Diso de s o he Ne ous Sys em
E ec s o Cannabidiol, Hypo he mia, and Thei
Combina ion in Newbo n Ra s wi h Hypoxic-
Ischemic Encephalopa hy
F ancisco J. Al a ez,
1,p
An onia A. Al a ez,
2,p
José. J. Rod íguez,
3,4,5
Hec o La uen e,
6
M. Josune Canduela,
7
William Hind,
8
José L. Blanco-B uned,
9
Daniel Alonso-Alconada,
2
and
En ique Hila io
2
h ps://doi.o g/10.1523/ENEURO.0417-22.2023
1
Bioc uces Bizkaia Heal h Resea ch Ins i u e, 48903 Ba akaldo, Spain,
2
Depa men o Cell Biology, Uni e si y o he
Basque Coun y, 48940 Leioa, Spain,
3
Func ional Neu oana omy G oup, Bioc uces Heal h Resea ch Ins i u e, 48903
Ba akaldo, Spain,
4
Basque Founda ion o Science (IKERBASQUE), 48009 Bilbao, Spain,
5
Depa men o
Neu osciences, Medical Facul y, Uni e si y o he Basque Coun y (UPV/EHU), 48940 Leioa, Spain,
6
Biodonos ia
Heal h Resea ch Ins i u e, 20014 Donos ia, Spain,
7
Depa men o Neu osciences, Uni e si y o he Basque Coun y,
48940 Leioa, Spain,
8
JazzPha maceu icals,Camb idgeCB249BZ,Uni edKingdom,and
9
Depa men o Pedia ic
Su ge y, C uces Uni e si y Hospi al, OSI-Ezke aldea Enka e i C uces, 48903 Ba akaldo, Spain
Abs ac
The apeu ic hypo he mia is well es ablished as a s anda d ea men o in an s wi h hypoxic-ischemic (HI) encephalop-
a hy bu i is only pa ially e ec i e. The po en ial o combina ion ea men s o augmen hypo he mic neu op o ec ion
has majo ele ance. Ou aim was o assess he e ec s o ea ing newbo n a s ollowing HI inju y wi h cannabidiol
(CBD) a 0.1 o 1 mg/kg, i.p., in no mo he mic (37.5°C) and hypo he mic (32.0°C) condi ions, om 7 d o age (neona al
phase) o 37 d o age (ju enile phase). Placebo o CBD was adminis e ed a 0.5, 24, and 48 h a e HI inju y. Two sen-
so imo o ( o a od and cylinde ea ing) and wo cogni i e (no el objec ecogni ion and T-maze) es s we e conduc ed
30 d a e HI. The ex en o b ain damage was de e mined by magne ic esonance imaging, his ologic e alua ion, mag-
ne ic esonance spec oscopy, ampli ude-in eg a ed elec oencephalog aphy, and Wes e n blo ing. A 37 d, he HI
insul p oduced impai men s in all neu obeha io al sco es (cogni i e and senso imo o es s), b ain ac i i y (elec oence-
phalog aphy), neu opa hological sco e ( empo opa ie al co exes and CA1 laye o hippocampus), lesion olume, mag-
ne ic esonance bioma ke s o b ain inju y (me abolic dys unc ion, exci o oxici y, neu al damage, and mi ochond ial
impai men ), oxida i e s ess, and in lamma ion (TNF
a
). We obse ed ha CBD o hypo he mia ( o a lesse ex en han
CBD) alone imp o ed cogni i e and mo o unc ions, as well as b ain ac i i y. When used oge he , CBD and hypo he -
mia amelio a ed b ain exci o oxici y, oxida i e s ess, and in lamma ion, educed b ain in a c olume, lessened he ex-
en o his ologic damage, and demons a ed addi i i y in some pa ame e s. Thus, coadminis a ion o CBD and
hypo he mia could complemen each o he in hei speci ic mechanisms o p o ide neu op o ec ion.
Key wo ds: cannabidiol; hypo he mia; hypoxic-ischemic encephalopa hy; neu op o ec ion; neona e; ju enile
Signi icance S a emen
Cannabidiol and hypo he mia ac on some common p ocesses ela ed o hypoxic-ischemic b ain damage,
modula ing exci o oxici y, in lamma ion, and oxida i e s ess. The wo he apies in combina ion do no com-
pe e agains each o he in modula ing hese p ocesses, bu a he p oduce addi i e neu op o ec i e e ec s.
Fu he mo e, in he ins ances whe e he e was no an addi i e e ec , he combina ion o cannabinoid wi h
hypo he mia o en esul ed in a signi ican ly supe io p o ile compa ed wi h hypo he mia alone, being a
p omising obse a ion o he clinic. These esul s jus i y in e es in cannabidiol o de eloping a combined
ea men wi h hypo he mia o inc ease he numbe o hypoxic-ischemic in an s ha bene i om ea men .
May 2023, 10(5) ENEURO.0417-22.2023 1–15
Resea ch A icle: New Resea ch
In oduc ion
Managemen o newbo n hypoxic-ischemic encephalop-
a hy (HIE) is de e mined by i s complex pa hophysiology,
and any neu op o ec i e s a egy mus ac on se e al ac-
o s, mainly exci o oxici y, oxida i e s ess, and in lamma-
ion (D u y e al., 2014). The apeu ic hypo he mia (HT)
demons a ed hese p ope ies, being he only app o ed
he apy wi h neu op o ec i e e icacy in human newbo ns
(Edwa ds e al., 2010;Jacobs e al., 2013;Heu sen e al.,
2017). Howe e , hese bene i s a e pa ial (Jacobs e al.,
2013), and he e idence om 11 andomized con olled i-
als (.1500 in an s) showed ha he apeu ic hypo he mia
p o ided some bene i o newbo ns wi h mode a e HIE,
bu hypo he mia was ine ec i e in in an s wi h se e e en-
cephalopa hy (Edwa ds e al., 2010;Heu sen e al., 2017).
Al hough he e is conside able e idence ha some in an s
wi h mode a e encephalopa hy can bene i o e all om
he apeu ic hypo he mia, cu en clinical p ac ice means
ha no ou ine ea men is e ec i e in se e e cases. On
his basis, cu en in es iga ion is ocused on combina ion
he apies wi h hypo he mia (Hobbs e al., 2008;La uen e e
al., 2016;Ba a a e al., 2019).
