Ci a ion: Belda ain, G.; Chillida, M.;
Hila io, E.; He e o de la Pa e, B.;
Ál a ez, A.; Alonso-Alconada, D.
URB447 Is Neu op o ec i e in Bo h
Male and Female Ra s a e Neona al
Hypoxia–Ischemia and Enhances
Neu ogenesis in Females. In . J. Mol.
Sci. 2024,25, 1607. h ps://doi.o g/
10.3390/ijms25031607
Academic Edi o s: C is o o o Sil es i
and Robe B. Lap ai ie
Recei ed: 15 Decembe 2023
Re ised: 15 Janua y 2024
Accep ed: 26 Janua y 2024
Published: 28 Janua y 2024
Copy igh : © 2024 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
In e na ional Jou nal o
Molecula Sciences
A icle
URB447 Is Neu op o ec i e in Bo h Male and Female Ra s a e
Neona al Hypoxia–Ischemia and Enhances Neu ogenesis in Females
Go ane Belda ain 1,† , Ma c Chillida 1,† , En ique Hila io 1, Bo ja He e o de la Pa e 2, An onia Ál a ez 1
and Daniel Alonso-Alconada 1,*
1Depa men o Cell Biology and His ology, School o Medicine and Nu sing, Uni e si y o he Basque
Coun y (UPV/EHU), 48940 Leioa, Spain
2Depa men o Su ge y and Radiology and Physical Medicine, Facul y o Medicine and Nu sing,
Uni e si y o he Basque Coun y (UPV/EHU), 48940 Leioa, Spain
*Co espondence: [email p o ec ed]
†These au ho s con ibu ed equally o his wo k.
Abs ac : The need o new and e ec i e ea men s o neona es su e ing om hypoxia–ischemia
is u gen , as he only implemen ed he apy in clinics is he apeu ic hypo he mia, only e ec i e
in 50% o cases. Cannabinoids may modula e neu onal de elopmen and b ain plas ici y, bu
u he in es iga ion is needed o be e desc ibe hei implica ion as a neu o es o a i e he apy a e
neona al HI. The cannabinoid URB447, a CB1 an agonis /CB2 agonis , has p e iously been shown
o educe b ain inju y a e HI, bu i is no clea whe he sex may a ec i s neu op o ec i e and/o
neu o es o a i e e ec . He e, URB447 s ongly educed b ain in a c , imp o ed neu opa hological
sco e, and augmen ed p oli e a i e capaci y and neu ogenic esponse in he damaged hemisphe e.
When analyzing hese e ec s by sex, URB447 amelio a ed b ain damage in bo h males and emales,
and enhanced cell p oli e a ion and he numbe o neu oblas s only in emales, hus sugges ing a
neu op o ec i e e ec in males and a double neu op o ec i e/neu o es o a i e e ec in emales.
Keywo ds: neona al hypoxia–ischemia; endocannabinoid sys em; neu op o ec ion; neu ogenesis
1. In oduc ion
Pe ina al hypoxia–ischemia (HI) o en causes a b ain condi ion known as hypoxic–
ischemic encephalopa hy (HIE), a ec ing a ound 2–3/1000 newbo ns in de eloped coun-
ies and 10–20/1000 newbo ns in low/middle-income coun ies [
1
–
3
]. Newbo ns who
unde go HI may de elop epilepsy, men al e a da ion, isual and hea ing p oblems and
cogni i e o beha io al diso de s, among o he hings, con ibu ing signi ican ly o o e all
disabili y wo ldwide [
2
]. As ecen epo s sugges ha males a e mo e ulne able o
su e ing om p ena al anoxia, hemo hage and in ec ion [
4
–
6
], sex dimo phic esponses
o HI mus be conside ed.
Cu en ly, he only he apy a ailable o ea HIE in hospi als is he apeu ic hypo he -
mia. Due o i s limi ed e icacy, al e na i e neu op o ec i e s a egies a e being es ed,
al hough none ha e been app o ed o clinics ye . In ecen yea s, he modula ion o he
endocannabinoid sys em (ECS) has been p oposed as a po en ial he apy, as his sys em
pa icipa es in p ocesses like b ain plas ici y, lea ning, memo y, neu onal de elopmen ,
ene gy balance and/o he mogenesis [
7
]. A e ha m ul e en s like HI, he ECS also plays
an impo an ole in egula ing glu ama e exci o oxici y, oxida i e s ess and in lamma ion,
mainly h ough he ac ion o he cannabinoid ecep o s 1 and 2 (CB1 and CB2) [8–10].
