Ci a ion: Co o-Vilcapoma, M.A.;
Cas illa-Silgado, J.; Fe nández-Ga cía,
B.; Pin o-He nández, P.; Cip iani, R.;
Cape illo-Za a e, E.; Menéndez-González,
M.; Ál a ez-Vega, M.; Tomás-Zapico,
C. New, Fully Implan able De ice o
Selec i e Clea ance o CSF-Ta ge
Molecules: P oo o Concep in a
Mu ine Model o Alzheime ’s
Disease. In . J. Mol. Sci. 2022,23, 9256.
h ps://doi.o g/10.3390/ijms23169256
Academic Edi o : S e anie Kü en
Recei ed: 29 July 2022
Accep ed: 13 Augus 2022
Published: 17 Augus 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
In e na ional Jou nal o
Molecula Sciences
A icle
New, Fully Implan able De ice o Selec i e Clea ance o
CSF-Ta ge Molecules: P oo o Concep in a Mu ine Model o
Alzheime ’s Disease
Ma ía Almudena Co o-Vilcapoma 1,2,† , Juan Cas illa-Silgado 1,2,†, Benjamín Fe nández-Ga cía2,3 ,
Paola Pin o-He nández 1, Ra aela Cip iani 4, Es ibaliz Cape illo-Za a e 4,5,6 ,
Manuel Menéndez-González 2,7,8,* , Ma co Ál a ez-Vega 9,10,‡ and C is ina Tomás-Zapico 1,2,‡
1Depa amen o de Biología Funcional, Á ea de Fisiología, Uni e sidad de O iedo, 33006 O iedo, Spain
2Ins i u o de In es igación Sani a ia del P incipado de As u ias, 33011 O iedo, Spain
3Depa amen o de Mo ología y Biología Celula , Á ea de Ana omía, Uni e sidad de O iedo,
33006 O iedo, Spain
4Achuca o Basque Cen e o Neu oscience, Depa amen o de Neu ociencias,
Uni e sidad del País Vasco (UPV/EHU), 48940 Leioa, Spain
5Cen o de In es igación en Red de En e medades, Neu odegene a i as (CIBERNED),
28029 Mad id, Spain
6IKERBASQUE, Basque Founda ion o Science, 48009 Bilbao, Spain
7Se icio de Neu ología, Hospi al Uni e si a io Cen al de As u ias, 33011 O iedo, Spain
8Depa amen o de Medicina Á ea de Medicina, Uni e sidad de O iedo, 33006 O iedo, Spain
9Se icio de Neu oci ugía, Hospi al Uni e si a io Cen al de As u ias, 33011 O iedo, Spain
10 Depa amen o de Ci ugía, Á ea de Ci ugía, Uni e sidad de O iedo, 33006 O iedo, Spain
*Co espondence: [email p o ec ed]
† These au ho s con ibu ed equally as i s au ho s.
‡ These au ho s con ibu ed equally as senio au ho s.
Abs ac :
We ha e p e iously p oposed a adical change in he cu en s a egy o clea pa hogenic
p o eins om he cen al ne ous sys em (CNS) based on he ce eb ospinal luid (CSF)-sink he apeu ic
s a egy, whe eby pa hogenic p o eins can be emo ed di ec ly om he CNS ia CSF. To his aim, we
designed and manu ac u ed an implan able de ice o selec i e and con inuous aphe esis o CSF
enabling, in combina ion wi h an i-amyloid-be a (A
β
) monoclonal an ibodies (mAb), he clea ance
o A
β
om he CSF. He e, we p o ide he i s p oo o concep in he APP/PS1 mouse model o
Alzheime ’s disease (AD). De ices we e implan ed in wen y- ou mice (se en een APP/PS1 and
se en W ) wi h low a es o complica ions. We con i med ha he aphe esis module is pe meable o
he A
β
pep ide and impe meable o mAb. Mo eo e , ou esul s showed ha con inuous clea ance
o soluble A
β
om he CSF o a ew weeks dec eases co ical A
β
plaques. Thus, we conclude ha
his in e en ion is easible and may p o ide impo an ad an ages in e ms o sa e y and e icacy.
Keywo ds:
immuno he apy; Alzheime ; be a-amyloid; CSF-sink; ce eb ospinal luid; implan able
de ice; blood–b ain ba ie ; nanopo ous memb anes
1. In oduc ion
The social impac o neu odegene a i e diseases (NDD) is undeniable. They comp ise
a wide ange o di e en neu opa hologies, which can be ei he spo adic o inhe i ed.
Howe e , mos o hem sha e he common hallma k o deposi s o disease-speci ic p o eins;
hus, NDD can be unde s ood as p o einopa hies.
In e es ingly, in he symp oma ic Hun ing on’s disease (HD) mouse model, blocking
mu an hun ing in exp ession a o s he disappea ance o agg ega es and amelio a es
he beha io al pheno ype [
1
]. This opened an insigh in o he pa hophysiology o NDD
whe e p o ein agg ega ion o e whelms he p o eos asis capaci y o neu ons (e.g., ubiqui in-
p o easome and au ophagy-lysosome sys ems), in e e ing wi h he abili y o neu ons
In . J. Mol. Sci. 2022,23, 9256. h ps://doi.o g/10.3390/ijms23169256 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2022,23, 9256 2 o 13
o cope wi h pa hogenic p o eins [
2
,
3
]. The o ma ion o agg ega es o hese p o eins
can ha e di e en o igins, including, among o he s, inc eased syn hesis o syn hesis o
s uc u ally abno mal o ms and/o dec eased deg ada ion, ei he by enzyma ic o cellula
sys ems [
4
,
5
]. A dec ease in hei clea ance o compa men s ou side he b ain pa enchyma
has also been desc ibed, which has been linked o he impai men o he blood–b ain ba ie
(BBB) [6], he ce eb ospinal luid (CSF) low [7], and he glympha ic sys em [8,9].