Cannabidiol (CBD) has been shown o educe b ain
damage in expe imen al neona al HIE models (Al a ez e
al., 2008;Cas illo e al., 2010;La uen e e al., 2011;Pazos
e al., 2012,2013). Some o he p ope ies associa ed
wi h he use o CBD, which may unde lie hese e ec s in
models o HIE, include, among o he s, an ioxidan and
an i-in lamma o y p ope ies, and educ ions o Ca
21
in-
lux and glu ama e elease, bu a di ec e ec o CBD on
hese pa ame e s has no been p o en ye (Al a ez e al.,
2008;Cas illo e al., 2010;La uen e e al., 2011;Pazos e
al., 2012,2013). Also, he neu op o ec i e ac ion o CBD
was no associa ed wi h signi ican side e ec s in hese
animal models, and ex ace eb al addi ional bene i s we e
associa ed wi h ca diac, hemodynamic, and en ila o y
e ec s (Al a ez e al., 2008).
A e HI insul , neona al neu op o ec ion induced by
CBD has been demons a ed in di e en animal models:
1 mg/kg, s.c., in neona al mice (Mohammed e al., 2017),
1 mg/kg, s.c., in neona al a s (Pazos e al., 2012), and 0.1
o 1 mg/kg, i. ., in pigle s (Al a ez e al., 2008;La uen e e
al., 2011,2016;Pazos e al., 2013;Ga be ge al., 2016).
Howe e , highe doses om 5 o 50 mg/kg, i. ., in global
HI pigle s did no o e neu op o ec ion in he i s hou s
a e se e e global hypoxia-ischemia (Ga be g e al.,
2017), bu , mo eo e , a posi i e co ela ion be ween he
deg ee o hypo ension and he plasma concen a ions o
CBD was obse ed a hese high doses (Ga be g e al.,
2017). In con as , a well ole a ed dose o CBD 1 mg/kg,
i. ., did no p oduce unwan ed ca dio ascula e ec s
in HI pigle s (Pazose al.,2013;Ga be g e al., 2016;
La uen e e al., 2016). Taken as a whole, hese s udies
suppo he neu op o ec i e ole o CBD a low doses
(,5mg/kg) as a pha macological agen o newbo ns wi h
HIE.
Howe e , he e a e limi ed da a on how CBD migh wo k
oge he wi h hypo he mia. Two s udies demons a ed ha
combining 1 mg/kg, i. ., CBD wi h hypo he mia in sho -
e m s udies (6 o 72 h) was sa e and p o ided neu op o-
ec ion in HI pigle s (La uen e e al., 2016;Ba a a e al.,
2019). The combined e ec o hypo he mia and CBD o e-
duce exci o oxici y, in lamma ion, and oxida i e s ess, as
well as cell damage, was g ea e han ei he ha o hypo-
he mia o CBD alone, indica ing addi i i y. Howe e , be-
o e conside ing he combina ion o bo h he apies o
clinical use, i is impo an o es his in mo e han one
species and o de e mine whe he hese adju an he a-
pies emain e ec i e h oughou he seconda y de e io-
a ion pe iod, which will equi e addi ional expe imen s
ex ending he moni o ing neona al pe iod beyond 72 h
a e hypoxia-ischemia, a leas o a ju enile phase (Juul
and Fe ie o, 2014). The cu en s udy is he i s one o
in es iga e he e ec s o combining CBD ( wo doses)
wi h HT in neona al a s (7 d old) and on long- e m ou -
comes in ju enile a s (37 d old).
Ma e ials and Me hods
E hical app o al
The expe imen al p o ocol sa is ies Eu opean and
Spanish egula ions o he p o ec ion o expe imen al
animals (86/609/EEC and RD 53/2013). The s udy p o-
ocol was e alua ed by he Animal Wel a e Body om
he Uni e si y o he Basque Coun y and was pe -
o med in i s expe imen al su gical hea es (pe mi no.
M20/2015/055).
All expe imen al p ocedu es we e designed and con-
duc ed by pe sonnel quali ied in Labo a o y Animal Science,
ollowing he Fede a ion o Eu opean Labo a o y Animal
Science Associa ions (FELASA) ecommenda ions on ca e-
go ies B and C. All su ge y was pe o med unde adequa e
anes hesia and analgesia, and all e o s we e made o mini-
mize su e ing and o educe he numbe o animals used.
Also, all expe imen al p ocedu es on animal wel a e (anes-
hesia and analgesia, d ug and subs ance adminis a ion)
and he humane killing o he animals we e conduc ed in
compliance wi h FELASA ecommenda ions.
Recei ed Oc obe 7, 2022; accep ed Ap il 7, 2023; Fi s published Ap il 18,
2023.
F.J.A. has a esea ch ag eemen wi h GW Resea ch L d, which is now a pa
o Jazz Pha maceu icals, om which he ecei es inancial suppo . W.H. is an
employee Jazz Pha maceu icals. The au ho s decla e no o he compe ing
inancial in e es s.
Au ho con ibu ions: F.J.A. and W.H. designed esea ch; F.J.A., H.L.,
M.J.C., and J.L.B.-B. pe o med esea ch; F.J.A., A.A.A., H.L., D.A.-A., and
E.H. con ibu ed unpublished eagen s/analy ic ools; F.J.A., A.A.A., J.J.R., H.L.,
M.J.C., W.H., J.L.B.-B., D.A.-A., and E.H. analyzed da a; F.J.A., A.A.A., J.J.R.,
H.L., M.J.C., W.H., J.L.B.-B., D.A.-A., and E.H. w o e he pape .
The p esen s udy was suppo ed by G an GWCRI1547 (GW Resea ch L d.,
now pa o Jazz Pha maceu icals); G an PI12/0852 (ISCIII-Gene al SubDi ec o a e
o Resea ch Assessmen and P omo ion and he Eu opean Regional De elopmen
Funds/Eu opean Social Fund: “Away obuildEu ope”); and G an UPV GIU 17/18
(Uni e si y o he Basque Coun y).
*F.J.A and A.A.A ha e con ibu ed equally o his wo k.
Co espondence should be add essed o F ancisco J. Al a ez a anciscojose.
al a ezdiaz@osakide za.eus
h ps://doi.o g/10.1523/ENEURO.0417-22.2023
Copy igh © 2023 Al a ez e al.