CB1 ecep o s a e mos ly exp essed in he cen al ne ous sys em and a e esponsi-
ble o he egula ion o exci o oxici y and oxida i e s ess a e HI, while CB2 ecep o s
appea mainly in immune sys em cells, egula ing he in lamma o y esponse caused by
he inju y [
11
]. P e ious s udies ha e ocused on ac i a ing/inac i a ing hese ecep o s o
enhance he neu op o ec i e e ec o he ECS, bu ha e ob ained con o e sial ou comes.
In . J. Mol. Sci. 2024,25, 1607. h ps://doi.o g/10.3390/ijms25031607 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2024,25, 1607 2 o 15
CB1 ecep o s imula ion, o example, was neu op o ec i e in some
in i o
and
in i o
s udies [
12
–
14
], bu neu o oxic in o he s [
15
]. Mo eo e , CB1 an agonism can also ha e ben-
e icial e ec s, as epo ed in a p eclinical s udy using middle ce eb al a e y occlusion [
16
].
The modula ion o he CB2 ecep o , in con as , has shown mo e consis en esul s, as i s ac-
i a ion esul ed in an i-in lamma o y esponses, whe eas i s an agonism has desc ibed no
bene icial e ec s [
17
]. Fu he , he ECS may play a ole in neu ogenesis a e HI [
11
], as i s
modula ion may compensa e cell dea h caused by he insul by p omo ing cell p oli e a ion
and issue epai .
URB447 ({[4-amino-1-(4-chlo obenzyl)-2-me hyl-5-phenyl-1H-py ole-3-yl](phenyl)
me hanone}) is a syn he ic cannabinoid ha binds o bo h CB1 and CB2 ecep o s, ac ing as
simul aneous CB1-an agonis and CB2-agonis [
18
]. In neona al a s, we p e iously showed
ha URB447 educed b ain inju y and whi e ma e demyelina ion [
19
], bu li le is known
abou i s neu op o ec i e e ec depending on sex a e HI. In p eclinical models o HI, sex
di e ences a e no ully clea : some au ho s ha e no obse ed any dimo phic e ec on he
inju y [
20
], whe eas o he s ha e ound sex dimo phism in speci ic b ain a eas a e HI, like
he co ex [
21
,
22
]. Clinically, esul s seem mo e consis en , and male sex is conside ed a
isk ac o [
23
]. Fu he , he neu op o ec i e e ec o he apies may di e depending on
sex [24], jus as may he neu ogenic esponse o some ea men s [25,26].
Gene a ing new neu al cells a e HI may also help o amelio a e he e ec s o b ain
inju y caused by HI, so disco e ing new he apies o enhance hese p ocesses is o g ea
in e es . Neu ogenesis only emains in wo a eas a e bi h: he sub en icula zone o he
la e al en icles and he subg anula zone o he den a e gy us o he
hippocampus [27–29]
.
As he neu ogenic po en ial o he hippocampus may be a ec ed a e HI due o i s pa icu-
la ulne abili y [30], i s p o ec ion migh help o main ain i s neu ogenic na u e.
In his wo k, we wan ed (i) o explo e he possible dimo phic e ec o sex on HI and
subsequen URB447 ea men and (ii) o e alua e he hippocampal neu ogenic esponse
a e he adminis a ion o he cannabinoid in male and emale neona al a s.
2. Resul s
2.1. Body and B ain Weigh
Da a om pos na al day 7 (P7) body weigh s and P14 body and b ain weigh s om
each expe imen al g oup a e shown in Table 1.