Accumula ion o amyloid-be a (A
β
) is he main pa hological hallma k o Alzheime ’s
disease (AD). In bo h ea ly-onse and la e-onse o ms o AD, A
β
clea ance seems al eady
impai ed a he p od omal s age o AD, and i is emo ed om he b ain by a ious
o e lapping and in e ac ing clea ance sys ems: deg ada ion, BBB anspo , in e s i ial
luid (ISF) bulk low, he glympha ic pa hway, and CSF abso p ion in o he ci cula o y and
lympha ic sys ems [10,11].
Di e en app oaches ha e been in es iga ed o emo e A
β
bo h biologically and
mechanically, om dec easing p oduc ion (i.e., BACE inhibi o s) o inc easing clea ance
in he pe iphe y (immuno he apy, plasmaphe esis, enzyma ic deg ada ion) [
12
]. Among
hem, immuno he apy wi h an i-A
β
monoclonal an ibodies (mAb) is he mos ex ensi ely
explo ed in humans, showing he capaci y o clea b ain plaques and es o e le els o
soluble A
β
in he CNS [
13
]. Howe e , none o hese he apies ha e shown clinically
ele an bene i s o AD pa ien s, and se ious side e ec s ha e been epo ed, including
amyloid- ela ed imaging abno mali ies (ARIA) a e an i-A
β
mAb he apies [
14
]. These
ailu es led o seek al e na i e me hods o elimina e pa hogenic p o eins om he b ain
using o he chemical o physical p inciples, such as hemodialysis o plasmaphe esis.
Among he esul s ob ained om he esea ch on hese in e en ions, i has been ound ha
blood dialysis and plasmaphe esis educe A
β
le els in plasma and CSF in AD pa ien s and
a enua e AD symp oms and pa hology in AD mouse models [
15
–
21
]. This sugges s ha
emo ing A
β
om he plasma migh be an e ec i e o m o gene a ing an e lux o b ain
Aβ h ough he BBB [22].
The BBB p e en s he ee mo emen o molecules be ween he in e s i ial luid
(ISF)/CSF and plasma [
11
], while ISF soluble molecules mo e in cons an equilib ium
be ween he CSF and he ISF, bo h being compa men s in di ec communica ion [
7
,
22
].
Gi en his, we ha e p e iously p oposed a adical change in he cu en pa adigm based
on clea ing a ge molecules om he CSF using implan able de ices [
23
]. Ou hypo hesis
elies on he CSF-sink he apeu ic s a egy, whe eby pa hogenic p o eins ha a e in equi-
lib ium be ween ISF and CSF can be emo ed di ec ly om CSF [
24
–
26
]. This in ol es
an al e a ion o his equilib ium, a o ing he clea ance o hese p o eins in hei soluble
s a e and hus dec easing hei a ailabili y o o m agg ega es in he b ain pa enchyma.
In e es ingly, he equilib ium be ween ISF and CSF emains s able in symp oma ic AD
models, in con as o he balance loss be ween he ISF and plasma [
22
]. Thus, he e is a
much mo e di ec way o emo ing a ge p o eins om he ISF han clea ing hem om
he plasma: clea ing hem om he CSF.
To his aim, we p opose o use a p ocedu e we named CSF aphe esis based on an
implan able de ice wi h a nanopo ous sys em allowing selec i e and con inuous aphe esis
o A
β
om he CSF. To e alua e he biological e icacy o he sys em, we ha e de eloped a
minia u ized p o o ype o conduc s udies in mu ine models o NDD. He e, we p esen he
i s p oo o concep in he APP/PS1 mouse model o AD.
2. Resul s
2.1. Implan a ion o he Minia u ized P o o ype Was Feasible in a Mu ine Model o Alzheime ’s Disease
Fo his p oo o concep , we ha e used a well-cha ac e ized mu ine model o AD
o which he ime a which A
β
plaques can be obse ed in he b ain is well known (six
mon hs) [
27
]. To be su e ha plaques would be p esen a he ime o he in e en ion, we
used se en-mon h-old mice. On he o he hand, a his age he plaques o med a e no as
dense as hey can be in olde mice, hus making i easie o iden i y he e ec o con inuous
il a ion ea men in a sho - e m s udy.
In . J. Mol. Sci. 2022,23, 9256 3 o 13
The minia u ized p o o ype o he implan able de ice consis ed o an aphe esis module
wi h a ese oi (Pa A; Figu e 1A) and a ca he e connec ing Pa A o a e minal cannula
o in ace eb o en icula implan a ion (Pa B; Figu e 1A). Pa A o he de ice was placed
subcu aneously on he back o he mouse, as was he cannula, and pa B was implan ed
in he la e al en icle (Figu e 1B,C). Due o he weigh (4.29
±
0.41 g), bu especially he
size o he de ice (3.5
×
1
×
1 cm), only male mice we e used in he s udy. In he case o
he se en-mon h-old APP/PS1 mice, he weigh o he de ice ep esen s 12.86
±
0.44%
o he mouse’s weigh , while in he case o W mice o he same age his pe cen age is
14.92 ±0.38%
. On he o he hand, he mos limi ing ac o was he leng h o he de ice,
which, in ela ion o he leng h o he back o he mice, ep esen s 66.51
±
1.77% o he
leng h in he case o he APP/PS1 mice and 69.56 ±1.86% in he case o he W mice.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 3 o 14
2. Resul s
2.1. Implan a ion o he Minia u ized P o o ype Was Feasible in a Mu ine Model o Alzheime ’s
Disease
Fo his p oo o concep , we ha e used a well-cha ac e ized mu ine model o AD o
which he ime a which Aβ plaques can be obse ed in he b ain is well known (six
mon hs) [27]. To be su e ha plaques would be p esen a he ime o he in e en ion, we
used se en-mon h-old mice. On he o he hand, a his age he plaques o med a e no as
dense as hey can be in olde mice, hus making i easie o iden i y he e ec o con inuous
il a ion ea men in a sho - e m s udy.