This is an open-access a icle dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion 4.0 In e na ional license, which pe mi s un es ic ed use,
dis ibu ion and ep oduc ion in any medium p o ided ha he o iginal wo k is
p ope ly a ibu ed.
Resea ch A icle: New Resea ch 2 o 15
May 2023, 10(5) ENEURO.0417-22.2023 eNeu o.o g
Animals
Twen y- i e p egnan -speci ic pa hogen- ee emale
Wis a a s (Ha lan) o iden ical age (8 weeks; weigh ,
149–191 g) a he s a o he s udy we e used. Se en
days a e bi h [pos na al day 7 (P7)], a pups we e used
o he expe imen al p ocedu e (N=262; Table 1). A
P21, male and emale a s we e sepa a ed in o small sin-
gle-sex social g oups (6 a s/cage) o a oid space e-
s ic ion and inb eeding.
Animals we e housed a a cons an empe a u e o
22 61°C, ela i e humidi y o 60 65%, and unde a 12 h
ligh /da k cycle wi h he ligh s u ned on a 8:00 A.M.
Food and wa e was supplied ad libi um, and no di e en-
ces we e obse ed in he amoun o s anda d ood ea en
by he di e en g oups o a s du ing he s udy pe iod.
Con ol wel a e- ela ed assessmen s we e conduc ed daily
du ing each ial. All animals we e weighed and ec al em-
pe a u e de e mined once a week.
Expe imen al p o ocol
The p o ocol was based on he model de eloped by
Vannucci’s g oup (1980), which has been ex ensi ely de-
sc ibed p e iously (Fe nández-López e al., 2007). B ie ly,
P7 is an age ha is ep esen a i e o a human in an bo n
a p e e m o nea e m (32–36 weeks) and has commonly
been used in NHIE s udies in he li e a u e, so P7 a pups
we e used in all expe imen s; howe e , since 2015 i has
now been shown ha P10 is he mos app op ia e age a
which o use a pups (Pa el e al., 2015). Only li e s wi h
11 o 12 pups we e used o ob ain 10 animals o andom
ea assignmen as well as 1 o 2 sen inel pups o em-
pe a u e con ol (Dubo ický e al., 2008;Chahoud and
Paumga en, 2009). The pups we e weighed on he day
o he expe imen o de e mine he co ec dose o d ug
ea men .
Pups we e anes he ized by inhaled iso lu ane (5% in-
duc ion, 1.5% main enance). The neck skin o he animal
was cleaned wi h 0.5% chlo hexidine be o e su ge y,
which was conduc ed in asep ic condi ions. The le com-
mon ca o id a e y was exposed, ca e ully isola ed om
su ounding issues, and cu by elec ocau e y (Low
Tempe a u e Mic o Fine Tip 454 Cau e y Pen, S oel ing).
Silk 3–0 su u es we e used o close he skin wound. A e
eco e y om anes hesia, pups we e e u ned o hei
dams o 3-4 h.
Pups we e placed in o 1000 ml ai igh glass con aine s
( i e animals each) in a wa e ba h a 36.5°C, and hen ex-
posed o 10% O
2
190% N
2
(hypoxic a mosphe e) o
120 min. A e he HI insul , pups we e esusci a ed when
needed by ca dio espi a o y s imula ion o a maximum o
10 min. Pups no esusci a ing wi hin 10 min we e no in-
cluded in he s udy ( o al numbe en e ing p ocedu e,
260; numbe o deceden s, 2). Al hough ea men wi h
10% O
2
could induce a mild insul compa ed wi h wha is
epo ed in he li e a u e, inc eased hypoxic in e al, om
75 o 90 min o 120 min, a e ela ed o a mo e se e e inju y
(Fe nández-López e al., 2007;Pazos e al., 2012).
Ra pups we e andomly assigned o be main ained in in-
di idual chambe s in an incuba o a 37.5°C [no mo he mia
(NT)] o 32.0°C (HT), based on p e ious s udies (Tho esen
e al., 1996;Wagne e al., 2002). Mo eo e , he “no mo-
he mic” empe a u e o 37.5°C measu ed ec ally was se-
lec ed by co esponding o he ac ual clinical p ac ice in
human newbo ns.
Pups we e main ained unde HT condi ions o 48 h and
ewa med o 12 h (a ;0.5°C/h). Co e empe a u es we e
eco ded in each chambe in “sen inel pups”(“p obe ani-
mals”) ca ying a ec al empe a u e p obe o moni o he
empe a u e du ing he whole expe imen al pe iod, and
hey we e la e excluded (K he mis o , Xinda ; p obe di-
ame e , ,1 mm) du ing p e iously ela ed phases (HT
ea men o 48 h and he ewa ming phase o 12 h).
Rec al empe a u e was main ained wi hin 60.2°C o he
a ge using a se o-con olled wa e ba h (P ecisdig, JP
Selec a) inside he chambe . In P7 a s, ec al empe a u e
co ela es wi hin 0.1°C wi h b ain empe a u e (Tho esen
e al., 1996). In all animals, ec al empe a u e was meas-
u ed du ing a ew seconds e e y 12 h du ing he i s 3 d.
Simila ly, NT pups we e main ained in indi idualized
cages o simila ime pe iods (48 h). All pups we e ed
e e y 6 h wi h a puppy milk eplace (10 ml/kg; Esbilac,
Pe Ag) by o ogas ic ga age (20 ga plas ic eeding ubes,
Ins ech Lab). Then, all pup a s we e e u ned o hei dams
and weaned a P21, as abo e desc ibed.