Table 1. P7 body and P14 body and b ain weigh om sham, hypoxic–ischemic (HI) and URB447-
ea ed (HI+URB447) animals. Da a a e shown as mean
±
s anda d de ia ion. * p< 0.05; ** p< 0.01;
*** p< 0.001; **** p< 0.0001 s. sham. # p< 0.05; ## p< 0.01 s. HI.
Bo h sexes
SHAM HI HI+URB447
P7 body weigh 16.09 ±0.38 15.51 ±1.6 15.4 ±1.20
P14 body weigh
29.45 ±0.67 23.56 ±4.25 **** 25.49 ±3.29 **
B ain weigh 1.12 ±0.02 0.89 ±0.08 **** 0.97 ±0.05 ** #
Males
P7 body weigh 16.33 ±0.31 16.29 ±1.08 15.36 ±1.57
P14 body weigh
29.78 ±0.50 24.86 ±4.30 * 25.4 ±3.66
B ain weigh 1.12 ±0.03 0.92 ±0.05 **** 0.97 ±0.05 ***
Females
P7 body weigh 15.74 ±0.03 13.94 ±1.29 * 15.44 ±0.73
P14 body weigh
28.95 ±0.06 22.10 ±3.92 ** 29.5 ±3.06
B ain weigh 1.11 ±0.02 0.86 ±0.08 **** 0.97 ±0.06 ** ##
The day o he su gical p ocedu e (P7), he weigh o he animals was simila in all
g oups. Se en days a e HI (P14), animals om he HI g oup had lowe body weigh s
when compa ed o sham (p< 0.0001 o bo h sexes; p< 0.05 o males and p< 0.01 o
emales). The HI+URB447 g oup showed lowe body weigh when compa ed o sham o
all he pups (p< 0.01), bu wi hou di e ences when e alua ed by sex o in compa ison
wi h HI animals. B ain weigh educ ion was e iden in bo h sexes, males and emales,
In . J. Mol. Sci. 2024,25, 1607 3 o 15
om he HI g oup compa ed o sham (p< 0.0001), and so was he HI+URB447 g oup
(
p< 0.01
in bo h sexes and emales; p< 0.001 in males). Di e ences be ween he HI and he
HI+URB447 g oups we e obse ed when compa ing bo h sexes oge he (p< 0.05) and in
emales (p< 0.01), wi h highe weigh s in he ea ed g oups.
2.2. B ain Inju y Assessmen
2.2.1. Hemisphe ic and Hippocampal A ea Ra ios
Hemisphe ic a ea a ios o bo h sexes oge he showed no signs o inju y in sham
animals, as he a io alues we e close o 1 (1.01
±
0.02). A e HI, he a eas o he ipsila e al
hemisphe es we e educed by a hal (0.50
±
0.26), being signi ican ly lowe han he sham
a ios (p< 0.0001). The hemisphe ic a ios o he URB447- ea ed animals e ealed simila
alues o he sham g oup (0.92
±
0.14), being signi ican ly highe han non- ea ed HI pups
(p< 0.001).
When sepa a ing da a by sex, he educ ion in he ipsila e al hemisphe e a ea was
e iden in bo h males (sham: 1.02
±
0.02 s. HI: 0.47
±
0.24; p< 0.0001) and emales (sham:
1.00
±
0.02 s. HI: 0.54
±
0.1; p< 0.01) om he HI g oup. Again, he hemisphe ic a ios o
URB447- ea ed animals (males: 0.87
±
0.17; emales: 0.96
±
0.10) we e close o sham, and
he e o e signi ican ly highe han HI-g oup a ios o bo h sexes (p< 0.05 o males and
p< 0.01 o emales). Resul s a e shown in Figu e 1A.
In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 3 o 15
The day o he su gical p ocedu e (P7), he weigh o he animals was simila in all
g oups. Se en days a e HI (P14), animals om he HI g oup had lowe body weigh s
when compa ed o sham (p < 0.0001 o bo h sexes; p < 0.05 o males and p < 0.01 o
emales). The HI+URB447 g oup showed lowe body weigh when compa ed o sham o
all he pups (p < 0.01), bu wi hou diffe ences when e alua ed by sex o in compa ison
wi h HI animals. B ain weigh educ ion was e iden in bo h sexes, males and emales,
om he HI g oup compa ed o sham (p < 0.0001), and so was he HI+URB447 g oup (p <
0.01 in bo h sexes and emales; p < 0.001 in males). Diffe ences be ween he HI and he
HI+URB447 g oups we e obse ed when compa ing bo h sexes oge he (p < 0.05) and in
emales (p < 0.01), wi h highe weigh s in he ea ed g oups.
