New Pe spec i es in Neu op o ec ion o Ischemic S oke
Ma ía Pé ez-Ma o
a
, Es eban López-A ias
a
, Ana Bugallo-Casal
a
, Cla a Co ea-Paz
a
, Susana A ias
b
,
Manuel Rod íguez-Yáñez
b
, Ma ía San ama ía-Cada id
b
, F ancisco Campos
a,c,
⇑
a
T ansla ional S oke Labo a o y G oup (TREAT), Clinical Neu osciences Resea ch Labo a o y (LINC), Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS), 15706 San iago
de Compos ela, Spain
b
S oke Uni , Depa men o Neu ology, Hospi al Clínico Uni e si a io, 15706 San iago de Compos ela, Spain
c
Uni e sidade de San iago de Compos ela, 15782 San iago de Compos ela, Spain
ARTICLE INFO
Key wo ds:
bioma ke s
ea ly diagnosis
ischemic s oke
neu op o ec ion
ecanaliza ion he apies
ABSTRACT
The cons an ailu e o new neu op o ec i e he apies o ischemic s oke has pa ially hal ed he sea ch o
new he apies in ecen yea s, mainly because o he high in es men isk equi ed o de elop a new ea men
o a complex pa hology, such as s oke, wi h a na ow in e en ion window and associa ed como bidi ies.
Howe e , owing o ecen p og ess in unde s anding he s oke pa hophysiology, imp o emen in pa ien ca e
in s oke uni s, de elopmen o new imaging echniques, sea ch o new bioma ke s o ea ly diagnosis, and
inc easingly widesp ead use o mechanical ecanaliza ion he apies, new oppo uni ies ha e opened o he
s udy o neu op o ec ion. This e iew summa izes he main p o ec i e agen s cu en ly in use, some o which
a e al eady in he clinical e alua ion phase. I also includes an analysis o how ecanaliza ion he apies, new
imaging echniques, and bioma ke s ha e imp o ed hei e ficacy.
INTRODUCTION
S oke is defined as “a neu ological defici a ibu ed o acu e ocal
inju y o he cen al ne ous sys em (CNS) om a ascula cause,
including ce eb al in a c ion, in ace eb al hemo hage, and suba ach-
noid hemo hage”and is a leading cause o disabili y and dea h wo ld-
wide, acco ding o he Ame ican S oke Associa ion/Ame ican Hea
Associa ion (Sacco e al., 2013). O he 15 million people wo ldwide
who expe ience s oke annually, 5 million die and 5 million a e le
wi h a ious disabili ies (Wo ld Heal h O ganiza ion da a). In men
and women younge han 55 yea s, he incidence emains simila ,
bu i is highe in men be ween he ages o 55 and 75 yea s
(Collabo a o s GBDLRoS e al., 2018; Collabo a o s GBDN, 2019).
Imp o emen s in he managemen o pa ien s wi h s oke in p ima y
ca e and s oke uni s, pa icula ly in de eloped coun ies, ha e con-
ibu ed o a educ ion in mo ali y in ecen yea s. Howe e , he inci-
dence does no ollow he same end owing o ac o s such as
demog aphic changes and inc eased p e alence o hype ension, obe-
si y, and o he como bidi ies (O biagele and Nguyen‐Huynh, 2011).
S oke can be di ided in o wo main ypes: hemo hagic and
ischemic. In hemo hagic s oke, he up u e o a blood essel causes
in ace eb al hemo hage owing o he ex a asa ion o blood in o
he b ain pa enchyma, whe eas in ischemic s oke, he occlusion o a
blood essel by a h ombus causes a dec ease in ce eb al blood flow
(Ama enco e al., 2009). F om a he apeu ic poin o iew, in in ac-
e eb al hemo hage, con ol o hemo hage and educ ion o in ac a-
nial p essu e a e he main he apeu ic s a egies o p e en neu onal
damage (Hemphill e al., 2015). In ischemic s oke, epe usion s a e-
gies (pha macological h ombolysis and mechanical h ombec omy
[MT]) a e he he apies aimed a es o ing ce eb al blood flow
(CBF). Pha macological he apy, based on he use o ecombinan is-
sue plasminogen ac i a o ( PA, also known as al eplase) o i s new
de i a i e enec eplase (TNK), is he only d ug ea men o acu e
ischemic s oke. These h omboly ics con e plasminogen in o plas-
min, which b eaks down he fib in ne wo k cons i u ing he h ombus.
This ea men has been in use since 1995, and i is e ec i e in imp o -
ing clinical ou comes, educing disabili y, and achie ing success ul
ecanaliza ion in app oxima ely 30% o pa ien s. Howe e , PA and
h ps://doi.o g/10.1016/j.neu oscience.2024.02.017
Recei ed 1 No embe 2023; Accep ed 16 Feb ua y 2024
A ailable online 21 Feb ua y 2024
0306-4522/© 2024 The Au ho (s). Published by Else ie Inc. on behal o IBRO.
This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
Abb e ia ions: ASo, an isense oligonucleo ides; CBF, ce eb al blood flow; CNS, cen al ne ous sys em; CT, compu ed omog aphy; FGF21, fib oblas g ow h ac o 21; GOT,
glu ama e oxaloace a e ansaminase; HT, hemo hagic ans o ma ion; LVO, la ge essel occlusion; MRI, magne ic esonance imaging; MT, mechanical h ombec omy; NA1,
ne ine ide; NMDA, N‐me hyl‐d‐aspa a e; NMDAR, NMDA glu ama e ecep o ; PET, posi on emission omog aphy; PA, ecombinan issue plasminogen ac i a o ; RBM3, RNA‐
binding mo i p o ein 3; S1P, sphingosine 1‐phospha e; SH, sys emic hypo he mia; SPAN, S oke P eclinical Assessmen Ne wo k; TNK, enec eplase; TLR, oll‐like ecep o .
⇑
Co esponding au ho .
E-mail add esses: [email p o ec ed],[email p o ec ed] (F. Campos).
Neu oscience 550 (2024) 30–42
Con en s lis s a ailable a ScienceDi ec
Neu oscience
jou nal homepage: www.else ie .com/loca e/nsc
TNK ha e many d awbacks ha limi hei use, such as a sho hal ‐li e
(a ew min), which implies ansien e ec s, neu o oxic side e ec s
owing o he modula ion o N‐me hyl‐
D
‐aspa a e (NMDA) glu ama e
ecep o s, isk o hemo hagic ans o ma ion (HT), and p oinflamma-
o y e ec s (Thiebau e al., 2018). These limi a ions esul in a sho
he apeu ic window o 4.5–6 h a e s oke onse , which subsequen ly
educes he numbe o pa ien s who can benefi om his he apy
(Tomsick e al., 2010). MT, ei he alone o combined wi h pha maco-
logical h ombolysis, has imp o ed ecanaliza ion a es (up o 80%)
and dec eased he isk o hemo hagic ans o ma ions (HTs), wi h a
gap be ween he inc eased ecanaliza ion a e and imp o ed neu olog-
ical unc ions. Howe e , his mechanical s a egy is use ul only when
h ombi a e loca ed in la ge essels and equi es specialized hospi al
acili ies (Gaube i e al., 2021; La edo e al., 2022).
Gi en ha he numbe o s oke candida es o ecanaliza ion ea -
men s o ischemic s oke is limi ed, he e is an u gen need o iden i y
al e na i e neu op o ec i e (o cy op o ec i e) agen s ha can be used
in a la ge numbe o pa ien s. I is gene ally accep ed ha a e ce e-
b al essel occlusion, he ischemic egion can be di ided in o wo
egions: an in e nal co e egion cha ac e ized by nec o ic cell dea h
wi hin he fi s ew hou s a e s oke and a po en ially sal ageable
egion su ounding he ischemic co e, defined as he penumb a. The e-
o e, i ep esen s a po en ial he apeu ic a ge o educing he e ec s
o ischemic inju y, as long as he changes esul ing om he ischemic
cascade do no cause i e e sible damage leading o issue dea h. Con-
side able esea ch has been de o ed o he de elopmen o no el he -
apies o p o ec he b ain om damage a e s oke, wi h pa icula
a en ion paid o he penumb al egion. Se e al neu op o ec i e ea -
men s, including glu ama e an agonis s, calcium blocke s, oxida i e
s ess‐ ee adicals, and immunomodula o s, ha e shown g ea po en-
ial in animal models o s oke. Howe e , almos all o hese ea -
men s ha e ailed o p o ide p o ec ion in human ials (Sa i z
e al., 2017).