The minia u ized p o o ype o he implan able de ice consis ed o an aphe esis mod-
ule wi h a ese oi (Pa A; Figu e 1A) and a ca he e connec ing Pa A o a e minal
cannula o in ace eb o en icula implan a ion (Pa B; Figu e 1A). Pa A o he de ice
was placed subcu aneously on he back o he mouse, as was he cannula, and pa B was
implan ed in he la e al en icle (Figu e 1B,C). Due o he weigh (4.29 ± 0.41 g), bu es-
pecially he size o he de ice (3.5 × 1 × 1 cm), only male mice we e used in he s udy. In
he case o he se en-mon h-old APP/PS1 mice, he weigh o he de ice ep esen s 12.86
± 0.44% o he mouse’s weigh , while in he case o W mice o he same age his pe cen age
is 14.92 ± 0.38%. On he o he hand, he mos limi ing ac o was he leng h o he de ice,
which, in ela ion o he leng h o he back o he mice, ep esen s 66.51 ± 1.77% o he
leng h in he case o he APP/PS1 mice and 69.56 ± 1.86% in he case o he W mice.
Figu e 1.
Componen s o he de ice o selec i e and con inuous aphe esis o CSF in mice. (
A
) Pa
A consis s o he aphe esis module whe e he nanopo ous memb ane o CSF il a ion is loca ed;
coupled o his module is he ese oi whe e he he apeu ic agen is con ained. Bo h he aphe esis
module and he ese oi a e p o ec ed by a capsule. Pa B is composed o a cannula connec ed o
he aphe esis module and, a he o he end, is coupled o a ca he e ha allows i s implan a ion in he
b ain. (
B
) Scheme o he loca ion o pa A on he back o he mouse (made in BioRende .com), as
well as i s connec ion h ough pa B o one o he ce eb al hemisphe es. (
C
) The whole sys em is
implan ed subcu aneously so ha i emains p o ec ed om he no mal ac i i y o he mouse.
In . J. Mol. Sci. 2022,23, 9256 4 o 13
Conside ing his, a o al o wen y- ou male mice we e success ully ope a ed on,
which se en een we e APP/PS1 and se en WT (Table 1). Rese oi s we e illed wi h mAb
in en mice ( wo W and eigh APP/PS1) and wi h ehicle (a i icial CSF, aCSF) in en
mice ( h ee W and se en APP/PS1). Among hem, ou mice we e eu hanized due o
se e e le ha gy ha appea ed 48–72 h a e su ge y in h ee cases, while ano he mouse
was sac i iced one week la e . In addi ion, one mouse showed signi ican weigh loss and
was eu hanized wo weeks a e su ge y. The emaining six een mice showed no appa en
complica ions and exhibi ed no mal cage beha io , such as nes o ma ion and g ooming,
eaching he end o he h ee-week in usion s udy.
Table 1. Summa y o he di e en p ocedu es o echniques ca ied ou on each o he mice used.
Mice ID Geno ype Con en
In used
Time
Implan ed Complica ions
AβPe meabili y mAb Impe meabili y
Aβ
Rese oi
Aβ
Plasma
Aβ
Co ex IHC IF Fluo escence
7 APP/PS1 mAb 48 h Le ha gy + +
20 W mAb 48 h Le ha gy
21 W aCSF 72 h Le ha gy +
5 APP/PS1 mAb 1 week Le ha gy +
11 APP/PS1 mAb 2 weeks Weigh loss + + +
26 APP/PS1 mAb 2 weeks Le ha gy +
27 APP/PS1 aCSF 2 weeks Le ha gy +
28 APP/PS1 aCSF 2 weeks Le ha gy +
2 APP/PS1 aCSF 3 weeks None + + +
8 APP/PS1 aCSF 3 weeks None + + + +
9 APP/PS1 aCSF 3 weeks None + + +
12 APP/PS1 aCSF 3 weeks None + + + +
4 APP/PS1 aCSF 3 weeks None + +
17 W aCSF 3 weeks None + +
19 W aCSF 3 weeks None + +
3 APP/PS1 mAb 3 weeks None + + +
6 APP/PS1 mAb 3 weeks None + + +
10 APP/PS1 mAb 3 weeks None + + + +
1 APP/PS1 mAb 3 weeks None + +
18 W mAb 3 weeks None +
13 APP/PS1 mAb 488 3 weeks None + +
14 APP/PS1 mAb 488 3 weeks None + +
15 W mAb 488 3 weeks None + +
16 W mAb 488 3 weeks None + + +
29 APP/PS1 naï e - None +
30 APP/PS1 naï e - None + +
31 APP/PS1 naï e - None + +
32 APP/PS1 naï e - None + +
33 APP/PS1 naï e - None + +
34 APP/PS1 naï e - None +
35 APP/PS1 naï e - None + +
36 W naï e - None +
37 APP/PS1 naï e - None +
2.2. The Aphe esis Module Is Pe meable o Aβand Impe meable o An i-AβAn ibody In Vi o
The nanopo ous memb anes (NPMBs) used in his s udy we e i s es ed
in i o
o de e mine hei e icacy in e ms o A
β
pe meabili y (Supplemen a y Figu e S1A,B).