2.2. B ain Inju y Assessmen
2.2.1. Hemisphe ic and Hippocampal A ea Ra ios
Hemisphe ic a ea a ios o bo h sexes oge he showed no signs o inju y in sham
animals, as he a io alues we e close o 1 (1.01 ± 0.02). A e HI, he a eas o he ipsila e al
hemisphe es we e educed by a hal (0.50 ± 0.26), being signi ican ly lowe han he sham
a ios (p < 0.0001). The hemisphe ic a ios o he URB447- ea ed animals e ealed simila
alues o he sham g oup (0.92 ± 0.14), being signi ican ly highe han non- ea ed HI pups
(p < 0.001).
When sepa a ing da a by sex, he educ ion in he ipsila e al hemisphe e a ea was
e iden in bo h males (sham: 1.02 ± 0.02 s. HI: 0.47 ± 0.24; p < 0.0001) and emales (sham:
1.00 ± 0.02 s. HI: 0.54 ± 0.1; p < 0.01) om he HI g oup. Again, he hemisphe ic a ios o
URB447- ea ed animals (males: 0.87 ± 0.17; emales: 0.96 ± 0.10) we e close o sham, and
he e o e signi ican ly highe han HI-g oup a ios o bo h sexes (p < 0.05 o males and p
< 0.01 o emales). Resul s a e shown in Figu e 1A.
We u he calcula ed he effec o he cannabinoid s. HI depending on sex: he pe -
cen age o imp o emen o he hemisphe ic a ea a ios o males was 45.98%, and 43.75%
o emales.
Figu e 1. E ec o HI and URB447 ea men on (A) hemisphe ic and (B) hippocampal a ios om
neona al a s. Da a analysis was i s pe o med in bo h sexes oge he and hen in males and emales
sepa a ely. HI educed he hemisphe ic and hippocampal a ios, bu subsequen URB447 ea men
es o ed a ios o sham-like alues in all cases. * p< 0.05; ** p< 0.01; *** p< 0.001; **** p< 0.0001 s. HI.
Rep esen a i e low-magni ica ion pho og aphs (C) o b ain sec ions om sham, HI and HI+URB447
ea ed animals. H&E s aining.
In . J. Mol. Sci. 2024,25, 1607 4 o 15
We u he calcula ed he e ec o he cannabinoid s. HI depending on sex: he
pe cen age o imp o emen o he hemisphe ic a ea a ios o males was 45.98%, and
43.75% o emales.
In he hippocampal a ea, sham animals also e ealed a ios close o 1 (1.02
±
0.09)
when analyzing males and emales oge he , a sign o no issue loss. As p e iously obse ed
o he ipsila e al hemisphe e om non- ea ed HI pups, he hippocampal a io was g ea ly
educed (0.28
±
0.22), being signi ican ly lowe han sham (p< 0.0001). On he con a y,
he hippocampal a ios o URB447- ea ed animals we e signi ican ly highe han he HI
ones (0.82
±
0.28; p< 0.0001), e ealing ewe signs o inju y and p oducing da a simila
o sham.
When analyzing he da a o males and emales sepa a ely, a simila p o ec i e pa e n
o URB447 was ob ained. In he sham males o emales, no signs o inju y we e obse ed
(males: 1.06
±
0.10; emales: 0.98
±
0.05), whe eas HI hippocampal a ios we e lowe ed by
mo e han a hal in bo h sexes (males: 0.24
±
0.19,
p< 0.001
; emales: 0.33
±
0.26;
p< 0.001
).
URB447 main ained hippocampal a ios o sham-like alues in bo h males (0.74
±
0.33) and
emales (0.90
±
0.21), being signi ican ly highe han hose om HI in bo h sexes (
p< 0.05
o males and p< 0.0001 o emales). Resul s a e shown in Figu e 1B.
As we did wi h he hemisphe ic a ios, we also calcula ed a pe cen age o imp o emen
o he hippocampal a ea a ios, being 67.57% o males and 63.33% o emales.