Many a gumen s can explain why neu op o ec i e d ugs ha e no
wo ked in pa ien s wi h s oke, such as ansla ional expe imen al lim-
i a ions o clinical issues, including p ope pa ien selec ion in clinical
ials and a na ow he apeu ic window (Su he land e al., 2012). All
hese p eclinical s udies ocused on he sea ch o new and e ec i e
p o ec i e agen s ha a e c ucial in defining and cha ac e izing he
molecula e en s ha occu a e ischemic onse . Howe e , he pe sis-
en ailu e in he sea ch o e ec i e neu op o ec i e he apies has
educed he en husiasm o pha maceu ical companies, mainly owing
o he high isk o in e sion. One o he bes examples is As aZeneca's
de elopmen o XY059, a ee adical sca enge (An onic e al., 2018).
Disodium 2, 4‐sulphophenyl‐N‐ e ‐bu ylni one (known as NXY‐059)
is a ee adical sca enge ha educes he in a c olume in oden
and p ima e s oke models. The ansla ional p omise o hese publica-
ions, oge he wi h he e idence o sa e y in con ol and acu e s oke
popula ions, led he company o conduc wo andomized, placebo‐
con olled phase III ials o NXY‐059. Al hough he fi s s udy showed
a weak posi i e signal in he p ima y and seconda y analyses, he sec-
ond, la ge s udy (3306 s. 1722 pa ien s) was neu al, p o iding no
e idence o he e ficacy o NXY‐059 in pa ien s wi h s oke (An onic
e al., 2018).
I we ex end hese analyses o o he s udies conduc ed in he ea ly
1990s, app oxima ely 500 clinical ials we e conduc ed o es mo e
han 200 new po en ial neu op o ec i e ea men s o ischemic
s oke. Howe e , as o 2018, only nine o hese ea men s ha e suc-
cess ully p og essed o phase III ials (Shi e al., 2018).
Recen ad ances in he unde s anding o s oke pa hophysiology,
imp o emen o pa ien ca e in s oke uni s, de elopmen o new imag-
ing modali ies, sea ch o new bioma ke s o ea ly diagnosis, and
inc easing use o mechanical ecanaliza ion he apies ha e opened
up new oppo uni ies o he s udy o new p o ec i e he apies
(Chamo o e al., 2021). This e iew p o ides an o e iew o cu en
and p omising app oaches in he field o neu op o ec i e s oke ea -
men (Chamo o e al., 2016, 2021; Sa i z e al., 2017; Shi e al., 2018;
Paul and Candela io‐Jalil, 2021; Fishe and Sa i z, 2022; Ghozy e al.,
2022; Haup e al., 2023; Liu e al., 2023) and analyzes how new
ad ances in he field o imaging, bioma ke s, and ecanaliza ion he -
apies ha e con ibu ed o imp o ing he e ficacy and u ili y o p o ec-
i e agen s (Kidwell e al., 2013; Rocha and Jo in, 2017; Shi e al.,
2018; D iga e al., 2021; Fishe and Sa i z, 2022).
EXPERIMENTAL PROCEDURES
The p esen e iew was pe o med by sea ching he MEDLINE da a-
base using he PubMed sea ch engine wi hou applying any es ic-
ions ela ed o he yea o publica ion, coun y, o ca ego y o
publica ion (e.g., o iginal a icle o e iew). Only a icles published
in English we e included. The sea ch c i e ia used we e keywo ds
ela ed o he p o ec i e he apies included in his manusc ip , which,
in he au ho s' opinion, ha e he g ea es impac in Eu ope in he field
o s oke and ha e eached he clinical analysis s age. In addi ion, a
manual sea ch was pe o med o include in o ma ion on clinical ials
in pa ien s wi h s oke whose esul s ha e been published in con e -
ences o cong esses bu no in jou nals de ec ed by PubMed.
NEUROPROTECTION AGAINST NEURONAL EXCITOTOXICITY
Glu ama e is one o he mos impo an exci a o y neu o ansmi -
e s in he CNS. Owing o i s high exci a o y e ec on neu onal cells,
i s elease mus be highly con olled o a oid an exci o oxic esponse
in neu onal issue. In con as o o he neu o ansmi e s, such as
dopamine and ace ylcholine, which use an enzyma ic sys em o con-
ol he neu onal esponse in he synap ic cle , he glu ama e gic sys-
em uses as e and mo e e ficien glu ama e anspo e s o p e en
excessi e ac i a ion o ecep o s by con inuously emo ing glu ama e
om he ex acellula fluid (Zhou and Danbol , 2014). The concen a-
ion o ex acellula glu ama e is igh ly egula ed by high‐ene gy
sodium‐dependen anspo e s (also known as exci a o y amino acid
anspo e s) loca ed mainly on as ocy es, which bind o and emo e
neu o ansmi e s o p ocessing and ecycling (Danbol , 2001). Glu a-
ma e exci o oxici y occu s when he homeos a ic balance o he neu o-
ansmi e is dis up ed and glu ama e le els inc ease in he
ex acellula fluid (Danbol , 2001). Failu e o he nu ien supply a e
ischemia causes neu onal depola iza ion, leading o a massi e elease
o glu ama e in o he ex acellula space. In addi ion, because glu a-
ma e up ake is a highly ene gy‐dependen p ocess, he es ic ion o
ene gy a e ischemia causes a d as ic dis up ion o glu ama e ans-
po e s, enhancing he exci o oxic e ec and igge ing neu onal dea h
(Cas illo e al., 2016). Real‐ ime analysis o glu ama e in he ischemic
egion indica es ha glu ama e elease occu s immedia ely (in min) in
he co e egion; he e o e, he he apeu ic in e en ional op ion o
block i s e ec s has a e y na ow ime ame. Con e sely, in he
penumb al egion, esidual blood flow allows he main enance o glu-
ama e up ake sys ems and ionic pumps. Howe e , he sp ead o glu a-
ma e om he ischemic co e o i s pe iphe y accele a es he
p og ession o damage in he s ill sal ageable penumb a egion
(Ramos‐Cab e e al., 2011). The e o e, ea ly con ol o glu ama e
exci o oxici y is an ambi ious he apeu ic s a egy o block he down-
s eam domino ischemic cascade; howe e , he na ow in e en ional
ime ame limi s he clinical applica ions o d ugs designed o block
his exci o oxic p ocess (Cas illo e al., 2016). We could define his
as he “unce ain y p inciple o glu ama e,” ha is, we canno ge p o-
ec ion and ime wi h pe ec accu acy.
Decades ago, clinical s udies epo ed ha glu ama e plays a c i i-
cal ole in neu onal damage ollowing ischemic s oke (Cas illo
e al., 1995; Cas illo e al., 1996; Cas illo e al., 1997; Cas illo e al.,
1999). The p e en ion o glu ama e‐media ed oxici y, mainly based
on he use o selec i e glu ama e an agonis s, has been a cen al he -
M. Pé ez-Ma o e al. Neu oscience 550 (2024) 30–42
31
apeu ic a ge in nume ous s udies; howe e , many o hese ha e
shown oxic side e ec s o ailed o show e ficacy when e alua ed in
clinical ials. Despi e hese disappoin ing esul s, he c i ical in ol e-
men o glu ama e‐media ed oxici y in unc ional ou comes a e
acu e ischemic s oke has no been igno ed, and ecen s udies in he
las yea s ha e expanded beyond he use o classical glu ama e an ag-
onis s o con ol glu ama e‐ ela ed downs eam signals. Examples o
hese new s a egic he apies include d ugs, such as ne ine ide
(NA1) and blood glu ama e sca enge s.
Ne ine ide
NA1 is a 20‐amino acid pep ide ha p e en s a achmen o pos sy-
nap ic densi y 95 (PDS‐95) p o ein o he NMDA glu ama e ecep o
(NMDAR) subuni . Ele a ed glu ama e le els in he ex acellula space
lead o NMDAR ac i a ion. This esul s in an influx o calcium, ul i-
ma ely leading o cellula damage in ischemic in a c s. The e o e,
blocking hese ecep o s has been p oposed as a po en ial ea men
s a egy o ischemic s oke. Howe e , he use o NMDA an agonis s
in ischemic s oke has been shown o induce side e ec s in heal hy
neu ons and has a e y na ow ime ame o inducing posi i e e ec s.