Thei impe meabili y o molecules la ge han he po e size o he NPMBs used he e
(9
±
2 nm) was also es ed wi h albumin (~68 kDa), which is smalle han mAb (~150 kDa),
as a e e ence molecule (Supplemen a y Figu e S1C,D). To de e mine whe he he soluble
A
β
pep ide p esen in CSF was able o pass h ough he NPMBs in he aphe esis module
in i o
, A
β
le els wi hin he ese oi we e quan i ied wi h SIMOA a e being explan ed
om sac i iced mice. One- hi d o he ese oi s analyzed showed he p esence o A
β
in
hei con en (Figu e 2A; Table 1).
On he o he hand, he impe meabili y o he sys em o he he apeu ic agen used
in his s udy, an i-A
β
mAb (clone 6E10), was s udied. Fo his pu pose, ou addi ional
mice ( wo APP/PS1 and wo WT) we e ea ed wi h he Alexa 488-conjuga ed e sion
o he same mAb (Table 1). The le els o luo escence emi ed by he mAb we e hen
de e mined bo h in he ese oi and in di e en issues o he mice. The able in Figu e 2B
shows luo escence (emission: 600 nm) de e mined in he ese oi , in pe iphe al issue
(li e homogena e), and sys emic blood (whole blood and plasma). As a con ol o he
luo escence emi ed by he mAb, a 1:200 dilu ion was measu ed. The same dilu ion was
In . J. Mol. Sci. 2022,23, 9256 5 o 13
also pe o med on he samples ob ained om he ese oi s. In his way, i was de e mined
ha hei luo escence was highe han he emission le els a 600 nm de ec ed in undilu ed
pe iphe al samples om he same mice. These samples also had simila emissions o he
alues de ec ed in naï e mice, which we e used as a con ol o basal au o luo escence.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 o 14
2.2. The Aphe esis Module Is Pe meable o Aβ and Impe meable o An i-Aβ An ibody In Vi o
The nanopo ous memb anes (NPMBs) used in his s udy we e i s es ed in i o o
de e mine hei e icacy in e ms o Aβ pe meabili y (Supplemen a y Figu e S1A,B). Thei
impe meabili y o molecules la ge han he po e size o he NPMBs used he e (9 ± 2 nm)
was also es ed wi h albumin (~68 kDa), which is smalle han mAb (~150 kDa), as a e -
e ence molecule (Supplemen a y Figu e S1C,D). To de e mine whe he he soluble Aβ
pep ide p esen in CSF was able o pass h ough he NPMBs in he aphe esis module in
i o, Aβ le els wi hin he ese oi we e quan i ied wi h SIMOA a e being explan ed
om sac i iced mice. One- hi d o he ese oi s analyzed showed he p esence o Aβ in
hei con en (Figu e 2A; Table 1).
Figu e 2. Pe meabili y o Aβ and impe meabili y o mAb analysis a e in i o s udy. (A) G aph
shows he le els o Aβ de ec ed (pg/mL) in he ese oi o W and APP/PS1 mice, which could be
loaded wi h ehicle (aCSF) o wi h an i-Aβ mAb. Da a a e p esen ed as mean ± SEM. Each do ep-
esen s one mouse. (B) Emi ed luo escence da a a 600 nm. Table shows he emission alues a 600
nm de ec ed in liquid samples om ou APP/PS1 mice. These samples we e ob ained om he
ese oi , whole blood, plasma, and li e homogena e om wo mice wi h he de ice and ea ed
wi h Alexa 488-conjuga ed mAb (clone 6E10) and wo mice ha had no been ope a ed (naï e). Da a
o hese mice can be ound in Table 1. The 600 nm emission alues o a 1:200 dilu ion o he mAb,
as well as o aCSF used as ehicle and li e homogeniza ion bu e a e also shown. (C) Rep esen a-
i e images o ce eb al co ex and li e sec ions om APP/PS1 mice ea ed wi h Alexa 488-conju-
ga ed mAb (clone 6E10). F om le o igh , immuno luo escence in consecu i e sec ions was de el-
oped using a p ima y an ibody o Aβ plaque de ec ion (clone 6C3), a p ima y an ibody mo e
Figu e 2.
Pe meabili y o A
β
and impe meabili y o mAb analysis a e
in i o
s udy. (
A
) G aph
shows he le els o A
β
de ec ed (pg/mL) in he ese oi o W and APP/PS1 mice, which could
be loaded wi h ehicle (aCSF) o wi h an i-A
β
mAb. Da a a e p esen ed as mean
±
SEM. Each do
ep esen s one mouse. (
B
) Emi ed luo escence da a a 600 nm. Table shows he emission alues a
600 nm de ec ed in liquid samples om ou APP/PS1 mice. These samples we e ob ained om he
ese oi , whole blood, plasma, and li e homogena e om wo mice wi h he de ice and ea ed
wi h Alexa 488-conjuga ed mAb (clone 6E10) and wo mice ha had no been ope a ed (naï e). Da a
o hese mice can be ound in Table 1. The 600 nm emission alues o a 1:200 dilu ion o he mAb, as
well as o aCSF used as ehicle and li e homogeniza ion bu e a e also shown. (
C
) Rep esen a i e
images o ce eb al co ex and li e sec ions om APP/PS1 mice ea ed wi h Alexa 488-conjuga ed
mAb (clone 6E10). F om le o igh , immuno luo escence in consecu i e sec ions was de eloped
using a p ima y an ibody o A
β
plaque de ec ion (clone 6C3), a p ima y an ibody mo e speci ic o
soluble A
β
(clone 6E10), only Alexa 488-conjuga ed seconda y an ibody, o no an ibody a all. As
shown in he images, no signal is de ec ed in he absence o any an ibody.