2.2.2. Neu opa hological Sco e
To u he assess HI-induced b ain inju y and he po en ial neu op o ec i e e ec o
he cannabinoid URB447, we examined he samples using a semi-quan i a i e inju y sco ing
sys em. In global/ o al neu opa hological sco e, he highes damage was es ablished when
ob aining 21 poin s o he whole b ain, whe eas a egional assessmen o co ical and
hippocampal sco e may each a maximum o 9 poin s. Resul s a e shown in Figu e 2.
In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 4 o 15
Figu e 1. Effec o HI and URB447 ea men on (A) hemisphe ic and (B) hippocampal a ios om
neona al a s. Da a analysis was i s pe o med in bo h sexes oge he and hen in males and e-
males sepa a ely. HI educed he hemisphe ic and hippocampal a ios, bu subsequen URB447
ea men es o ed a ios o sham-like alues in all cases. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p <
0.0001 s. HI. Rep esen a i e low-magni ica ion pho og aphs (C) o b ain sec ions om sham, HI
and HI+URB447 ea ed animals. H&E s aining.
In he hippocampal a ea, sham animals also e ealed a ios close o 1 (1.02 ± 0.09)
when analyzing males and emales oge he , a sign o no issue loss. As p e iously ob-
se ed o he ipsila e al hemisphe e om non- ea ed HI pups, he hippocampal a io
was g ea ly educed (0.28 ± 0.22), being signi ican ly lowe han sham (p < 0.0001). On he
con a y, he hippocampal a ios o URB447- ea ed animals we e signi ican ly highe
han he HI ones (0.82 ± 0.28; p < 0.0001), e ealing ewe signs o inju y and p oducing
da a simila o sham.
When analyzing he da a o males and emales sepa a ely, a simila p o ec i e pa e n
o URB447 was ob ained. In he sham males o emales, no signs o inju y we e obse ed
(males: 1.06 ± 0.10; emales: 0.98 ± 0.05), whe eas HI hippocampal a ios we e lowe ed by
mo e han a hal in bo h sexes (males: 0.24 ± 0.19, p < 0.001; emales: 0.33 ± 0.26; p < 0.001).
URB447 main ained hippocampal a ios o sham-like alues in bo h males (0.74 ± 0.33)
and emales (0.90 ± 0.21), being signi ican ly highe han hose om HI in bo h sexes (p <
0.05 o males and p < 0.0001 o emales). Resul s a e shown in Figu e 1B.
As we did wi h he hemisphe ic a ios, we also calcula ed a pe cen age o imp o e-
men o he hippocampal a ea a ios, being 67.57% o males and 63.33% o emales.
2.2.2. Neu opa hological Sco e
To u he assess HI-induced b ain inju y and he po en ial neu op o ec i e effec o
he cannabinoid URB447, we examined he samples using a semi-quan i a i e inju y sco -
ing sys em. In global/ o al neu opa hological sco e, he highes damage was es ablished
when ob aining 21 poin s o he whole b ain, whe eas a egional assessmen o co ical
and hippocampal sco e may each a maximum o 9 poin s. Resul s a e shown in Figu e 2.
Figu e 2. Effec o HI and URB447 on neu opa hological sco e o o al b ain (A), co ex (B) and hip-
pocampus (C). Da a analysis was i s pe o med in bo h sexes oge he and hen in males and e-
males sepa a ely. Non- ea ed HI animals showed he highes sco es, whe eas subsequen URB447
ea men es o ed sco es o sham-like alues in all cases. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p <
0.0001 s. HI. Rep esen a i e mic opho og aphs o he CA1 a ea o he hippocampus om sham
Figu e 2. E ec o HI and URB447 on neu opa hological sco e o o al b ain (A), co ex (B) and
hippocampus (C). Da a analysis was i s pe o med in bo h sexes oge he and hen in males and
emales sepa a ely. Non- ea ed HI animals showed he highes sco es, whe eas subsequen URB447
ea men es o ed sco es o sham-like alues in all cases. * p< 0.05; ** p< 0.01; *** p< 0.001;
**** p< 0.0001 s.
HI. Rep esen a i e mic opho og aphs o he CA1 a ea o he hippocampus om
sham (D), HI- (E) and HI+URB447- ea ed (F) animals. H&E s aining. All images we e aken a 40
×
magni ica ion. Scale ba : 50 µm.