NA1 ac s by inducing selec i e inhibi ion o he PDS‐95 p o ein and
educing neu onal oxici y, and mo e impo an ly, i does no block
he no mal synap ic ac i i y o NMDARs. In e en ion s udies pe -
o med be o e endo ascula aneu ysm su ge y ha e shown ha p e-
ea men wi h NA1 dec eases he equency o ia ogenic s oke
(ENACT ial) (Hill e al., 2012). This compound has also been es ed
in macaques, and he esul s showed posi i e beha io al findings and
magne ic esonance imaging (MRI) esul s a e ischemic s oke (Cook
e al., 2012). These p e ious p eclinical findings led o a mul icen e ,
andomized clinical ial wi h mo e han 1000 pa icipan s using NA1
as a po en ial neu op o ec i e agen in combina ion wi h PA ecanal-
iza ion he apy (ESCAPE‐NA1) (Hill e al., 2020). Un o una ely, he
adminis a ion o NA1 did no imp o e neu ological ou comes in
pa ien s who ha e unde gone h ombolysis compa ed wi h he placebo
g oup. A seconda y sub‐analysis showed ha NA1 is suscep ible o p o-
eoly ic clea age when adminis e ed a e PA and he e o e loses i s
e ec in pa ien s ecei ing h omboly ic he apy (Mayo ‐Nunez e al.,
2021). Two new ials o NA1, ESCAPE‐NEXT and FRONTIER, we e
de eloped o es NA1 in pa ien s who ha e no unde gone h omboly-
sis and o p e‐hospi al adminis a ion in pa ien s wi h suspec ed
s oke, espec i ely (Ghozy e al., 2022; Haup e al., 2023).
Bo h we e ecen ly concluded; he EXCAPE‐NEXT ial showed neu-
al esul s, whe eas he FRONTIER ial showed some benefi s in
pa ien s who we e ea ed in a p e‐hospi al se ing (ambulance) e y
ea ly a e s oke onse , eflec ing he impo ance o blocking he exci-
o oxic cascade e y ea ly a e s oke onse ( esul s p esen ed in he
2023 Wo ld S oke Cong ess in To on o, Canada).
Blood/b ain glu ama e sca enge s
Blood/b ain glu ama e g abbing o sca enging p ocesses a e
eme ging as no el p o ec i e s a egies o educe he exci o oxic
e ec s o excess ex acellula glu ama e ha accumula es in he b ain
a e ischemic inju y (Leibowi z e al., 2012; Cas illo e al., 2016;
Kaplan‐A abaci e al., 2022). The blood/b ain glu ama e g abbing
mechanism is based on he educ ion o blood glu ama e concen a-
ion, which leads o a la ge na u al glu ama e g adien be ween he
b ain and blood, hus acili a ing he e flux o ex acellula glu ama e
in o he blood. The e o e, he manipula ion o his mechanism may
ha e a neu op o ec i e e ec a e a s oke. The main ad an age o
his no el he apeu ic s a egy is ha i occu s in he blood ci cula ion
and he e o e does no in e e e wi h no mal b ain neu ophysiology,
as has been desc ibed o o he d ug ea men s (such as glu ama e
an agonis s) (Cas illo e al., 2016). This new app oach has been ali-
da ed in se e al ischemic animal models by independen labo a o ies
(Rink e al., 2011; Boyko e al., 2012a, 2012b; Rink e al., 2017)
and has also been success ully es ed in o he pa hological models
associa ed wi h inc eased glu ama e le els in he b ain, such as au-
ma ic b ain inju y (Zhang e al., 2019), glioma (Ruban e al., 2012),
Alzheime disease (Zhang e al., 2017), and amyo ophic la e al scle o-
sis (Ruban e al., 2015; Roge s e al., 2023), wi h success ul esul s.
The fi s in e en ional clinical ial designed o demons a e he
beneficial e ficacy o blood glu ama e sca enging in s oke used dialy-
sis o fil e he blood and emo e excess glu ama e om pa ien s. Pe i-
oneal dialysis in oden models o ischemia esul ed in a dec ease in
blood glu ama e le els, wi h a co esponding inc ease in glu ama e
in he dialysis solu ion, ollowed by a significan educ ion in in a c
size (Godino Mdel e al., 2013). Howe e , he use o pe i oneal dialysis
(Eud aC Numbe : 2012‐000791‐42) o hemodialysis (NCT04297345)
in pa ien s wi h s oke had o be discon inued because o he side
e ec s o his in asi e p ocedu e.
To demons a e he clinical e ficacy o blood glu ama e sca enging
he apy in pa ien s wi h s oke, he glu ama e sca enging p ope ies o
ibofla in ( i amin B2) we e es ed in a p oo ‐o ‐concep phase IIb
clinical ial (NCT02446977) wi h 25 con ol (placebo) and 25 ibofla-
in (20 mg)‐ ea ed pa ien s wi h s oke (da Sil a‐Candal e al., 2018).
This analysis confi med a educ ion in blood glu ama e le els in he
ea ed pa ien s and a end owa d unc ional eco e y compa ed wi h
he placebo g oup. Despi e he p omising clinical esul s obse ed wi h
ibofla in, he main limi a ion o his clinical app oach is he highly
wa e ‐soluble na u e o he i amin, eflec ed in i s apid blood clea -
ance, which equi es con inuous adminis a ion o high doses o
achie e significan esul s.
One o he mos explo ed and e ec i e pha macological s a egies
o educing blood glu ama e le els is he exogenous adminis a ion o
glu ama e oxaloace a e ansaminase (GOT) enzyme, also known as
aspa a e amino ans e ase. GOT plays a c i ical ole in he egula ion
o blood glu ama e le els by ca alyzing he e e sible con e sion o
oxaloace a e and glu ama e o aspa a e and α‐ke oglu a a e, espec-
i ely. Thus, he adminis a ion o he pu ified ecombinan o m o
human GOT ( GOT) in ischemic animal models leads o he me abo-
lism and educ ion o glu ama e in he blood and lowe ing o glu a-
ma e in he ce eb al pa enchyma, which is associa ed wi h a
educ ion o he ischemic lesion and senso imo o eco e y (Pe ez‐
Ma o e al., 2014). In addi ion, h ough bioconjuga ion wi h polye hy-
lene glycol, i was possible o inc ease he hal ‐li e o GOT in he
blood a e adminis a ion; a single adminis a ion o his new o mu-
la ion had a p o ec i e e ec simila o ha o GOT adminis e ed ou
imes (Zhang e al., 2019). The inhibi ion o blood GOT ac i i y also
esul s in highe b ain glu ama e le els and g ea e damage (Dopico‐
Lopez e al., 2021). These expe imen al findings a e also consis en
wi h wo e ospec i e s udies wi h mo e han 400 pa ien s wi h
ischemic s oke, indica ing ha endogenous blood GOT ac i i y le els
g ea e han 17 U/L a hospi al admission we e associa ed wi h lowe
blood glu ama e concen a ions and good ou comes 3 mon hs a e
s oke, suppo ing he clinical use o GOT as a he apy (Campos
e al., 2011a, 2011b).
The he apeu ic mechanisms pos ula ed o he p o ec i e e ec o
GOT a e mainly ela ed o he me abolism and educ ion o blood glu-
ama e; howe e , o he s udies ha e also epo ed ha GOT me abo-
lizes b ain glu ama e o use as an ene gy subs a e in anae obic
condi ions, such as ischemia (Rink e al., 2011; Rink e al., 2017). This
hypo hesis is suppo ed by he ac ha GOT o e exp ession in he
b ain educes glu ama e le els and p e en s ATP loss unde ischemic
condi ions. Recen s udies ha e epo ed he abili y o GOT o escue
he ac i i y o mi ochond ia a ec ed by ischemia and p o ec agains
ene gy ailu e (Xu e al., 2020).
The adminis a ion o an endogenous se um enzyme, such as GOT,
as a new p o ec i e ea men agains ischemia is an in e es ing s a -
egy because he adminis a ion o GOT is unlikely o induce oxic
e ec s in humans, as he le els o his enzyme a y in heal hy indi id-
M. Pé ez-Ma o e al. Neu oscience 550 (2024) 30–42
32
uals (7–45 U/L) and ha e been shown o inc ease mo e han 10‐ old in
pa ien s wi h li e damage. Fu he human s udies a e equi ed o
demons a e he po en ial he apeu ic e ec s o his enzyme.
