic opening o! the BBB, and c"to-ines


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%rug resistance remains one of the primarycauses of suboptimal outcomes in cancer therapy

A#P'inding cassette =ABC> transporters

A#P'dependent drug e!!lu4 pumps,oere4pression in chemo'resistant tumorsand upregulation with disease progression

A large !amil", e.g., P'gp, MRP1, ABC0$

8nhiitors e!!ectie in animal studies hae notdemonstrated clinical e!!icac"


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http://theoncologist.alphamedpress.org/cgi/content/full/8/5/411/F1


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Another e4ample C"to-ine transc"tosis across the BBB


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&h' is it im!ortant to stud' the interations&h' is it im!ortant to stud' the interationso 'toines *ith the BBB+o 'toines *ith the BBB+

Peripheral c"to-ines modulateneuroendorine untionsin the C(

Peripheral c"to-ines a!!ect CNS regeneration C"to-ines in the rain and circulation

in!luence !eeding ehaior, sleep, mood,cognition, and a lot o! other important C(actiities


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0hat $e focus in our laboratory(teaching /europharm is just my side job)

)unctional implications o! c"to-ine transport

across the BBB

#ra!!ic-ing o! c"to-ines within cereral

microessel endothelial cells

Regulator" !actors !or c"to-ine transport and

targeted drug delier"


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Studies with Anesthetized mice:

Eample '. multiple-time regression analysis

?"pothesis in!lu4 is linear during stud" interal

Design i olus, di!!erent time o! circulation,each mouse represents one time point

Controls ascular mar-ers stailit" studies

compartmental distriution in'situ per!usion

Data anal"sis e4posure time, in!lu4 trans!ercoe!!icient, Supta-e


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% : & 5 1%2.3

:.%

:.3

3.%

#ime =min>

Log

=serum

radioacti1it">


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% 3 1% 13 $% $3 2% 23 :%

%

1%%%

$%%%

2%%%

:%%%

3%%%

&%%%


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parench"ma,no per!usion parench"ma, per!used capillar", no per!usion capil lar", per!used%

1

$

2

:

3

&

The bottom line? The behavior of itsinteractions with the """ may determine the

CNS effects &or side-effects' of a com!ound(


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SC*. ,ll in due course

)mmediately after lumbar contusion:suarachnoid hemorrhage petechial hemorrhages in gra" and white matter !irinoidnecrosis o! essels

*+ min - , h after traumanecrotic essels, aneur"smal dilitation, periascular hemorrhage, neuronal and glial

necrosis

- * d after traumapol"morphonuclear leu-oc"te and macrophage in!iltration a4onal spheroids

w% - m after traumareduced in!lammator" changesc"stic changes and gliosis

Balentine 9.D.,Balentine 9.D., .ab/ )nvest.ab/ )nvest. 1+


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%ynamic changes of the BSCB

after SC* Reersile

Biphasic Correlation with seerit" o! inKur"

(el!'repair !unction o! the B(CB is also

re!lected " the regulator" changes o!speci!ic transport s"stems !or c"to-ines


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Brain #) Brain Alumin (pinal cord #) (pinal cord Alumin%

1%

$%

2%

1 w- e4cess #)

NNN NNNN

1 w-

control

NNN NNNN

Right L1 hemisection 8ncreased #)upta-e in all C( regions is still saturale.

Tissue/s

erumR

atio

oRadioativit'(l/g)


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Brain Cerical #horacic Lumar (pinal cord%

3

1%

13

W# cont

W# (C8

DMA contDMA (C8

NNN NNN

NN NN NNN

N

++#c'Alumin#issue*(erum

Ratioo!Radioac

ti1it"=

l*g>


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Brain Cerical #horacic Lumar (pinal cord%

3

1%

13

$%

$3

W# cont

W# (C8

DMA cont

DMA (C8

NNN

NNN

NNN

1$38'#B)#issue*(erum

Ratioo!Ra

dioactiit"=

l*g>


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% 1% $% 2% :% 3% &%%

$

:

&

5

1%

D apical to asolateral

cont apical to asolateral

D asolateral to apical

cont asolaterl to apical

#ime =min>

1$38'

#B

)

R

atio

=4

1%

2>


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nderstand and modulate the transport

s"stems Modulate general permeailit"

Design analogs !or etter penetration

se ehicles e.g., iral ectors, liposomes B"pass the BBB

0hat $e can do to improve C/S drug

delivery


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Summary

0emember #he BBB*B(CB as a d"namic

regulator" inter!ace

0emember #he morphological*cellular asisand sustrates o! the arrier

1#!osure to Anal"tical methods

2nderstand )unctional implications in normal

and pathological conditions

Thin% about Pharmacological manipulations


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#emory test Concepts ' "es Applications ' ma"e #hin- o! the BBB in case o! drug delier" to the C(

Re!erences =supplied !or "our interest, not !or !inal e4am>Begle" and Brightman, (tructural and !unctional aspects o! the lood'rain arrier. Prog.

Drug Res., $%%2

Pan and astin, Wh" stud" transport o! peptides and proteins at the neuroascularinter!ace. Brain Res Re, $%%:

9ohanson C et al., nhanced prospects !or drug delier" and rain targeting " thechoroid ple4us'C() route. Pharm. Res., $%%3

Boo-s

Dason and (egal, Ph"siolog" o! the C() and lood'rain arriers. CRC Press.

(harma and Westman, Blood'spinal cord and rain arriers in health and disease. lseier.


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Blood'Brain Barrier 0roup

L:%2%, Pennington Biomedical Research Center

Baton Rouge, LA

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