In . J. Mol. Sci. 2024,25, 1607 5 o 15
In he o al sco e o bo h sexes, he sham g oup ecei ed a ma k o 0, as he e was
no isible issue damage in hese samples. In con as , he HI g oup ob ained a ma k o
18.26 ±2.60
, signi ican ly highe han sham (p< 0.0001). Animals ea ed wi h URB447
showed low signs o inju y and ecei ed a sco e o 4.40
±
7.78 poin s, signi ican ly lowe
han he non- ea ed HI ones (p< 0.001) and simila o he alues ob ained o sham.
Simila ly, when s udying he o al sco es o male and emale animals sepa a ely, HI-
males (19.40
±
3.10) and HI- emales (17.00
±
6.87) ob ained o al sco es signi ican ly highe
han hose om sham animals (p< 0.001 o males and p< 0.05 o emales). Consis en
wi h p e ious da a, he bene icial e ec o URB447 was obse ed in bo h sexes, as he
sco es we e signi ican ly lowe in bo h males (6.40
±
8.67; p< 0.05 s. HI) and emales
(2.40
±
6.60;
p< 0.01 s.
HI) when compa ed o HI, and simila o sham. When e alua ing
he e ec o he ea men compa ed o HI, we ound ha emales educed hei sco es
mo e subs an ially (85.88%) han males (67.01%), al hough bo h sexes bene i ed om
he ea men .
We hen assessed he neu opa hological sco e in he main a ec ed b ain a eas a e HI:
he co ex and he hippocampus.
In he co ex, he HI g oup ecei ed a sco e o 7.47 ±1.65 poin s, signi ican ly highe
han sham (p< 0.0001, which ecei ed 0 poin s), and also han he URB447- ea ed g oup
(
p< 0.0001
, which ecei ed 1.65
±
3.31 poin s). Sham and URB447- ea ed co ical sco es
we e no di e en . When s udying he wo sexes sepa a ely, he co ical sco es o HI-males
(7.90
±
166) and o HI- emales (7.00
±
3.50) we e highe han sham (p< 0.001 o males and
p< 0.05 o emales). URB447- ea ed males and emales, howe e , ob ained signi ican ly
lowe neu opa hological sco es s. HI (males: 2.40
±
3.72; p< 0.05; emales: 0.90
±
2.85;
p< 0.01
). As we ound wi h he o al sco e, he e ec o he cannabinoid compa ed o HI in
he co ex sco es was 87.14% o emales and 69.62% o males.
Las ly, he hippocampal neu opa hological sco e o bo h sexes e ealed ha a pups
om he HI g oup had signi ican ly highe neu opa hological sco e alues (8.10
±
2.23;
p< 0.0001
) when compa ed o sham animals. Again, URB447- ea ed hippocampal sco es
we e signi ican ly lowe han he HI ones (2.00
±
3.29; p< 0.0001), wi h no di e ence
o sham.
When e alua ing males and emales sepa a ely, he same pa e n was obse ed: sham
animals showed no inju y signs in any sexes, whe eas HI animals e ealed high signs o
damage, eaching nea ly he maximum sco e in males (8.80
±
0.63; p< 0.001) and emales
(7.33
±
3.08; p< 0.05). URB447- ea ed a s, on he con a y, did no show signs o damage
in he hippocampus, wi h signi ican ly lowe sco es han he HI g oup in bo h males
(
2.80 ±3.61
;p< 0.05) and emales (0.90
±
2.85; p< 0.01), and no di e ence wi h sham pups.
Resul s om he ea ed g oup and he HI g oup we e compa ed, showing hippocampal
sco e imp o emen s in bo h emales (87.72%) and males (68.18%).
2.3. Cell P oli e a ion
We nex used he nuclea ma ke Ki67 o e alua e cell p oli e a ion.
Fi s , we analyzed he di e ences in he numbe o p oli e a ing Ki67+ cells be ween
he con ala e al and he ipsila e al den a e gy us o each g oup (i.e., Sham-C s. Sham-I;
HI-C s. HI-I; HI+URB447-C s. HI+URB447-I) o bo h sexes oge he (Figu e 3A).