In addi ion, an immuno luo escence s udy was pe o med on c yop ese ed sec ions
o bo h he b ain and li e (Figu e 2C). Fi s , we used a p ima y an ibody ha de ec s
oligome ic o ms o A
β42
(clone 6C3) and a seconda y an ibody conjuga ed wi h Alexa 488.
Resul s showed ha , in he ce eb al co ex, high abundance o A
β
plaques we e de ec ed,
while no signal was ound in he li e o hese mice. In consecu i e sec ions, ano he

In . J. Mol. Sci. 2022,23, 9256 6 o 13
immuno luo escence was pe o med using he same p ima y an ibody as o ea men
(clone 6E10), wi h highe a ini y o soluble and in acellula o ms o A
β
[
28
]. In his
case, sligh labelling was obse ed in he ce eb al co ex bu no in he li e . Howe e ,
incuba ion o he sec ions wi h only labelled seconda y an ibody o no an ibody showed
no signal in ei he he ce eb al co ex o in he li e . All in all, hese esul s indica e ha
he mAb con ained in he ese oi s is no able o pass h ough he il a ion sys em and
he e o e does no appea in b ain issue o pe iphe al issues.
2.3. Con inuous CSF Aphe esis o Rapidly Dec eased A ea Co e ed by A
β
Plaques in he Ce eb al Co ex
Once we de e mined ha A
β
eaches he ese oi while he mAb emained e ained
in he il a ion sys em
in i o
, we assessed he e ec o he h ee-week in e en ion. To
his aim, we quan i ied he a ea occupied by A
β
plaques in ela ion o he o al assessed
a ea o he ce eb al co ex in APP/PS1 mice (Figu e 3). Resul s showed ha con inuous
aphe esis o CSF wi h an i-A
β
mAb signi ican ly educed he a ea occupied by plaques
ela i e o aCSF APP/PS1 mice (100
±
18.80% s. 58.84
±
7.01%; Figu e 3A,B,D). In some
o hese mice, we also de e mined soluble A
β42
le els in ce eb al co ex homogena es and
in plasma (Figu e 3E,F). Howe e , we obse ed no di e ences be ween mice ea ed wi h
aCSF o mAb in ela ion o soluble A
β42
le els in APP/PS1 mice o he same age and ha
had no unde gone su ge y a his le el. Thus, hese esul s indica ed ha con inuous CSF
aphe esis educes he su ace a ea occupied by A
β
plaques in he ce eb al co ex wi h no
changes in b ain o plasma soluble Aβ42 le els a e his in e en ion ime.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 o 14
Figu e 3. E alua ion o he e ec o selec i e and con inuous CSF aphe esis in i o. (A–C) Rep e-
sen a i e images o immunohis ochemis y de eloped o he quan i ica ion o Aβ plaques in
APP/PS1 mice wi h he de ice and ea ed wi h aCSF (A) o mAb (B) and hei compa ison wi h a
W mouse (C). Scale ba : 100 µm. (D) Quan i ica ion o he a ea occupied by plaques wi h espec o
he o al su ace a ea o he co ex is ep esen ed as he pe cen age o change wi h espec o aCSF-
ea ed mice. Da a a e p esen ed as mean ± SEM. Each do ep esen s one mouse. S a is ical com-
pa ison be ween he wo g oups was pe o med using unpai ed S uden ’s - es . *, p < 0.05. (E)
G aphical ep esen a ion o he quan i ica ion o Aβ le els (pg/mL) in ce eb al co ex homogena es
om APP/PS1 mice wi h he de ice, con aining aCSF o mAb, and in age-ma ched APP/PS1 mice
ha ha e no unde gone su ge y (naï e). Da a a e p esen ed as mean ± SEM. Each do ep esen s
one mouse. (F) G aphical ep esen a ion o he quan i ica ion o Aβ le els (pg/mL) in plasma om
APP/PS1 mice implan ed wi h he de ice con aining aCSF o mAb and in in age-ma ched APP/PS1
mice ha ha e no unde gone su ge y (naï e). Da a a e p esen ed as mean ± SEM. Each do ep e-
sen s one mouse.
3. Discussion
In his wo k, we p esen a new implan able de ice o clea ing selec i ely a ge ed
molecules di ec ly om he CSF. The inno a i e mechanism o ac ion o his de ice is
based on he p ope y o selec i e molecula pe meabili y o NPMBs. Thus, we bioengi-
nee ed a modula de ice whe e he key componen is he aphe esis module endowed wi h
ailo ed NPMBs wi h speci ic physicochemical p ope ies. Addi ionally, we in eg a ed he
aphe esis module wi h o he componen s o access o he CSF and o in usion o he a-
peu ic agen s/aCSF. As he mechanism o ac ion is based on he p ope y o selec i e mo-
lecula pe meabili y o NPMBs, we i s es ed he sys em bo h in i o and in i o,
demons a ing he e lux o small molecules, such as Aβ, and he impe meabili y o mol-
ecules la ge han he memb ane po e size, such as albumin o immunoglobulins. Albei
we showed ha memb anes a e pe meable o Aβ, he e we e some inconsis encies among
he di e en samples aken om he in i o s udy ha may ela e o di e en a iables,
such as he use o he apeu ic agen o aCSF, he espec i e doses, ime o de ice im-
plan ed, and sample p ocessing. Impo an ly, we ha e no assessed he le el o bio ouling
ha may ha e occu ed in memb anes and po en ially a ec he pe meabili y p ope ies
o e ime [29]. Fu he s udies a e needed o be e unde s and bio ouling o memb anes
and he ime-dependen molecula dynamics o Aβ and mAb h ough NPMBs in i o.