NEUROPROTECTION AGAINST STRESS OXIDATIVE DAMAGE
Oxida i e and ni osa i e s ess a e c i ical playe s in he p ocess o
ischemic inju y. Bo h p ocesses a e igge ed a e exci o oxic ischemic
inju y and occu as a consequence o an inc ease in seconda y messen-
ge sys ems coupled wi h enzyma ic gene a ion o ee adicals (e.g.,
ia cyclooxygenase o ni ic oxide syn hases) (Chamo o e al.,
2016). Such mechanisms a e pa icula ly exace ba ed a e delayed
ecanaliza ion, p oducing la ge amoun s o oxida i e and ni osa i e
ee adicals, including supe oxide (O
2
−
), hyd ogen pe oxide (H
2
O
2
),
ni ic oxide (NO), hypochlo ous acid (HClO), and pe oxyni i e
(ONOO
−
) in he CNS and pe iphe al sys ems, ac i a ing adhesion
molecules, and p omo ing inflamma o y and immune cell infil a ion
in ischemic egions (Chen e al., 2020).
NXY-059
Neu aliza ion o oxida i e and ni osa i e s ess has been explo ed
as an ambi ious he apeu ic s a egy because he ischemic b ain is
highly suscep ible o oxida i e damage owing o i s high oxygen con-
sump ion, high i on and unsa u a ed lipid con en , and ela i ely low
endogenous an ioxidan capaci y. Oxida i e and ni osa i e adicals
a e mainly gene a ed in he ischemic penumb a, and p e en ing he
e ec s o hese compounds has been shown o be an e ec i e measu e
o limi he expansion o ischemic lesions in p eclinical s oke models
(Chamo o e al., 2016). Pe oxyni i e, gene a ed om he in e ac ion
be ween ni ic oxide and supe oxide, has pa icula he apeu ic alue
because i p omo es mul iple cell‐ ela ed s ess p ocesses such as lipid
pe oxida ion, mi ochond ial and DNA damage, p o ein ni a ion, and
oxida ion o deple ion o an ioxidan ese es (Lei a e al., 2023).
One o he widely known pe oxyni i e sca enge s es ed was NXY‐
059. NXY‐059 ini ially showed posi i e esul s in oden models o
s oke and p omising p o ec i e e idence in he clinical ial SAINT I
bu no in he la ge ial SAINT II (Chamo o e al., 2016).
U ic acid
A e NXY‐059, new oxida i e and ni osa i e ee adical sca -
enge s ha e eme ged such as u ic acid. U ic acid is he final oxida ion
p oduc o pu ine ca abolism in humans and accoun s o up o wo‐
hi ds o he o al an ioxidan capaci y o he plasma (Chamo o
e al., 2016, 2021; Lei a e al., 2023). In p eclinical app oaches, i s p o-
ec i e e ficacy has been widely epo ed o p e en glu ama e‐induced
cell dea h in i o, whe eas in ansien o pe manen ischemia in
oden s, i supp esses oxida i e and ni osa i e adicals and
pe oxyni i e‐media ed damage, educes in a c olume, and imp o es
unc ional ou comes (Romanos e al., 2007; One i e al., 2015).
New ends aimed a imp o ing he ep oducibili y o p eclinical da a
and demons a ing he ansla ional alue o new candida es o he
ea men o acu e ischemic s oke ecommend he de elopmen o
mul i‐labo a o y p eclinical ials by independen labo a o ies be o e
mo ing o clinical assays. Wi h his aim, a S oke P eclinical Assessmen
Ne wo k (SPAN) es ed six ea men candida es (including u ic acid) in
ou s ages in di e en oden models o ischemic s oke (Lyden e al.,
2023). In his analysis, u ic acid supplemen a ion was he only ea men
ha showed benefi s in male and emale animals, young mice, young
a s, aging mice, obese mice, and spon aneously hype ensi e a s
(Lei a e al., 2023), suppo ing i s po en ial applicabili y in humans.
The e ficacy o u ic acid ea men has been widely assessed in se -
e al clinical ials, such as he phase IIb/III URICOICTUS ial, which
confi med he sa e y o a combina ion o u ic acid and al eplase
(s a ed wi hin 4–5 h o symp om onse ) in pa ien s wi h acu e
ischemic s oke. The ial did no demons a e he e ficacy o u ic acid
on he p ima y ou come (modified Rankin sco e a he 90‐day ollow‐
up) (Chamo o e al., 2014). Howe e , u ic acid educed he incidence
o ea ly clinical wo sening, and mo e pa ien s ea ed wi h u ic acid
achie ed ull independence a ollow‐up han hose who ecei ed pla-
cebo. U ic acid has also been shown o educe in a c g ow h and
imp o e unc ional ou comes in p edefined pa ien subg oups such
as women o pa ien s wi h p e ea men hype glycemia o ea ly
ecanaliza ion (Ama o e al., 2015; Llull e al., 2015). Because ee ad-
ical agen s a e eleased a e ecanaliza ion and p oduce la ge amoun s
o ee adicals, u u e di ec ions sugges combining u ic acid wi h MT
o educe oxida i e s ess and imp o e mic oci cula o y hypope usion
(Lei a e al., 2023). Conclusi e clinical ials o u ic acid ea men a e
equi ed o alida e he clinical p o ec i e e ec s o his p omising ee
adical sca enge .
Eda a one
Ano he an ioxidan agen ha has shown g ea a fini y as a chela-
o o hyd oxyl adicals wi h clea benefi s in expe imen al models o
ischemia is eda a one (also known as MCI‐186) (Nishi e al., 1989;
Oishi e al., 1989), which is manu ac u ed by Mi subishi Tanabe
Pha ma Co po a ion (Osaka, Japan). Fa o able p eclinical da a led
o his compound o be es ed in clinical ials, wi h sa is ac o y
esul s, allowing i s app o al in coun ies such as Japan, China, and
India (Kobayashi e al., 2019). In Japan, nea ly hal o he pa ien s
wi h ischemic s oke ecei e eda a one o acu e ea men
(Kobayashi e al., 2019). Becasuse eda a one is known o ha e po en
an ioxidan e ec s, Japanese au ho i ies ha e also alida ed i s clinical
applica ion in combina ion wi h PA (Kimu a e al., 2012). Al hough
he da a a ailable hus a ha e confi med i s sa e y in Asian coun ies,
i s applica ion is no ye widely accep ed o ea ing ischemic s oke
in Wes e n coun ies. A ecen me a‐analysis o andomized con olled
clinical ials conduc ed o da e wi h his compound showed ha
al hough he a ailable clinical da a a e limi ed, eda a one appea s
o imp o e neu ological impai men , wi h a su i al benefi a he 3‐
mon h ollow‐up, ega dless o he mean age and ea men cou se
(Chen e al., 2021). Howe e , la ge s udies a e equi ed o de e mine
he benefi s o eda a one in pa ien s be o e i can be app o ed in Wes-
e n coun ies.
INFLAMMATION
The inflamma o y esponse ollowing acu e ischemic s oke is one o
he mos complex p ocesses media ing he p og ession o in a c lesions.
This complexi y is caused by a ious ac o s, including local and ci cula -
ing immune cells and cy okines (inflamma o y and immunomodula o y
ac o s). I is well known ha he egion o ischemic b ain inju y eleases
se e al damage‐associa ed molecules, such as hea shock p o eins, ATP,
S100 p o eins, hepa an sul a e, DNA, and RNA, which subsequen ly ig-
ge an inflamma o y esponse and infil a ion o immune cells in o he
b ain pa enchyma, acco ding o he empo al pa e n o chemoa ac an
and adhesion molecules (El zschig and Eckle, 2011; Chamo o e al.,
2016, 2021). A e an ischemic e en , he fi s immune cells o espond
a e b ain‐in insic mic oglia, which elease media o s ha u he a ac
neu ophils, monocy es, and lymphocy es (El zschig and Eckle, 2011;
Kolaczkowska and Kubes, 2013). Once in he b ain pa enchyma, he
immune cells igge a cascade o seconda y e en s ha con ibu e o
inflamma o y esponses, b ain inju y, and subsequen neu ological
defici s.