In sham animals, he e we e no di e ences in cell coun s be ween he con ala e al
(
389 ±136 cells/mm2)
and ipsila e al (452
±
191 cells/mm
2
) sides. The HI g oup, in
con as , showed educed Ki67+ cells on he ipsila e al hippocampi (85
±
246 cells/mm
2
),
when compa ed o he con ala e al side (641
±
239 cells/mm
2
;p< 0.0001). In URB447-
ea ed animals, he e we e no di e ences be ween he con ala e al (478
±
111 cells/mm
2
)
and ipsila e al (384 ±197 cells/mm2) hippocampi.
In . J. Mol. Sci. 2024,25, 1607 6 o 15
In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 6 o 15
We hen compa ed he esul s o he diffe en expe imen al g oups o assess he effec
o HI and he ea men o URB447 in cell p oli e a ion om con ala e al (i.e., Sham-C s.
HI-C s. HI+URB447-C) and ipsila e al (Sham-I s. HI-I s. HI+URB447-I) hippocampi o
bo h sexes oge he .
In he con ala e al hippocampi, HI animals had highe Ki67+ cells (641 ± 239
cells/mm
2
) compa ed o sham (389 ± 136 cells/mm
2
; p < 0.01). The numbe o p oli e a ing
cells was simila in he con ala e al den a e gy i o HI and HI+URB447 g oups.
When e alua ing he ipsila e al hippocampi, HI educed he Ki67+ cells (85 ± 246
cells/mm
2
; p < 0.001 s. Sham). This dec ease in cell p oli e a ion induced by HI in he
ipsila e al hippocampi was es o ed a e URB447 ea men , showing highe Ki67+ coun s
in HI+URB447 animals (384 ± 197 cells/mm
2
; p < 0.001 s. HI) and simila i y o sham.
Figu e 3. Effec o HI and URB447 on cell p oli e a ion e ealed by Ki67+ cell coun s. Da a analysis
was i s pe o med in bo h sexes oge he and hen in males and emales sepa a ely. G aphs show
Ki67+ cells pe mm
2
in bo h sexes (A), males (B) and emales (C). * p < 0.05; ** p < 0.01; *** p < 0.001;
**** p < 0.0001 s. HI. Rep esen a i e mic opho og aphs showing Ki67 exp ession in he subg anula
zone (SGZ) o he ipsila e al hippocampi om sham (D), HI (E) and HI+URB447- ea ed (F) animals.
Ki67+ cells appea in b own. All images we e aken a 40× magni ica ion. Scale ba : 50 µm.
Nex , we compa ed he effec o sex in cell p oli e a ion in he h ee expe imen al
g oups.
In males (Figu e 3B), he numbe o p oli e a ing cells in he sham g oup was simila
in bo h hemisphe es (con ala e al: 348 ± 125; ipsila e al: 440 ± 246). In he HI g oup, ipsi-
la e al cell coun s (HI-I: 112 ± 311) e ealed a s a is ically signi ican educ ion in p oli e -
a ing cells compa ed o con ala e al (HI-C: 548 ± 150; p < 0.001). URB447- ea ed pups
e ealed no signi ican diffe ences in con ala e al (482 ± 111) and ipsila e al (300 ± 234)
Ki67+ cells.
We also compa ed he effec o HI and URB447 in cell p oli e a ion om con ala e al
(i.e., Sham-C s. HI-C s. HI+URB447-C) and ipsila e al (Sham-I s. HI-I s. HI+URB447-
I) hippocampi o males. The numbe o p oli e a ing cells was signi ican ly highe in HI
animals compa ed o sham (Sham-C: 348 ± 126 s. HI-C: 548 ± 150 cells/mm
2
; p < 0.05). The
numbe o p oli e a ing cells was simila in he con ala e al den a e gy i o HI and
HI+URB447. In he male ipsila e al hippocampi, on he con a y, we ound educed Ki67+
cells in he HI g oup compa ed o sham (Sham-I: 440 ± 246 s. HI-I: 112 ± 311 cells/mm
2
; p
Figu e 3. E ec o HI and URB447 on cell p oli e a ion e ealed by Ki67+ cell coun s. Da a analysis
was i s pe o med in bo h sexes oge he and hen in males and emales sepa a ely. G aphs show
Ki67+ cells pe mm
2
in bo h sexes (A), males (B) and emales (C). * p< 0.05; ** p< 0.01; *** p< 0.001;
**** p< 0.0001 s. HI. Rep esen a i e mic opho og aphs showing Ki67 exp ession in he subg anula
zone (SGZ) o he ipsila e al hippocampi om sham (D), HI (E) and HI+URB447- ea ed (F) animals.