T ea men
Thi y minu es a e HI insul , a pups we e i s
andomized by sealed en elope o no mo he mia o hypo-
he mia ea men . Then, HI-inju ed pups ea ed wi h NT
o HT we e again andomized o d ug adminis a ion by
Table 1: To al numbe o animal used o pha macokine ic and pha macodynamic s udies as well as sex assignmen (male/
emale) pe g oup
a
P7 animals assigned o
PKS
b
o al, n(male/ emale)
P14 animals assigned o NS and
NIS o al, n(male/ emale)
P37 animals assigned o NFS, NS, NIS,
and BS o al, n(male/ emale)
SHAM 0 (0/0) 10 (6/4) 10 (6/4)
VEH-NT 0 (0/0) 10 (4/6) 10 (7†/4)
VEH-HT 0 (0/0) 10 (6/4) 10 (6†/5)
CBD 0.1-NT 30 (14/16) 10 (5/5) 10 (6/4)
CBD 0.1-HT 30 (16/14) 10 (6/4) 10 (4/6)
CBD 1-NT 30 (17/13) 10 (5/5) 10 (5/5)
CBD 1-HT 30 (16/14) 10 (5/5) 10 (6/4)
To al 120 (63/57) 70 (37/33) 70 (38/32)
PKS, PK s udies; NS, neu opa hological s udies; NIS, neu al inju y s udies; NFS, neu ologic unc ional s udies; BS, biochemical s udies.
a
One animal excluded by sudden unexpec ed dea h.
b
Fi e addi ional animals used as a e e ence o analy ical pu poses a e no included in he able.
Resea ch A icle: New Resea ch 3 o 15
May 2023, 10(5) ENEURO.0417-22.2023 eNeu o.o g
sealed en elope. All neona al a s ecei ed placebo solu-
ion, 0.1 o 1 mg/kg CBD (GW Resea ch, now pa o Jazz
Pha maceu icals) by in ape i oneal injec ion, 30 min,
24 h, and 48 h a e insul . B ain damage and neu op o-
ec i e e ec s we e assessed by his ologic, neu oimag-
ing, biochemical, and neu obeha io al s udies. A sham-
ope a ed g oup o animals wi hou hypoxia-ischemia o
d ug ea men was included as e e ence.
Fo he pha macokine ic (PK) analysis o CBD in no mo-
he mia and hypo he mia, a s ecei ed CBD 0.1 o 1 mg/kg,
i.p., 30 min a e hypoxia-ischemia. Pups we e humanely
killed by decapi a ion a 0.5, 1, 6, 12, 24, o 36 h a e ini ial
d ug adminis a ion. B ain and plasma samples we e col-
lec ed and immedia ely ozen o de e mine d ug concen a-
ion by liquid ch oma og aph wi h liquid ch oma og aphy-
andem mass spec ome y (analysis conduc ed a LGC).
B ains om i e animals wi hou HI insul o d ug ea men
we e used as e e ence o analy ical pu poses.
Neu ologic unc ional es s
All animals we e acclima ized o he es oom be o e
ials du ing p e ious days. P37 animals unde wen wo
mo o and wo cogni i e es s, wi h 1 h la ency be ween
wo consecu i e es s.
The o a od es assesses locomo o de ici in neu ode-
gene a i e disease models in oden s. The accele a ing
p o ocol p o ides a mo e disc imina i e es o co ela e
locomo o de ici s agains lesion size (Mon ille e al.,
2006). Ra s we e placed on a cylinde wi h linea accele a-
ion om 4 o 40 pm o 5 min. The ime un il alling down
was eco ded. Mo o coo dina ion was assessed by com-
pa ing he la ency o all in seconds on he i s ial be-
ween ea men g oups.
The cylinde ea ing es (CRT) examines la e al bias o
senso imo o de ici s (G ow e al., 2003). Ra s we e placed
in o a glass cylinde o 5 min, and he numbe o imes he
le and/o he igh o epaw was placed on he cylinde
inne su ace was eco ded. Righ de ici (le p e e ence)
was assessed as ollows: ([no. le o epaw placemen s] –
[no. igh o epaw placemen s])/(no. le , igh , and bo h
o epaw placemen s) 100.
The no el objec ecogni ion es explo es nonspa ial
wo king memo y (De e e al., 2007;B oadben e al.,
2010). Ra s we e exposed o wo iden ical objec s o
5 min. One hou la e , one o he objec s was eplaced o
a new one (di e en in shape, colo , and size), and all ani-
mals we e eco ded o 5 min. The ime spen by each ani-
mal o explo e he no el and he amilia objec was used
o calcula e he disc imina ion index (DI). DI uses he di -
e ence in explo a ion ime o amilia objec s (TF) and
ime o no el objec s (TN), di ided by he o al amoun o
explo a ion o bo h ypes o objec s, as ollows: DI = (TN –
TF)/(TN 1TF) (De e e al., 2007). This esul can a y be-
ween 11 and 1, whe e a posi i e sco e indica es mo e
ime spen wi h he no el objec , a nega i e sco e indi-
ca es mo e ime spen wi h he amilia objec , and a ze o
sco e indica es a null p e e ence (An unes and Biala,
2012).
Spon aneous al e na ion in a T-maze is used o s udy
spa ial lea ning and memo y (Deacon and Rawlins, 2006).
The ull expe imen consis ed o he ollowing h ee pa s
du ing 3 consecu i e days: habi ua ion, aining, and es -
ing. Two hou s be o e he aining and es ing phases,
daily ood a ioning was pe o med and eplaced by
50 mg o goodie ewa ds (F ui C unchies, Biose ) as in-
dica ed by p o ocol (Deacon and Rawlins, 2006). The da a
ob ained om he T-maze consis s o he numbe o co -
ec e sus inco ec a m en ies in each ial (a leas a
i s ee un and i e uns pe animal), o no longe han
15 min. The pe cen ages o co ec a m choices we e
g aphed and compa ed ac oss con ol and ea ed dis-
ease g oups (Deacon and Rawlins, 2006).
Ampli ude-in eg a ed elec oencephalog aphy
B ain ac i i y was moni o ed using a wo-channel bed
elec oencephalog aphy (EEG) moni o (EEG-SMT, Olimex)
wi h i e needle elec odes placed in on al and pos e io
bipa ie al placemen s o ou eco ding elec odes and
one e e ence elec odes (Tucke e al., 2009). The “ aw”
EEG signal was pe o med a 100 Hz sampling equency
and in he equency domain om 0 o 60 Hz (low-pass il-
e , 0.2 Hz; high-pass il e , 59 Hz; Zayachki skye al.,
2013). The aw EEG signal was egis e ed only a P37,
unde gene al anes hesia wi h iso lu ane, as desc ibed
abo e, o a du a ion o 30 min. The ampli ude in eg a ed
EEG signal was ob ained ia a s ep-by-s ep signal-p oc-
essing me hod (Zhang e al., 2011). Six s eps we e ap-
plied o calcula e a compac ampli ude-in eg a ed EEG
(aEEG) acing and he uppe /lowe ma gin using aw
EEG da a: (1) asymme ical da a il e ing ( o p o ide
equal weigh o he ene gy o non hy hmic componen s
a each equency); (2) absolu e alue e alua ion ( o ac-
qui e heampli udein o ma iono EEGda awi habi-
phasic na u e); (3) en elope de ec ion ( o moni o he
b ain unc ion by displaying he ampli ude end o b ain
ac i i y); (4) acing comp ession ( o ob ain a bi d’s-eye-
iew o he ce eb al unc ion o e a long du a ion); (5)
segmen a ion and e minal poin ex ac ion ( o ob ain a
compac aEEG, simpli ying he ull acing in o a se ies o
e ical lines); and (6) ma gin calcula ion ( o ob ain a
smoo h and ep esen a i e ma gin, he median ampli-
ude o e e y successi e 20 e minal poin s was used).