The fi s wa e o immune/inflamma o y esponse is essen ial o
emo e dead cells and cell deb is; howe e , exace ba ed inflamma o y
esponse may con ibu e o he de elopmen o inju y h ough he
elease o highly oxidan and cy o oxic compounds (Chamo o e al.,
M. Pé ez-Ma o e al. Neu oscience 550 (2024) 30–42
33
2016, 2021; B ea, 2023). The e o e, he apeu ic in e en ions mus be
p ope ly designed o ac a a specific ime poin a e s oke and modu-
la e he balance be ween inflamma o y and immunomodula o y ac o s
o p omo e p o ec i e and es o a i e p ocesses (Ceulemans e al., 2010).
Rapid ad ances in ou unde s anding o he inflamma o y esponse a e
s oke, much o which ha e ocused on desc ibing pa e ns o infil a ing
immune cells and he elease o inflamma o y molecules, ha e opened
he doo o new p o ec i e he apies wi h highly selec i e ac i i y on
key playe s associa ed only wi h he beneficial e ec o he immune
esponse. We e iew some o he mos ele an pha maceu ical
app oaches used o modula e he inflamma o y esponse a e a s oke
ha has achie ed o clinical analysis, including fingolimod, na alizumab,
in e leukin (IL)‐1Ra, and he no el ApTOLL agen .
Fingolimod
Fingolimod (also known as FTY720) is a high‐a fini y sphingosine
1‐phospha e (S1P) ecep o agonis ha blocks lymphocy e elease
om lymph nodes, he eby limi ing lymphocy e infil a ion in o he
b ain and inhibi ing local ac i a ion o mic oglia and mac ophages.
The ac i e (phospho yla ed) o m o fingolimod binds o lymphocy e
S1P ecep o s and causes hei in e naliza ion and deg ada ion, esul -
ing in lymphopenia (Naseh e al., 2021). Lymphocy es play impo an
oles in he pa hogenesis o s oke and issue inju y. They unc ion as
modula o s o leukocy e and pla ele adhesion ollowing an ischemic
s oke. Ac i a ed T lymphocy es appea as ea ly as 24 h a e epe u-
sion in ischemic b ain issue, and hey p oduce inflamma o y cy oki-
nes such as IL‐17 and in e e on γin he si e o inju y. In pa icula ,
lymphocy es CD4
+
helpe , CD8
+
cy o oxic, and γδT cells play damag-
ing oles in expe imen al s oke. Thus, he deficiency o CD4
+
helpe /
CD8
+
cy o oxic T cells leads o a dec eased numbe o adhe en leuko-
cy es and lymphocy es, esul ing in smalle ischemic in a c sizes and
amelio a ion o neu ological ou comes ollowing ischemic damage in
mice. Roden models o b ain ischemia ha e shown ha lymphopenia
caused by fingolimod ea men is associa ed wi h smalle in a c size,
neu ological defici s, and edema (Wei e al., 2011; Liu e al., 2013). In
a mouse model o h omboembolic s oke, in which he beneficial
e ec o PA‐induced epe usion and HT associa ed wi h delayed
adminis a ion was simila o ha occu ing in humans, fingolimod
was obse ed o educe he in a c size and isk o hemo hage o
he PA h omboly ic d ug (Campos e al., 2013; Naseh e al.,
2021). Howe e , in he ecen mul i‐labo a o y p eclinical ial, SPAN,
fingolimod did no show e ec i e esul s, con a y o he esul s
obse ed wi h u ic acid (Lyden e al., 2023).
As fingolimod is an o al d ug app o ed o he ea men o di e -
en ypes o scle osis, i can be easily eposi ioned o es ing in s oke,
sa ing c i ical s eps in he de elopmen o he fi s sa e y phase I s udy.
Wi h his aim in mind, an open‐label pilo s udy es ed he e ficacy o
o al fingolimod in imp o ing unc ional ou comes a e s oke (Fu
e al., 2014). When adminis e ed wi hin 72 h o s oke onse , he ea -
men limi ed seconda y issue inju y, achie ing less mic o ascula pe -
meabili y, imp o ing neu ological defici s, and p omo ing eco e y; in
addi ion, i did no cause se e e ad e se e ec s (Fu e al., 2014). Fu -
he mo e, when adminis e ed in combina ion wi h PA, wi hin 4.5 h
o s oke onse , he e we e ewe ci cula ing lymphocy es, smalle
lesion olumes, less hemo hage, and be e neu ological ou comes
han wi h al eplase alone (Zhu e al., 2015; Chamo o e al., 2016,
2021). Howe e , despi e hese p omising esul s, he p o ec i e e ec
o fingolimod in s oke s ill needs o be es ed in double‐blind s udies,
and u he ex ensi e wo k is needed be o e i can be app o ed as a
ea men o s oke (Cao e al., 2023).
Na alizumab
Na alizumab is a humanized CD49d an ibody ha blocks he α4
in eg in, he eby educing he en y o leukocy es in o he CNS. I
has been app o ed as an in a enous d ug o mul iple scle osis. In
he field o s oke, some p eclinical s udies ha e es ed he e ficacy
o na alizumab in oden models, wi h con o e sial esul s hus a :
one s udy showed posi i e esul s, whe eas he o he , despi e educing
T‐cell and neu ophil infil a ion o he CNS, did no show e ficacy in
p o ec ing mice om unc ional defici s a e s oke (Liesz e al.,
2011). Ano he mul icen e p eclinical s udy in mice showed a educ-
ion in in a c size in models o mild ischemia, bu no in la ge in a c s
(Llo e a e al., 2015). The ACTION ial, designed o es he e ec o
na alizumab in s oke wi hin 9 o 24 h, did no show any e ec on
in a c olume g ow h ( he p ima y s udy endpoin ), bu imp o e-
men s in se e al p especified seconda y and e ia y endpoin s o unc-
ional ou come a 30 and 90 days we e obse ed, compa ed wi h
placebo (Elkind e al., 2020). Cu en ly, as a as we know, he e a e
no new clinical analyses on he e ec o na alizumab in pa ien s wi h
s oke.
In e leukin 1Ra
The p oinflamma o y cy okine IL‐1 is closely ela ed o he immune
esponse a e an ischemic e en , and an inc ease in i s le els is asso-
cia ed wi h g ea e ischemic damage, as obse ed in p eclinical mod-
els. The close ela ionship be ween IL‐1 le els and poo p ognosis
has led o he de elopmen o an agonis s agains he IL‐1 ecep o
(IL‐1Ra), which has high e ficacy in blocking he esponse o his cy o-
kine (Smi h e al., 2018). Subcu aneous o in a enous adminis a ion
in expe imen al models has shown sa is ac o y esul s in e ms o long‐
e m unc ional eco e y (Maysami e al., 2016). A subsequen me a‐
analysis o exis ing p eclinical da a o 1283 animals alida ed he e fi-
cacy o IL‐1Ra ea men in e ms o educ ion o in a c olume and
suppo ed i s subsequen ansla ion o he clinic (McCann e al.,
2016). I s in a enous adminis a ion in pa ien s wi h acu e s oke in
a phase II placebo‐con olled s udy demons a ed sa e y and high e fi-
cacy in educing inflamma o y ma ke le els (Emsley e al., 2005). IL‐
1Ra has been app o ed o subcu aneous ea men o heuma oid
a h i is, making i easy o adminis e . A subsequen phase II s udy,
SCIL‐STROKE (Subcu aneous In e leukin‐1 Recep o ‐An agonis in
Ischemic S oke), analyzed he e ec o IL‐1Ra adminis e ed subcu a-
neously a e acu e ischemic s oke and demons a ed he e ficacy o
he ea men o significan ly educe inflamma o y ma ke s associa ed
wi h a wo se ou come a e s oke, bu no significan e ec ela ed o
unc ional imp o emen in he modified Rankin scale was obse ed;
hus, u he s udies a e s ill equi ed o demons a e i s clinical bene-
fi in acu e s oke (Smi h e al., 2018).
ApTOLL
ApTOLL is an unmodified single‐s anded DNA ap ame designed
o an agonize oll‐like ecep o 4 (TLR4) and has a high specifici y
o blocking he inflamma o y esponse p oduced a e di e en
insul s, such as acu e ischemic s oke and acu e myoca dial in a c ion.