Figu e 3.
E alua ion o he e ec o selec i e and con inuous CSF aphe esis
in i o
. (
A
–
C
) Rep esen a i e
images o immunohis ochemis y de eloped o he quan i ica ion o A
β
plaques in APP/PS1 mice
wi h he de ice and ea ed wi h aCSF (
A
) o mAb (
B
) and hei compa ison wi h a W mouse (
C
).
Scale ba : 100
µ
m. (
D
) Quan i ica ion o he a ea occupied by plaques wi h espec o he o al su ace
a ea o he co ex is ep esen ed as he pe cen age o change wi h espec o aCSF- ea ed mice. Da a
a e p esen ed as mean
±
SEM. Each do ep esen s one mouse. S a is ical compa ison be ween he
wo g oups was pe o med using unpai ed S uden ’s - es . *, p< 0.05. (
E
) G aphical ep esen a ion
o he quan i ica ion o A
β
le els (pg/mL) in ce eb al co ex homogena es om APP/PS1 mice wi h
he de ice, con aining aCSF o mAb, and in age-ma ched APP/PS1 mice ha ha e no unde gone
su ge y (naï e). Da a a e p esen ed as mean
±
SEM. Each do ep esen s one mouse. (
F
) G aphical
ep esen a ion o he quan i ica ion o A
β
le els (pg/mL) in plasma om APP/PS1 mice implan ed
wi h he de ice con aining aCSF o mAb and in in age-ma ched APP/PS1 mice ha ha e no
unde gone su ge y (naï e). Da a a e p esen ed as mean ±SEM. Each do ep esen s one mouse.
In . J. Mol. Sci. 2022,23, 9256 7 o 13
3. Discussion
In his wo k, we p esen a new implan able de ice o clea ing selec i ely a ge ed
molecules di ec ly om he CSF. The inno a i e mechanism o ac ion o his de ice is based
on he p ope y o selec i e molecula pe meabili y o NPMBs. Thus, we bioenginee ed a
modula de ice whe e he key componen is he aphe esis module endowed wi h ailo ed
NPMBs wi h speci ic physicochemical p ope ies. Addi ionally, we in eg a ed he aphe esis
module wi h o he componen s o access o he CSF and o in usion o he apeu ic
agen s/aCSF. As he mechanism o ac ion is based on he p ope y o selec i e molecula
pe meabili y o NPMBs, we i s es ed he sys em bo h
in i o
and
in i o
, demons a ing
he e lux o small molecules, such as A
β
, and he impe meabili y o molecules la ge han
he memb ane po e size, such as albumin o immunoglobulins. Albei we showed ha
memb anes a e pe meable o A
β
, he e we e some inconsis encies among he di e en
samples aken om he
in i o
s udy ha may ela e o di e en a iables, such as he
use o he apeu ic agen o aCSF, he espec i e doses, ime o de ice implan ed, and
sample p ocessing. Impo an ly, we ha e no assessed he le el o bio ouling ha may ha e
occu ed in memb anes and po en ially a ec he pe meabili y p ope ies o e ime [
29
].
Fu he s udies a e needed o be e unde s and bio ouling o memb anes and he ime-
dependen molecula dynamics o Aβand mAb h ough NPMBs in i o.
E en when implan a ion o he minia u ized p o o ype in mice is easible and globally
sa e, complica ions may p esen in he immedia e and de e ed pos -ope a o y. Some
complica ions, such as weigh loss and le ha gy, a e mo e likely o be de i ed om he
su gical p ocess i sel o om he side e ec s o pos -su gical medica ion han om he
de ice o he he apeu ic agen . Howe e , he size o pa A in he de ice is wi hin he
uppe limi o wha is easible o an implan able subcu aneous de ice in mice. The e o e,
we will wo k o ine- une he de ice by mainly educing he size o pa A o u u e s udies.
In addi ion, we a e cu en ly conduc ing a ho ough e iew o he p o ocols o inc ease
sa e y and imp o e he wel a e o he mice.
The main inding o his s udy is ha con inuous selec i e aphe esis o soluble A
β
om
he CSF apidly clea s amyloid plaques. This educ ion could be due o he en apmen o
Aβin o he aphe esis module, hus gene a ing an e lux om he ISF o he CSF. P e ious
s udies ha e al eady epo ed ha CNS-deli e ed immuno he apies wi h an ibodies agains
A
β
, including he one used in his s udy, was e ec i e in educing bo h A
β
plaques and
in acellula A
β
[
28
,
30
]. Those esul s we e ob ained ei he a e an in ahippocampal
injec ion in which case he educ ion occu ed on he ipsila e al side [
30
] o a e an injec ion
in o he hi d en icle wi h educ ion obse ed bila e ally in he hippocampus bu no
in he amygdala [
28
]. Ne e heless, in bo h cases he e ec s we e obse ed a se en days
pos -injec ion and we e los 30 days a e injec ion. In his p oo o concep , he educ ion
obse ed in up o h ee weeks o selec i e CSF aphe esis con i ms ha his sys em elici s
in he sho ime he same e ec on A
β
plaques as A
β
-immuno he apies and o he A
β
-
clea ing me hods. Howe e , heo e ically, he mo e sus ained he A
β
clea ance e ec , he
mo e p o ound and ex ensi e he e e se in he neu opa hological and subsequen clinical
changes will be in he long e m. Consequen ly, sys ems o con inuous and sus ained
clea ance o a ge molecules in he CNS a e needed, and he de ice he e p esen ed may
con ibu e o his aim.