Fu he mo e, aw EEG aces we e manually e iewed
o he p esence o seizu es, which a e desc ibed as pe-
iods o a sudden inc ease in ol age, accompanied by a
na owing o he band o aEEG ac i i y, and ollowed by
a b ie pe iod o supp ession (Al a ez e al., 2008).
B ain samples
Animals we e killed by decapi a ion a P14 and P37.
The b ains o he animals we e immedia ely emo ed om
he skull, and he b ains o hal o he animals in each
g oup we e ozen wi h liquid ni ogen (,1 min), while he
b ains o he o he hal we e ixed by imme sion in pa a-
o maldehyde 4%. All b ains we e s o ed indi idually. The
pa a o maldehyde- ixed b ains (N= 5/g oup a P14 and
P37) we e used o his ologic s udies, and he ozen
b ains (N= 5/g oup a P14 and P37) we e used o bo h
magne ic esonance imaging (MRI) and p o on magne ic
esonance spec oscopy (H
1
-MRS).
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His ologic s udies
The b ains we e embedded in pa a in, and co onal sec-
ions (4 mm) we e cu and moun ed on glass slides o he-
ma oxylin-eosin and Nissl s aining. Th ee consecu i e
sec ions co esponding o coo dina es in he a b ain
a las ( om b egma, 4.44; in e au al, 4.56; Paxinos and
Wa son, 2007, hei Figu e 70) we e selec ed o analysis
by an examine blinded o he expe imen al g oup o he
animal. The deg ee o b ain damage (neu opa hological
sco e) in he CA1 a ea o he ipsila e al hippocampus and
he ipsila e al empo opa ie al co ex we e sco ed as ol-
lows (Mohammed e al., 2017;Ba a a e al., 2019):
0 = no mal; 1 = ew neu ons damaged (1–5%); 2 = se e al
neu ons damaged (6–25%); 3 = mode a e numbe o neu-
ons damaged (26–50%); 4 = mo e han hal o neu ons
damaged (51–75%); and, 5 = majo i y o neu ons dam-
aged (.75%) o absen hippocampus. The mean o h ee
sec ions om each animal was de e mined. The empo o-
pa ie al co ex includes he ollowing a eas: pa ie al co -
ex, pos e io a ea, do sal pa ; pa ie al co ex, pos e io
a ea, os al pa ; p ima y soma osenso y co ex; p ima y
audi o y co ex; seconda y audi o y co ex, do sal a ea;
and seconda y audi o y co ex, en al a ea.
P o on magne ic esonance spec oscopy
Ipsila e al samples ( empo opa ie al co ex and hippocam-
pus) om ozen b ains o he se en g oups (N= 5 animals/
g oup) a P14 and P37 we e analyzed in he Biomedical
Resea ch Ins i u e Albe o Sols (Au ónoma Uni e si y,
Mad id) as p e iously desc ibed (Pazose al.,2012). H
1
-
MRS was pe o med a 500.13 MHz using an 11.7 T
spec ome e (AMX500, B uke ) ope a ing a 4°C on o-
zen samples (3 mg) placed wi hin a 50-l zi conium oxide
o o wi h a cylind ical inse and spun a 4000 Hz. All spec-
a we e p ocessed using TOPSPIN so wa e, e sion 1.3
(B uke ). Spec a we e phased, baseline co ec ed, and e -
e enced o he sodium (3- ime hylsilyl)2,2,3,3- e adeu e -
iop opiona e single a
d
0 ppm. Lac a e/N-ace ylaspa a e
(Lac/NAA; me abolic dys unc ion), glu ama e/NAA (Glu/
NAA; exci o oxici y), NAA/choline (NAA/Cho; neu al
damage), and lac a e/c ea ine (Lac/C ; mi ochond ial
impai men ) a ios we e calcula ed.
Magne ic esonance imaging
Whole ixed b ains o he se en g oups (N= 5 animals/
g oup) a P14 and P37 we e ans e ed o he Biomedical
Resea ch Ins i u e Albe o Sols (Au ónoma Uni e si y,
Mad id), whe e an MRI scan was pe o med on an MRI
(BioSpec BMT 47/40, B uke -Medical) ope a ing a 4.7 T,
equipped wi h an a oid ac i ely shielded g adien inse
wi h 11.2 cm bo e, a maximal g adien s eng h o 200 mT/m
and 80 ms ise ime, and a home-made 4 cm su ace coil, as
p e iously desc ibed (Pazos e al., 2012). T2-weigh ed
images (T2WIs) we e acqui ed wi h a mul islice apid ac-
quisi ion (TR = 3.4 s; apid acquisi ion wi h elaxa ion
enhancemen ac o = 8; in e echo in e al = 30 ms; e -
ec i e TE = 120 ms; ma ix size, 256 256; pixel dimen-
sions, 117 117 mm; FOV = 3 cm
2
).The slice package
consis ed o 13 consecu i e slices o 1.0 mm slice
hickness in he axial plan in e lea ed by a 0.2 mm gap,
co e ing he en i e b ain. The ac ional ex en o b ain in-
a c ion was calcula ed om MR images (Pazos e al.,
2012), using ImageJ e sion 1.43u so wa e (NIH). In each
slice, he a ea o each hemisphe e was manually ou lined,
and he size o his a ea calcula ed by mean alues om
ImageJ. The combined a ea o he 13 consecu i e slices
was used o calcula e he o e all olume. A ela ionship o
0.97 be ween le hemisphe e olumes (LHVs; ipsila e al)
and igh hemisphe e olumes (RHVs; con ala e al) in
sham animals was used as p e iously epo ed (Pazos e
al., 2012). In HI animals, he olume o lesion was calcu-
la ed by sub ac ing he olume o in ac b ain issue in he
le hemisphe e (ipsila e al) om he heo e ical LHV, calcu-
la ed as RHV 0.97. The bounda y o each lesion was iden-
i ied by a well de ined hype in ense and/o in a c ed a ea.