Toll‐like ecep o s a e a amily o highly conse ed inna e immune
ecep o s ha ecognize highly p ese ed s uc u es in pa hogens,
called pa hogen‐associa ed molecula pa e ns. Howe e , hese
immune ecep o s also ecognize damage‐associa ed molecula pa -
e ns om endogenous molecules eleased as a esul o issue inju y
such as ce eb al ischemic s oke. The le el o ac i a ion o TLRs medi-
a ed by issula endogenous ligands can also be simila o ha caused
by ligands om in ec ious agen s, in a way ha p omo es he ec ui -
men o se e al adap i e p o eins o ac i a e nuclea ac o ‐κB, which
induces he exp ession o p oinflamma o y genes, inflamma o y
cy okines, and adhesion molecules and ac i a ion o adap i e immu-
ni y. Up o 14 ypes o TLRs ha e been desc ibed; howe e , one o
he mos equen ly associa ed wi h he inflamma o y esponse a e
s oke is TLR4, which is widely co ela ed wi h poo ou comes. This
associa ion has been well demons a ed in TLR4 knockou mice, which
M. Pé ez-Ma o e al. Neu oscience 550 (2024) 30–42
34

showed smalle in a c sizes and imp o ed neu ological es sco es
han wild‐ ype mice (Caso e al., 2007; B ea e al., 2011), whe eas
up egula ion co ela ed wi h ischemic s oke se e i y (Yang e al.,
2008).
Based on he c i ical ole o TLR4 in he immune esponse a e
s oke and i s co ela ion wi h a poo p ognosis, ApTOLL was designed
o specifically modula e he immune esponse h ough his ecep o ,
he eby p e en ing he fi s wa e o he inflamma o y cascade om
occu ing a e s oke onse . Expe imen al models o ischemic s oke
ha e shown ou s anding neu op o ec i e e ec s, educing b ain dam-
age by up o 65% wi hin an e ec i e ime window o 12 h (Caso e al.,
2007). To e alua e he clinical e ec s o ApTOLL in pa ien s wi h
s oke, a fi s ‐in‐human s udy was conduc ed o assess he sa e y and
pha macokine ics o ApTOLL in heal hy indi iduals (He nandez‐
Jimenez e al., 2022). The da a de i ed om his s udy showed an
excellen sa e y p ofile in heal hy humans. A subsequen assessmen
o he sa e y and e ficacy o ApTOLL in combina ion wi h MT in
pa ien s wi h ischemic s oke (APRIL s udy) showed ha 0.2 mg/kg
ApTOLL adminis e ed wi hin 6 h o onse in combina ion wi h MT
was sa e and associa ed wi h a po en ially meaning ul clinical e ec ,
educing mo ali y and disabili y a 90 days compa ed wi h placebo.
These p elimina y esul s ha e led o he de elopmen o la ge pi o al
ials o e i y he p oposed e ec s o his immunosupp essan
(He nandez‐Jimenez e al., 2023a, 2023b).
NON-PHARMACOLOGICAL PROTECTIVE APPROACHES
Neu op o ec ion agains s oke is usually associa ed wi h he use o
pha maceu ical d ugs designed o selec i ely in e e e wi h o block
key molecula a ge s in he ischemic cascade o educe he p og es-
sion o b ain inju y. Howe e , in he long sea ch o e ec i e p o ec-
i e he apies agains ischemic s oke, al e na i e non‐
pha maceu ical app oaches ha e eme ged wi h a ying deg ees o suc-
cess. He e, we epo h ee examples o clinically es ed non‐
pha maceu ical app oaches: hypo he mia, ischemic p econdi ioning,
and oxygen supplemen a ion.
Hypo he mia
Hypo he mia is one o he mos e ec i e neu op o ec i e he apies
epo ed a he p eclinical le el o ce eb al ischemia (Campos e al.,
2012). Howe e , al hough e ec i e in pedia ic uni s (e.g., o e al
hypoxia), i s clinical ansla ion (body o sys emic hypo he mia
[SH]) in adul pa ien s wi h s oke is hampe ed by side e ec s such
as shi e ing, hypo ension, a hy hmia, and inc eased isk o pneumo-
nia, which usually equi e seda ion o anes hesia (Geu s e al., 2017).
Non‐in asi e ocal hypo he mia applied locally (o e he skull) o he
ce eb al ischemic egion dec eases he side e ec s o cooling s ess in
awake animals while e aining benefi s simila o hose o SH.
Undoub edly, he clinical ansla ion o his app oach has c i ical lim-
i a ions because he human skull e ec i ely insula es he b ain, equi -
ing he use o p olonged skin‐damaging cold o achie e a ge
in ace eb al empe a u es (Viei es‐P ado e al., 2016).
Despi e he challenges ha in ol e he use o hypo he mia as an
al e na i e he apy, analysis o he physiological mechanisms unde ly-
ing i s he apeu ic e ec s has p o ided new molecula p o ec i e can-
dida es o educing ischemic lesions (Han e al., 2012). Fo example,
al hough hypo he mia down egula es global p o ein syn hesis and cell
me abolism, i induces he up egula ion o o he known cold shock
p o eins (CSPs). The wo main CSPs iden ified in mammals a e cold‐
inducible RNA‐binding p o ein (CIRP) and RNA‐binding mo i p o ein
3 (RBM3). Al hough CIRP is de imen al o enhancing he inflamma-
o y esponse, in e es in RBM3 has significan ly inc eased because
o i s c i ical ole in he p o ec i e e ec s o hypo he mia (Zhu e al.,
2016).
The exp ession o RBM3 has been well in es iga ed in s oke mod-
els subjec ed o sys emic and ocal b ain hypo he mia and in pa ien s
wi h s oke subjec ed o body he apeu ic cooling in he phase III
Eu oHYP‐1 ial. P eclinical da a om ischemic animals subjec ed o
sys emic and ocal hypo he mia confi med an inc ease in b ain
RBM3 exp ession, which was selec i ely highe in cooled hemisphe es
o animals unde going ocal b ain hypo he mia, confi ming he di ec
e ec o hypo he mia on RBM3 exp ession. Consis en wi h hese
expe imen al findings, blood samples om pa ien s wi h s oke who
unde wen hypo he mic ea men in he Eu oHYP‐1 ial showed an
associa ion be ween RBM3 and cooling. These esul s sugges ha
he pha macological induc ion o RBM3 could be a po en ial means
o neu op o ec ion agains s oke in he absence o hypo he mia. How-
e e , a d ug o agonis ha di ec ly a ge s RBM3 exp ession o ac i -
i y has no ye been de eloped o es his hypo hesis (A ila‐Gomez
e al., 2020).
In he absence o a specific agonis , he ups eam induc ion o
RBM3, media ed by opomyosin ecep o kinase B (T kB), has been
e alua ed as an al e na i e neu obiological mechanism o he apeu ic
hypo he mia. T kB is a ansmemb ane ecep o o b ain‐de i ed neu-
o ophic ac o , which is in ol ed in neu al de elopmen , p oli e a-
ion, and su i al and is also associa ed wi h inc eased exp ession o
RBM3 media ed by cooling. T kB agonism has been epo ed o induce
RBM3 wi hou exposu e o cold, he eby p e en ing neu odegene a-
i e damage. These da a clea ly demons a e ha pha macological
ac i a ion o RBM3 can be used he apeu ically wi hou inducing
hypo he mia (Pe e i e al., 2021).
Clinical s udies ha e also epo ed ha fib oblas g ow h ac o 21
(FGF21) is an obesi y‐and empe a u e‐ ela ed ho mone ha up egu-
la es he exp ession o RBM3. Clinical e ospec i e s udies ha e
shown ha highe concen a ions o FGF21 on admission and RBM3
a 72 h we e associa ed wi h good ou comes bu we e in e sely ela ed
o he maximum empe a u e du ing he fi s 24 h a e s oke (A ila‐
Gomez e al., 2022). This associa ion be ween FGF21 and RMB3 led o
he pos ula ion ha FGF21 is an induce o RBM3 exp ession and a
p o ec i e d ug agains s oke. This g ow h ac o has al eady been
used as a ecombinan ea men unde di e en expe imen al pa ho-
logical condi ions (Do doe e al., 2021); howe e , he main limi a ion
o he use o ecombinan FGF21 o induce RBM3 exp ession is he side
e ec s obse ed in clinical ials wi h pa ien s wi h s oke (Do doe
e al., 2021). New gene ic s a egies based on he use o an isense
oligonucleo ides (ASO) ha e been demons a ed o manipula e
RBM3 le els independen o cooling. Indeed, his ea men led o
ema kable neu op o ec ion in animal models o Alzheime disease,
wi h he p e en ion o neu onal loss and spongiosis despi e high le els
o disease‐associa ed p ion p o eins (P eussne e al., 2023). These
esul s in mice suppo he possibili y ha RBM3‐inducing ASOs also
deli e neu op o ec ion in s oke pa hology.