One o he main limi a ions o his s udy is ha we ha e no pe o med unc ional s ud-
ies o de e mine whe he con inuous and selec i e aphe esis no only educes A
β
plaques
bu also has cogni i e bene i s. Howe e , a educ ion o bo h plaques and in acellula A
β
has p e iously been epo ed o be associa ed wi h some cogni i e impai men s in mu ine
models o AD [
28
]. Ne e heless, his is no he case in clinical ials wi h sys emically
adminis e ed an i-A
β
mAb which, while showing e ec i e emo al o plaques, no signi i-
can clinical bene i was ound [
13
,
14
]. Indeed, plaque o ma ion i sel could be a de ense
mechanism [
4
], and densely deposi ed A
β
may no esul as oxic as p e iously hough .
Fu he mo e, he mos oxic species o A
β
a e oligome ic soluble o ms [
31
,
32
], which is
now pe cei ed as an ad an age o mAb speci ically add essed agains hem [
13
]. Al hough
In . J. Mol. Sci. 2022,23, 9256 8 o 13
in his s udy we ha e no de ec ed changes in soluble Aβin b ain pa enchyma o plasma,
i is possible ha longe aphe esis imes may ha e he capaci y o modi y he le els o
hese mo e oxic A
β
species. The e o e, in u u e long- e m s udies, a mo e comp ehensi e
analysis o he di e en compa men s in o which soluble o ms o A
β
may mo e will
be needed o ully unde s and he dynamics o A
β
be ween plaques, ISF, CSF, and he
aphe esis module.
Al hough he implan able de ice o CSF con inuous and selec i e aphe esis is an
in asi e p ocedu e in small animal models, in i s cu en o m, he esul s ob ained in his
p oo -o -concep s udy o e a sho pe iod o ime a e p omising. Cos /bene i implica ions
o his he apeu ic s a egy o NND pa ien s may be e y high as his ou e p o ides
se e al ad an ages o e “s anda d” pe iphe ally adminis e ed d ugs, including: 1, he
sys em allows ac ing on he CSF di ec ly and con inuously, p o iding a mo e powe ul
clea ance e ec han he apies based on pe iphe al clea ance; 2, being a selec i e he apy
p e en s po en ial sa e y issues ha may occu i he le els o o he molecules in he CSF
we e modi ied; 3, immunoisola ion o mAb impedes immune esponses, ully a oiding one
o he mos se ious side e ec s epo ed wi h mAb sys emically adminis e ed. All in all, he
inno a ion he e p esen ed opens up he doo o a subs an ial imp o emen in he ea men
o de as a ing NDD o which he e is cu en ly no cu e.
4. Ma e ials and Me hods
4.1. Mice
Se en een se en-mon h-old male APP/PS1 mice, ansgenic o mouse/human amy-
loid p ecu so p o ein (Mo/HuAPP695swe) and a mu an human p esenilin 1 (PS1-dE9) in
129S backg ound and se en non- ansgenic li e ma es (W ) we e used o de ice implan-
a ion. These mice we e andomly di ided in o wo g oups: con ol ( ehicle, aCSF) and
ea men (mAb). Nine addi ional male mice o he same age, bu which had no unde gone
su ge y, we e also used (eigh APP/PS1 and one W ; naï e). The dis ibu ion o hese mice
in he di e en g oups and hei use o he espec i e p ocedu es can be seen in Table 1.
Mice we e main ained wi h ood and wa e ad libi um on a 12 h ligh /da k cycle (onse
a 8:00 a.m.) and unde con olled empe a u e (22
±
2
◦
C). All p ocedu es we e conduc ed
du ing he ligh po ion o he cycle be ween 8:30 and 14:00 a.m. a he Animal Facili y o
Uni e sidad de O iedo and we e pe o med in acco dance wi h ins i u ional guidelines
app o ed by The Resea ch E hics Commi ee o he Uni e si y o O iedo (PROAE 32/2020).
4.2. Implan able De ice o Mice
We ha e designed a p o o ype o an implan able de ice o con inuous and selec i e
aphe esis o CSF (Figu e 1A). The de ice was minia u ized o adap i o he size o mice
and o manu ac u e in biocompa ible ma e ials. I consis s o wo main componen s, a
subcu aneous ese oi and an aphe esis module endowed wi h ailo ed
NPMBs [33,34]
,
which we e encapsula ed oge he in a capsule made by Neu oscience Inno a i e Technolo-
gies (Figu e 1A, pa A). Fo his s udy, NPMBs ha e a po e size o 9
±
2 nm. This allows
he passi e low o smalle subs ances, such as soluble A
β
(~4 kDa in i s monome ic o m),
on bo h sides while p e en ing he passage o molecules la ge han 10 nm, e.g., an ibodies
(immunoglobulin G, ~150 kDa) o albumin (~68 kDa) ha may ac as he apeu ic agen s
(Supplemen a y Ma e ial). These agen s a e e ained in he de ice wi hin he subcu aneous
ese oi which has a olume o 100
µ
L. A he op o he ese oi is a sel -sealing po ,
which allows illing using a 50
µ
L sy inge (800 se ies mic oli e sy inges; Hamil on, Reno,
NV, USA). This design allows d ugs con ined in he ese oi o in e ac wi h he a ge
molecule ha is il e ed h ough he NPMBs.