The lesion olume was exp essed as a pe cen age o he he-
o e ical o e all b ain olume, calcula ed as RHV 1 heo e ical
LHV (= RHV 0.97). The e o e, he inal o mula olume o le-
sion (%) = 49.23 –100 in ac le olume/(RHV 1.97).
Biochemical s udies
Le els o oxidized p o eins we e quan i ied by Wes e n
blo analysis o assess p o ein ca bonyla ion in ipsila e al
empo opa ie al co exes. A de ec ion ki (Millipo e Ibé ica)
was used acco ding o he manu ac u e p o ocol. Oxidized
p o ein le els we e quan i ied ia measu emen o he op i-
cal densi y, using he NIH ImageJ analysis so wa e. Resul s
we e no malized by o al p o ein loading (Red Ponceau
s aining) and exp essed as he OXYBLOT/Red Ponceau
a io.
TNF
a
Wes e n blo assays we e pe o med wi h b ain
samples om ipsila e al empo opa ie al co exes, con-
aining 20 mg o o al p o ein. The p o ocol used was as
p e iously desc ibed (La uen e e al., 2016). P o ein le els
(1:1000; abbi an i-TNF
a
; Abcam) we e quan i ied using
densi ome y analysis no malized by
b
-ac in (1:500;
Abcam).
S a is ics
The sample size was calcula ed using G*Powe 3.1
(Faul e al., 2007) op o ide.80% powe o de ec a e-
duc ion in beha io al ou comes o 45% ( ange, 40–50%) be-
ween heg oups.JMP e sion6.0so wa e(SASIns i u e)
was used o all s a is ical analyses. Resul s we e compa ed
wi h a B own–Fo sy he es o con i m he homogenei y o
a iance be ween he di e en ea men s, and he dis ibu-
ion o da a was assessed using he Shapi o–Wilk es ,
wi h he ou come o each da ase desc ibed in he igu e
legends. No mal da a a e p esen ed by his og ams (mean 6
SEM), and non-no mal his og ams a e p esen ed as box
plo s (median wi h in e qua ile ange).
Fo no mal da a, compa isons be ween he g oups
we e analyzed using one-way ANOVA wi h Bon e oni–
Dunn’s co ec ion as unc ion o g oup, disease, o ea -
men . Two-way ANOVA ( ime s g oup) ollowed by
Bon e oni–Dunn’spos hoc es was applied o he
g ow h cu e (weigh s ime). Fo non-no mal da a, com-
pa isons we e es ed wi h he K uskal–Wallis es and
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Dunn’s es o mul iple compa isons, as a unc ion o
g oup, disease, o ea men . A p alue ,0.05 was ac-
cep ed as signi ican .
Resul s
Al hough an o e all mo ali y a e o 10% (62 o 631)
was measu ed on p e ious ials o expe imen s, only wo
animals in no mo he mic and hypo he mic ehicle- ea ed
g oups (one pe g oup) died du ing he HI insul o du ing
HT ea men (Table 1). Only su i ing animals a P37
we e included in he s udy. A P7, all g oups demon-
s a ed simila weigh (Fig. 1A). A P37, g ow h cu es
we e obse ed wi h signi ican di e ences (Fig. 1B).
Basal ec al empe a u e was 37.7 60.1°C in all pups
(N=10,F
(6,63)
= 0.413, p= 0.868), which was main ained in
no mo he mic animals a e he equi alen in e al o HT
ea men [a 48 h: VEH, 37.5 60.2°C; HI CBD 0.1mg/kg
(CBD0.1)- ea ed g oup, 37.6 60.2°C; CBD 1 mg/kg
(CBD1)- ea ed g oup, 37.5 60.2°C; N=10,F
(2,27)
= 0.133,
p= 0.876] and he ewa ming phase (a 60 h: VEH g oup,
37.6 60.2°C; CBD0.1 g oup, 37.560.2°C; CBD1 g oup,
37.5 60.2°C; N=10,F
(2,27)
= 0.062, p= 0.940). In hypo he -
mic g oups, he a ge empe a u e (32.1 60.2°C) was
eached a e 18.4 66.5 min. Tempe a u e emained s able
h oughou HT ea men (48 h) in all g oups wi hou di e en-
ces (VEH g oup, 32.1 60.1°C; CBD0.1 g oup, 32.0 60.2°C;
CBD1 g oup, 32.0 60.2°C; N= 10, F
(2,27)
= 0.082, p= 0.921).
A e he ewa ming phase (60 h), he hypo he mic g oups did
no demons a e di e ences in empe a u e (VEH g oup,
37.7 60.2°C; CBD0.1 g oup, 37.8 60.2°C; CBD1 g oup,
37.7 60.2°C; N= 10, F
(2,27)
= 0.154, p= 0.858). The SHAM
g oup was main ained a cons an empe a u e du ing all ex-
pe imen al phases (37.1 60.1°C).
Neu ologic unc ional s udies
The HI insul caused ipsila e al (le ) b ain damage, e-
sul ing in igh mo o de ici as well as cogni i e impai -
men s (Fig. 2A). Vehicle- ea ed animals demons a ed a
p e e ence o he ipsila e al o epaw (inju ed side), a
de ici ha was no al e ed by HT ea men (Fig. 2A). CBD
ea men signi ican ly a enua ed his asymme y a all
doses es ed o an ex en ha was also signi ican ly g ea -
e han ha achie ed wi h HT (Fig. 2A).
The HI insul caused a signi ican educ ion in ime
spen explo ing he new objec s (Fig. 2B), a de ici ha
was imp o ed by HT ea men alone and 1 mg/kg CBD
alone. Fu he mo e, a s ea ed wi h 1 mg/kg CBD spen
signi ican ly mo e ime explo ing he no el objec han did
a s ea ed wi h HT 1VEH (Fig. 2B).