Ischemic p econdi ioning
Ischemic p econdi ioning was fi s desc ibed in he field o ca diol-
ogy and e e s o he abili y o a issue o be e esis ischemic damage
when p e iously subjec ed o sho pe iods o ischemic lesions. This
phenomenon was la e ansla ed o expe imen al ce eb al ischemia,
in which unde con olled ansien ischemia in he ca o id and middle
ce eb al a e ies, inju y was significan ly associa ed wi h smalle ce e-
b al in a c sizes (S agliano e al., 1999). Al hough his in e en ion
p esen ed low clinical applicabili y, i has allowed us o explain o jus-
i y why pa ien s wi h ecen ansien ischemic a acks (TIA) p io o
ce eb al in a c ion ha e be e clinical and adiological e olu ion han
hose who ha e no expe ienced TIA (Sol e al., 2022).
Thus, a e he fi s desc ip ion o ischemic p econdi ioning, a new
and mo e p ac ical app oach consis ing o emo e p econdi ioning by
ansien and mild ischemia o he emo al a e y was sugges ed. The
fi s demons a ion o his phenomenon was pe o med in co ona y
M. Pé ez-Ma o e al. Neu oscience 550 (2024) 30–42
35
ischemia by inducing epe i i e infla ion and defla ion o a blood p es-
su e cu on he limb. Remo e ischemic p econdi ioning be o e hospi al
admission sa ely was obse ed o inc ease myoca dial sal age (Bo ke
e al., 2010; Hess e al., 2015).
Remo e condi ioning, applied be o e, du ing, o a e ischemia, ep-
esen s a new pa adigm o neu op o ec ion wi h mul iple mechanisms
o ac ion media ed by he neu ohumo al pa hways. Local ischemia
induces he elease o humo al ac o s such as adenosine, b adykinin,
and opioids, which ac i a e a e en ne es o a e eleased in o he
bloods eam. The agus ne e is also ac i a ed, which is inhibi ed by
pa asympa he ic inflamma o y p ocesses induced by he li e and
spleen. Simul aneously, i ac i a es he sphenopala ine ganglion and
o he pa asympa he ic pa hways esponsible o inc easing ce eb al
blood flow (Bo ke e al., 2010; Pu oy e al., 2020a, 2020b; To es‐
Que ol e al., 2021).
Se e al ials ha e es ed he e ec o emo e p econdi ioning in
he acu e phase o s oke, usually by applying b ie episodes o an-
sien ischemia o a limb, demons a ing i s ease o applica ion and
sa e y (An e al., 2020; Pico e al., 2020; Pu oy e al., 2020a,
2020b; Poalelungi e al., 2021; Landman e al., 2023), whe he applied
o pa ien s wi h ischemic s oke o o hose wi h hemo hagic s okes
(Hougaa d e al., 2014). Howe e , none ha e demons a ed clinical
benefi s, p obably owing o me hodological limi a ions (numbe o
cycles o loca ion o cycles) and insu ficien sample size. New modifi-
ca ions o his he apy ha e been op imizing by es ing emo e
ischemic pos condi ioning; o example, pa ien s wi h mino s oke
in he RICAMIS s udy demons a ed a be e e olu ion in hose who
ecei ed a emo e ischemic pos condi ioning s a egy wice daily o
10–14 days (Chen e al., 2022). This app oach seems o be mo e a o -
able o e alua ing and op imizing he clinical applica ion o ischemic
p econdi ioning.
Oxygen supplemen a ion
In clinical p ac ice, he occu ence o hypoxia du ing he acu e
phase o s oke is common and is associa ed wi h a wo se p ognosis,
neu ological de e io a ion, and highe mo ali y a e. The impo ance
o his pa ame e is e idenced by he ac ha moni o ing and con ol-
ling oxygen sa u a ion is associa ed wi h imp o ed ou comes (B a a a
e al., 2010). Based on he ac ha dec eased oxygen deli e y is one o
he main causes o ischemic damage, hypoxia con ol has long been
pos ula ed as a way o p o ec issue a isk o cell dea h (Ronning
and Guld og, 1999). Howe e , oxygen deli e y p esen s some isks,
such as asocons ic ion, oxici y, and e en ai way in ec ion, when
applied o pa ien s wi h s oke. A leas h ee s udies ha e es ed he
use o high‐flow (45 L/min) he apeu ic oxygen o less han 12 h
wi hou obse ing imp o emen s in pa ien s (Singhal e al., 2005;
Padma e al., 2010; Wu e al., 2012). A la ge ial (n = 550) using
low‐dose supplemen al oxygen (3 L/min o 24 h) did no show any
benefi ei he ; howe e , ea ly neu ological eco e y imp o ed when
low‐dose oxygen was adminis e ed o 72 h (Ronning and Guld og,
1999).
One o he mos comp ehensi e s udies designed o analyze he
he apeu ic con ibu ion o oxygen is he S oke Oxygen S udy. I s
main objec i e was o de e mine whe he low‐dose oxygen he apy
du ing he fi s 3 days a e an acu e s oke imp o ed p ognosis com-
pa ed wi h usual ca e (oxygen only when needed). Since oxygen can
es ic mobili y and in e e e wi h day ime ac i i ies, i is adminis-
e ed only a nigh , when hypoxia is mo e likely (Ro e e al., 2017).
A s udy o 148 ea ed pa ien s s. 141 con ols showed ha oxygen
supplemen a ion ini ia ed wi hin 24 h o hospi al admission o acu e
s oke p oduced a sligh imp o emen in pa ien eco e y, bu he ben-
efi o his he apy emains inconclusi e un il a la ge , longe ‐ e m
s udy is pe o med.
NEW ADVANCES IN THE IMPROVEMENT OF NEUROPROTECTIVE
THERAPIES
As p e iously epo ed, hund eds o new p o ec i e agen s o he -
apies ha e been es ed o educe he p og ession o neu onal damage
a e ischemic e en s, wi h us a ing esul s o da e. Se e al explana-
ions o his pe sis en ailu e ha e been p oposed. One o he mos
widely accep ed easons is he app op ia e selec ion o pa ien s
included in he ials. Pa ien s wi h s oke usually ha e mul iple como -
bidi ies; he ime a e s oke onse is o en unknown, he use o
ecanaliza ion he apies is o en no been well conside ed, and sal age-
able penumb a egions ha e no been e alua ed. In addi ion, o sa e y
easons, many clinical ials o neu op o ec ion in pa ien s wi h s oke
mus s a a e hospi al a i al, which significan ly educes he e ficacy
o he d ugs, as he hey a e designed o in e e e wi h he ini ial pa h-
ways o he ischemic cascade (G upke e al., 2015). The inc easing use
o MT, combined wi h he analysis o new bioma ke s o ea ly diagno-
sis and imp o emen s in high‐ esolu ion imaging, has con ibu ed sig-
nifican ly o he selec ion o candida es acco ding o d ug e ficacy
design. The ele ance o ecanaliza ion he apy, imaging, and bioma k-
e s in imp o ing neu op o ec ion is desc ibed below (Fig. 1).
Recanaliza ion he apies
Since mos clinical ials we e designed o compa e a placebo wi h a
ea ed g oup, he numbe o en olled pa icipan s who also ecei ed
h ombolysis was e y low; he e o e, he possibili y o epe usion
be o e o a e ea men adminis a ion is unlikely. His o ical ailu e
in he sea ch o an e ec i e p o ec i e d ug has led o he assump ion
ha he lack o ecanaliza ion p e en s he d ug om e ec i ely each-
ing he lesion egion, hus impeding i s e ec (Fishe and Sa i z, 2022)
(Fig. 2). Addi ionally, many neu op o ec an s ha e been s udied in p e-
clinical models o ansien ischemia, bu wi hou adequa e epe usion
in pa ien s wi h s oke (Fishe and Sa i z, 2022). Based on his p e ious
e idence, in he “ ecanaliza ion e a,”new clinical ials ha e s a ed o
design he e ficacy analysis o new neu op o ec i e agen s in combina-
ion wi h epe usion he apies ( h ombolysis, h ombec omy, o bo h),
as a fi s s ep owa d d ug analysis.