The aphe esis module is connec ed h ough a b ain in usion cannula (B ain In usion
ki 2
;
Alze , Cupe ino, CA, USA) o one o he la e al en icles o he b ain (Figu e 1A, pa B).
In . J. Mol. Sci. 2022,23, 9256 9 o 13
4.3. Filling he Rese oi wi h An i-AβmAb o Vehicle
The ese oi was illed unde s e ile condi ions wi h an an i-A
β1–42
mAb, which
ecognizes human bu no mu ine A
β1–42
. To es ima e he mos app op ia e dose o
an ibody, we conside ed p e ious s udies in which a single dose o an ibody was injec ed
in ace eb o en icula ly, and A
β
clea ance was e alua ed [
28
]. Since hese s udies showed
ha he dose applied, 10
µ
g, had an e ec up o se en days bu no up o hi y days, we
conside ed a dose o 50
µ
g o an ibody and a s udy ime o h ee weeks in ou expe imen
o assess he changes associa ed wi h con inuous CSF aphe esis in he p esence o his
he apeu ic agen . Thus, en de ices (Table 1) we e loaded wi h a solu ion o non-conjuga ed
an i-
β
-Amyloid an ibody (clone 6E10; Biolegend, San Diego, CA, USA) dilu ed in a i icial
CSF (aCSF; ACSF, Toc is Bioscience, B is ol, UK). Addi ionally, ano he ou we e illed
wi h luo opho e-conjuga ed an i-A
β
an ibody (Alexa Fluo
®
488 an i-
β
-Amyloid, clone
6E10; Biolegend). Final mAb concen a ion wi hin he de ice was 500
µ
g/mL. As con ol,
en de ices we e illed wi h he same olume o aCSF.
4.4. Su ge y and Mice Wel a e
The su gical p ocedu e was de eloped acco ding o he p o ocol desc ibed in [
35
].
B ie ly, anes he ized mice (2% iso luo ane and 0.4 L/min O
2
) we e placed in he s e eo axic
ame (Digi al Compac Mouse S e eo axic Ins umen ; Ha a d Appa a us, Hollis on, MA,
USA), and he su ge y a ea was hen sha ed. To expose he skull, an incision was made
om he middle o he skull o he base o he neck om whe e a subcu aneous pocke was
opened, and he de ice was inse ed. Finally, he cannula was implan ed in o he le la e al
en icle. Su ge y coo dina es, wi h espec o B egma, we e AP—1.26 mm, ML—0.7 mm,
and—2.5 mm o he skull. The cannula was glued wi h cyanoac yla e (Cicas ik Su u e ;
Chemical Ibe ica, Salamanca, Spain), and he incision was su u ed using 5–0 su u e h ead
(S e ile Su gical Su u e; Lo caMa ín, Mu cia, Spain).
A e su ge y, all he mice we e placed in an in ensi e ca e cage wi h exhaus i e
empe a u e con ol (28–30
◦
C; Ve a io S50, Kanal e , Bizkaia, Spain) du ing he i s 24 h
o ensu e p ope eco e y. All mice ecei ed p e- and pos -su gical analgesic and an ibio ic
medica ion; addi ionally, hey we e housed indi idually a e su ge y o p e en damage
o he su u es.
Te mina ion was scheduled a 21 days. Th oughou he s udy, he mice we e moni o ed
daily, obse ing hei weigh , cage beha io (nes o ma ion), ood, and wa e consump ion.
4.5. Tissue and Fluids Collec ion and P ocessing
Th ee weeks a e su ge y—o ea lie i se e e complica ions p esen ed—mice we e
deeply anaes he ized wi h ke amine (100 mg/kg) and xylazine (10 mg/kg) in saline solu ion.
The de ice was ca e ully emo ed and ho oughly cleaned ex e nally wi h 70% e hanol.
Subsequen ly, he luid was emo ed om he ese oi and s o ed a
−
80
◦
C o u he
s udy. Pe iphe al blood samples we e collec ed om he in e io ena ca a (~0.5–1 mL).
Fo his, EDTA (as an icoagulan ; 1.5 mg/mL blood) p e ea ed 1 mL sy inges (BD, F anklin
Lakes, NJ, USA) and in a enous ca he e s (BD Insy e-W, 21 GA, BD) we e used. Blood
was immedia ely cen i uged a 2000 g, and plasma was s o ed a
−
80
◦
C un il use. A e
blood ex ac ion, he abdominal ao a was cu a he same le el o he ena ca a, and s e ile
ice-cold phospha e bu e saline (PBS; ~30 mL) was pe used o clean issues.
4.5.1. Tissue P ocessing o Biochemical Analysis
In mice used o biochemical analysis, he b ains we e sagi ally dissec ed, and he
ce eb al co exes we e ex ac ed om he igh hemisphe es and subsequen ly ozen a
−
80
◦
C. In addi ion, li e po ions (equal o 200 mg) we e emo ed and spli in o wo
hal es, one o which was also ozen un il use a −80 ◦C.
The ce eb al co exes and li e s (100 mg) we e homogenized in lysis bu e (20 mM
HEPES pH 7.4, NaCl 100 mM, NaF 50 mM, EDTA 5 mM, T i on X-100 1%) wi h p o ease
inhibi o s (cOmple e
™
, Mini P o ease Inhibi o Cock ail; Roche, Basel, Swi ze land) in