Locomo o unc ion was impai ed as a consequence o
hypoxia-ischemia (Fig. 2C), and his was signi ican ly im-
p o ed in all ea men g oups (Fig. 2C). Animals ea ed
wi h CBD 1 mg/kg demons a ed a wo old inc ease o he
la ency o all in ela ion o ehicle- ea ed animals, and
showed a signi ican ly supe io mo o coo dina ion com-
pa ed wi h HT 1VEH- ea ed a s. In he g oups ea ed
wi h 0.1 mg/kg CBD, he combined use wi h hypo he mia
signi ican ly inc eased he la ency o all compa ed wi h
no mo he mic g oups ecei ing a simila dose, bu was
no signi ican ly di e en om HT 1VEH (Fig. 2C).
In ela ion o he abili y o choose he co ec esponse
in he T-maze, hypoxia-ischemia esul ed in signi ican ly
mo e e o s (Fig. 2D). Hypo he mia alone did no a ec
pe o mance, bu CBD 1 mg/kg ea men signi ican ly im-
p o ed he pe cen age o co ec esponses in bo h no -
mo he mia and hypo he mia (Fig. 2D).
A P37, he aEEG mean alues o ju enile a s showed a
signi ican impai men in he VEH-NT g oup (Fig. 3). All
ea ed g oups showed an imp o emen o mean aEEG al-
ues, and 1 mg/kg CBD combined wi h HT was signi ican ly
supe io o hypo he mic ehicle- ea ed a s (Fig. 3). A P37,
nei he hypoxic-ischemic no sham animals demons a ed
con ulsi e pa e ns (seizu es) based on he aw EEG aces.
His ologic s udies
Hypoxia-ischemia led o an ipsila e al a ea o in a c ion
wi h se e e damage o he su ounding issues, including
he ipsila e al empo opa ie al co ex a P14 (Fig. 4A)o P37
Figu e 1. Body weigh s. A,B, G aph ep esen s he mean body weigh s a P7 (A) and P37 (B) o he ollowing h ee NT and HT
g oups: HI VEH- ea ed g oup (VEH); CBD0.1 g oup; and HI CBD1 g oup. The SHAM g oup was used as a e e ence. Da a a e p e-
sen ed by his og ams (mean 6SEM) and one-way ANOVA as a unc ion o g oup, disease o ea men was used. Fo each g oup,
N= 10. JMP e sion 6.0 was used o all s a is ical analyses. A p alue ,0.05 was conside ed o be signi ican . A P7, all g oups
demons a ed simila weigh (N= 10; one-way ANOVA: F
(6,63)
= 0.7142, p= 0.6395). A P37: *p,0.05 e sus VEH-NT; #p,0.05 e -
sus VEH-HT; ¶p,0.05 e sus CBD0.1-NT; ‡p,0.05 e sus CBD0.1-HT; §p,0.05 e sus CBD1-NT.
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(Fig. 4B) and he hippocampus a P14 (Fig. 4C)o P37(Fig.
4D). Thus, many neu ons in he b ains o VEH animals ap-
pea ed se e ely damaged (neu opa hological sco e exceed-
ing 3 poin s), bo h a P14 (Fig. 4A,C)andP37(Fig. 4B,D).
His ologic e alua ion demons a ed ha ea men wi h
CBD educed he ex en o b ain damage in almos all
g oups a P37 in a dose-dependen manne . O e all, he e
was a mean neu opa hological sco e educ ion o 1 poin
when compa ed wi h he b ains o he espec i e VEH
g oups. Hypo he mic ehicle- ea ed animals did no display
bene i o e no mo he mic ehicle animals. Addi i e e ec s
we e obse ed in he combina ion o 1 mg/kg CBD wi h HT
a P37 in he hippocampus (Fig. 4D). Rep esen a i e images
o ipsila e al empo opa ie al co exes and CA1 hippocam-
pal laye s a e shown in Figu es 5 and 6.
MRI
Ra s exposed o hypoxia-ischemia had de ec able le-
sion olumes o ;25% a P14 and P37. A P14, he only
ea men ha signi ican ly educed he lesion olume
was CBD 1 mg/kg combined wi h HT (Fig. 7A). A P37, ani-
mals ea ed wi h CBD 1mg/kg signi ican ly dec eased he
olume o lesion. Combina ion o CBD 1 mg/kg wi h HT e-
sul ed in signi ican ly supe io e ec s compa ed wi h HT
alone (Fig. 7B). Rep esen a i e images o all g oups a his
de elopmen al age a e included in Figu e 8.
H
1
-MRS
Ra s exposed o hypoxia-ischemia showed inc eased
Lac/NAA a ios (me abolic dys unc ion), which we e e i-
den a P14 and P37 (Table 2). A P14, his e ec was no
al e ed by ea men wi h CBD o HT, bu CBD 1 mg/kg 1
HT had signi ican ly lowe ed Lac/NAA a ios compa ed
wi h HT alone (Table 2). A P37, ea men wi h HT o CBD
1 mg/kg signi ican ly educed he Lac/NAA a io. (Table 2).
Exposu e o HI inju y inc eased he Glu/NAA a io (exci-
o oxici y) a P14 and P37. A P14, he HT and CBD ea -
men s alone did no al e he Glu/NAA a io, bu he
combina ion o CBD and HT educed his a io a bo h
doses, indica ing addi i e e ec s (Table 2). A P37, all
Figu e 2. Neu ologic unc ional s udies. A–D, Cylinde ea ing es (A), disc imina ion index o no el objec ecogni ion es (B), o a -
od es (C), and spon aneous al e na ion in a T-maze es (D). G aphs ep esen he mean o median alues o he ollowing NT and
HT h ee g oups a P37 pos na al age: HI VEH- ea ed g oup; HI CBD0.1 g oup; and HI CBD1 g oup. The SHAM g oup was used as
a e e ence. Da a in Aand Da e p esen ed as box plo s (median wi h in e qua ile ange), and he K uskal–Wallis es was used as a
unc ion o g oup, disease, o ea men . Da a in Band Ca e p esen ed by his og ams (mean 6SEM), and one-way ANOVA was
used as a unc ion o g oup, disease, o ea men . Fo each g oup, N=10. JMP e sion 6.0 was used o all s a is ical analyses. A p
alue ,0.05 was conside ed o be signi ican . *p,0.05 e sus VEH-NT; #p,0.05 e sus VEH-HT; ¶p,0.05 e sus CBD0.1-NT;
‡p,0.05 e sus CBD0.1-HT; §p,0.05 e sus CBD1-NT.