Howe e , clinical analyses o neu op o ec i e agen s in combina ion
wi h ecanaliza ion he apies ha e e ealed some limi a ions (mainly in
combina ion wi h PA he apy) ha should be conside ed when design-
ing u u e clinical ials. One o hese limi a ions was he possible in e -
ac ion be ween he d ug unde s udy and PA. Since PA is a se ine
p o ease ha clea es pep ide bonds in p o eins, i may deg ade new
compounds o annula e hei ac i i y. A ecen example is he clinical
analysis o NA1 in he phase III ESCAPE‐NA1 ial. In his s udy, pa ien s
in he NA1 g oup who unde wen PA h ombolysis showed no signi -
ican benefi . Howe e , in pa ien s who did no ecei e PA, 59.3% o
pa ien s who ecei ed NA1 had a a o able ou come (Hill e al.,
2020). A subsequen analysis confi med an in e ac ion be ween NA1
and PA, a oiding he he apeu ic e ec o his new compound
(Mayo ‐Nunez e al., 2021). These da a eflec he impo ance o e alu-
a ing he compa ibili y o new h omboly ic d ugs du ing he p eclinical
phase, be o e mo ing o human alida ion.
The second conside a ion is p ope selec ion o he unc ional inde-
pendence alue o de ec he addi ional benefi s o new p o ec i e
d ugs when combined wi h ecanaliza ion he apies. Fo ins ance, in
he case o PA, he e is 33% es ima ed pa ial o comple e a e ial
ecanaliza ion a e ea ly adminis a ion; howe e , his a e has
imp o ed significan ly wi h MT ( a ying be ween 63.7% and 95%,
mainly in s okes wi h la ge essel occlusion [LVO], depending on
he s udy e iewed) (Mo eu e al., 2023). Al hough success ul ecanal-
iza ion is no always associa ed wi h a o able ou comes (defined as
u ile epe usion), many pa ien s (wi h success ul epe usion) usually
a e asymp oma ic and unc ionally independen . In he la e case, he
M. Pé ez-Ma o e al. Neu oscience 550 (2024) 30–42
36
addi ional benefi o a new p o ec i e d ug could be nullified o masked
by epe usion in e en ions. An example o his si ua ion was he clin-
ical analysis o he p o ec i e d ug ci icoline used in pa ien s who
unde gone h ombolysis ea ed wi h PA. Ci icoline, o CDP‐
choline, is a d ug ha combines neu o ascula p o ec ion and epai
e ec s. I has been used o ea acu e ischemic s oke and o he neu o-
Fig. 1. Schema ic ep esen a ion o he con ibu ion o new ecanaliza ion he apies, imp o emen s in high- esolu ion imaging and bioma ke s o ea ly
diagnosis. App op ia e segmen a ion o pa ien s allows selec ion o candida es acco ding o d ug e ficacy design. Images c ea ed wi h BioRende .com.
Fig. 2. Schema ic ep esen a ion o p o ec i e he apies wi h and wi hou ecanaliza ion. Lack o ecanaliza ion p e en s he d ug om e ec i ely eaching he
lesion egion, esul ing in ailu e o educed e ficacy o he he apy. Images c ea ed wi h BioRende .com.
M. Pé ez-Ma o e al. Neu oscience 550 (2024) 30–42
37
logical diso de s and has an excellen sa e y p ofile. In 2002, a o mal
me a‐analysis o ials o CDP‐choline in acu e and subacu e s oke sug-
ges ed a beneficial and subs an ial ea men e ec , wi h absolu e
educ ions o 10%–12% in he a es o long‐ e m dea h and disabili y.
Howe e , a new ial (ICTUS ial) on ci icoline did no demons a e
e ec i i y in he ea men o mode a e o se e e acu e ischemic s oke
(Da alos e al., 2012). Pos e io analyses epo ed ha he inclusion o
pa ien s ea ed e y ea ly wi h PA in a mul idisciplina y s oke uni
could mask he p o ec i e benefi s o ci icoline (Secades e al., 2016).
Howe e , he e ficacy and use o MT ha e inc eased significan ly in
ecen yea s, pa icula ly in s oke cases whe e h ombi a e loca ed in
accessible LVOs. Labo a o y and clinical imaging s udies ha e shown
ha ea ly a e LVO, he co e o ischemia is s ill educed, whe eas
he su ounding in a c ed issue co esponds o a sal ageable penum-
b a, an ideal condi ion o p o ec ion analyses. The e o e, pa ien s
wi h LVO awai ing h ombec omy appea o be excellen candida es
o ials, as he fi s s ep in es ing he p o ec i e e ec s o new agen s.
(Shi e al., 2018; Fishe and Sa i z, 2022).
Imaging app oaches
Ea ly epe usion a e s oke aims o p e en expansion o he co e
egion in o he penumb al a ea and subsequen ly educe he neu olog-
ical sequelae o he inju y. The g ow h o he ischemic co e a ies
be ween pa ien s and depends on colla e al flow capaci y and
pa enchymal ischemic ole ance. Based on his p emise, pa ien s wi h
educed colla e al flow capaci y and pa enchymal ischemic ole ance
can be classified as “ as p og esso s,”wi h wo se clinical ou comes,
la ge in a c s, and an inc eased isk o malignan ce eb al edema
and HT. In con as , pa ien s wi h supe io colla e al flow and ischemic
ole ance, defined as “slow p og esso s,” end o exhibi a smalle
ischemic co e se e al days a e s oke onse . These pa ien s ypically
expe ience supe io clinical ou comes in esponse o epe usion he -
apy (Rocha and Jo in, 2017; Shi e al., 2018)(Fig. 3).
The e o e, in as p og esso s, adminis a ion o epe usion he -
apy and/o p o ec i e d ugs as ea ly as possible (fi s hou s a e s oke
onse ) is c ucial, whe eas slow p og esso s can ecei e significan ben-
efi s e en wi h delayed epe usion (and p o ec i e d ugs), as demon-
s a ed in he DAWN and DEFUSE‐3 ials (Shi e al., 2018). The speed
o in a c p og ession is a c i ical pa ame e in selec ing op imal can-
dida es, defining he ange o in e en ions, and selec ing he mos
con enien p o ec i e he apy. In his ega d, he use and de elopmen
o neu oimaging echniques based on mul ipa ame ic compu ed
omog aphy (CT) and MRI a e powe ul ools o measu e he colla e al
blood flow capaci y and speed o in a c p og ession and imp o e he
selec ion o pa ien s o ial en ollmen (Goyal e al., 2022).
The combina ion o posi on emission omog aphy (PET) wi h adi-
olabeled O
2
was ini ially he gold s anda d (a leas in expe imen al
p ac ice) o isualizing colla e al blood flow and penumb a p og es-
sion. As his imaging echnique is no easible in he acu e clinical se -
ing, MRI pe usion‐and di usion‐weigh ed imaging misma ch and CT
pe usion ha e been p oposed as he mos accessible me hods o iden-
i ying he penumb a in pa ien s wi h acu e ischemic s oke (Chale
e al., 2022). In he li e a u e, mul iple no el me hods based on he
use o PET, CT, and MRI echniques o define he penumb al egion
and measu e ischemic p og ession seem o ocus on he de elopmen
o au oma ed image analysis wi h he suppo o machine lea ning
o a ificial in elligence‐ ela ed algo i hms o assis physicians in
ob aining mo e accu a e and s anda dized in e p e a ions o b ain
images, which may imp o e s oke managemen and pa ien selec ion
o app op ia e ea men (Mokli e al., 2019). The apid p ocessing o
Pe usIon and Di usion so wa e (iSchemaView) is now one o he
mos common ools used o iden i y he ischemic penumb a in many
ischemic s oke clinical s udies (Zhang e al., 2022).
Bioma ke u ili y
Fo sa e y easons, many clinical ials o neu op o ec ion agains
s oke equi e ini ia ion a e hospi al admission and neu oimaging
diagnos ics, which significan ly educes he in e en ion ime and e fi-
cacy o he d ugs (G upke e al., 2015). Ea ly ea men o pa ien s is
c ucial o maximize he benefi s a e s oke; he e o e, p e‐hospi al
Fig. 3. Schema ic ep esen a ion o he g ow h o ischemic inju y in as and slow p og esso s. In as p og esso s, he co e g ows apidly and he penumb a
diminishes wi h inc easing ime om s oke onse , he eby educing he ime o medical in e en ion. In slow p og esso s, he ischemic co e g ows e y slowly,
allowing he use o p o ec i e d ugs in a longe he apeu ic window. Images c ea ed wi h BioRende .com.
M. Pé ez-Ma o e al. Neu oscience 550 (2024) 30–42
38