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ea men g oups demons a ed signi ican educ ions in
exci o oxici y compa ed wi h ehicle (Table 2).
Also, we obse ed ha hypoxia-ischemia educed he
NAA/Cho a io (neu al damage) a bo h P14 and P37 (Table
2). A P14, his e ec was signi ican ly amelio a ed in all
ea men g oups. Combina ion o HT wi h CBD 0.1mg/kg
esul ed in signi ican ly supe io e ec s o HT alone (Table
2). A P37, he NAA/Cho a io was signi ican ly inc eased
wi h CBD 0.1 mg/kg and CBD 1 mg/kg, bu o a lesse ex-
en han HT alone. When HT was combined wi h CBD
1 mg/kg he e was an addi i e e ec (Table 2).
The Lac/C a io (mi ochond ial impai men ) was in-
c eased in HI animals a P14 and P37 (Table 2). A P14,
his e ec was no signi ican ly al e ed in CBD- ea ed
g oups. Indeed, ea men wi h hypo he mia esul ed in an
exace ba ion o his, an e ec ha was signi ican ly e-
duced when gi en in combina ion wi h CBD 1 mg/kg
(Table 2). A P37, all ea men g oups signi ican ly e-
duced he Lac/C a io (Table 2).
Biochemical s udies
Exposu e o hypoxia-ischemia signi ican ly ele a ed
TNF
a
le els (Fig. 9A). HT and CBD 1 mg/kg ea men
led o a educ ion o TNF
a
con en in he ipsila e al
Figu e 3. Ampli ude-in eg a ed elec oencephalog aphy: he
g aph ep esen s he mean alues ollowing NT and HT o
h ee g oups a P37: HI VEH- ea ed g oup; HI CBD0.1 g oup;
and HI CBD1 g oup. The SHAM g oup was used as a e e ence.
Da a a e p esen ed by his og ams (mean 6SEM), and one-way
ANOVA was used as a unc ion o g oup, disease, o ea men .
Fo each g oup, N=10. JMP e sion 6.0 was used o all s a is-
ical analyses. A p alue ,0.05 was conside ed o be signi ican .
*p,0.05 e sus VEH-NT; #p,0.05 e sus VEH-HT; ¶p,0.05
e sus CBD0.1-NT; ‡p,0.05 e sus CBD0.1-HT; §p,0.05
e sus CBD1-NT (Ex ended Da a Fig. 3-1).
Figu e 4. Neu opa hological sco e. A–D, G aph ep esen s he neu opa hological sco es in he empo opa ie al co ex a P14 (A)
and P37 (B), and in he CA1 a ea o he hippocampus a P14 (C) and P37 (D), o h ee g oups ollowing NT and HT: HI VEH- ea ed
g oup, HI CBD0.1 g oup, and HI CBD1 g oup. The SHAM g oup was used as a e e ence. Da a a e p esen ed as box plo s (median
wi h in e qua ile ange), and K uskal–Wallis es was used as a unc ion o g oup, disease, o ea men . Fo each g oup, N= 5. JMP
e sion 6.0 was used o all s a is ical analyses. A p alue ,0.05 was conside ed o be signi ican . *p,0.05 e sus VEH-NT;
#p,0.05 e sus VEH-HT; ¶p,0.05 e sus CBD0.1-NT; ‡p,0.05 e sus CBD0.1-HT; §p,0.05 e sus CBD1-NT.
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Figu e 5. Rep esen a i e images o ipsila e al empo opa ie-
al co exes a P37. On op, he SHAM g oup was used as a
e e ence (neu opa hological sco e, 0). The le column co -
esponds o NT g oups, and he igh column co esponds
o HT g oups, espec i ely. A–F, HI ehicle g oups [VEH-NT
(A) and VEH-HT (B), neu opa hological sco es 4 and 2, e-
spec i ely]; HI CBD0.1-NT (C)andCBD0.1-HT(D) (neu o-
pa hological sco es 1 and 2, espec i ely); and CBD1-NT (E)
and CBD1-HT (F) g oups (neu opa hological sco es 1 and 1,
espec i ely). Many neu ons in he b ains o VEH- ea ed an-
imals appea ed se e ely damaged. Animals ea ed wi h
CBD (bo h 0.1 and 1 mg/kg) educed he ex en o b ain
damage. Hypo he mic VEH g oup did no signi ican ly dis-
play educ ion o e he no mo he mic VEH one. Howe e ,
addi i e imp o emen was obse ed in he combina ion o
1 mg/kg CBD and HT. O iginal magni ica ion: 100(scale
ba , 50 mm); inse s, 400(scale ba , 10 mm).
Figu e 6. Rep esen a i e images o he CA1 a ea o he ipsi-
la e al hippocampus a P37. On op, he SHAM g oup was
used as a e e ence (neu opa hological sco e, 0). The le
column co esponds o NT g oups, and he igh column co -
esponds o HT g oups, espec i ely. A–F, HI VEH g oups [VEH-
NT (A) and VEH-HT (B) (neu opa hological sco es 4 and 2, e-
spec i ely); HI CBD0.1-NT (C) and CBD0.1-HT (D) g oups (neu o-
pa hological sco es 1 and 1, espec i ely); and CBD1-NT (E)and
CBD1-HT (F) g oups (neu opa hological sco es 0 and 1, espec-
i ely). Neu ons in CA1 hippocampal laye o he b ains o VEH
animals appea ed damaged. The damaged a ea o he hippo-
campi o CBD- ea ed animals (bo h 0.1 and 1 mg/kg concen-
a ions) was educed. The hypo he mic VEH g oup did no
signi ican ly display educ ion o e he no mo he mic VEH
g oup. Howe e , addi i e imp o emen was obse ed in he
combina ion o 1 mg/kg CBD and HT. O iginal magni ica ion:
100(scale ba , 50 mm); inse s, 400(scale ba , 10 mm).
Resea ch A icle: New Resea ch 9 o 15
May 2023, 10(5) ENEURO.0417-22.2023 eNeu o.o g