Ki67+ cells appea in b own. All images we e aken a 40×magni ica ion. Scale ba : 50 µm.
We hen compa ed he esul s o he di e en expe imen al g oups o assess he e ec
o HI and he ea men o URB447 in cell p oli e a ion om con ala e al (i.e., Sham-C s.
HI-C s. HI+URB447-C) and ipsila e al (Sham-I s. HI-I s. HI+URB447-I) hippocampi o
bo h sexes oge he .
In he con ala e al hippocampi, HIanimalshad highe Ki67+cells (
641 ±239 cells/mm2)
compa ed o sham (389
±
136 cells/mm
2
;p< 0.01). The numbe o p oli e a ing cells was
simila in he con ala e al den a e gy i o HI and HI+URB447 g oups.
When e alua ing he ipsila e al hippocampi, HI educed he Ki67+ cells (85
±
246
cells/mm
2
;p< 0.001 s. Sham). This dec ease in cell p oli e a ion induced by HI in he
ipsila e al hippocampi was es o ed a e URB447 ea men , showing highe Ki67+ coun s
in HI+URB447 animals (384 ±197 cells/mm2;p< 0.001 s. HI) and simila i y o sham.
Nex , we compa ed he e ec o sex in cell p oli e a ion in he h ee expe imen-
al g oups.
In males (Figu e 3B), he numbe o p oli e a ing cells in he sham g oup was simila in
bo h hemisphe es (con ala e al: 348
±
125; ipsila e al: 440
±
246). In he HI g oup, ipsila -
e al cell coun s (HI-I: 112
±
311) e ealed a s a is ically signi ican educ ion in p oli e a ing
cells compa ed o con ala e al (HI-C: 548
±
150; p< 0.001). URB447- ea ed pups e ealed
no signi ican di e ences in con ala e al (482
±
111) and ipsila e al (
300 ±234)
Ki67+ cells.
We also compa ed he e ec o HI and URB447 in cell p oli e a ion om con ala e al
(i.e., Sham-C s. HI-C s. HI+URB447-C) and ipsila e al (Sham-I s. HI-I s. HI+URB447-I)
hippocampi o males. The numbe o p oli e a ing cells was signi ican ly highe in HI
animals compa ed o sham (Sham-C: 348
±
126 s. HI-C: 548
±
150 cells/mm
2
;p< 0.05).
The numbe o p oli e a ing cells was simila in he con ala e al den a e gy i o HI and
HI+URB447. In he male ipsila e al hippocampi, on he con a y, we ound educed Ki67+
cells in he HI g oup compa ed o sham (Sham-I: 440
±
246 s. HI-I: 112
±
311 cells/mm
2
;
In . J. Mol. Sci. 2024,25, 1607 7 o 15
p< 0.05). URB447 ea men was no able o inc ease he numbe o p oli e a ing cells in
he ipsila e al hippocampi (300 ±234 cells/mm2) when compa ed o HI.
In emales (Figu e 3C), he numbe o p oli e a ing cells in he sham g oup was simila
in bo h hemisphe es (con ala e al: 450
±
146; ipsila e al: 469
±
91). In he HI g oup,
ipsila e al cell coun s educed signi ican ly (p< 0.0001) compa ed o con ala e al. Again,
URB447- ea ed pups e ealed no signi ican di e ences in con ala e al (473
±
117) and
ipsila e al (469 ±106) Ki67+ cells.
We also compa ed he e ec o HI and URB447 in cell p oli e a ion om con ala e al
(i.e., Sham-C s. HI-C s. HI+URB447-C) and ipsila e al (Sham-I s. HI-I s. HI+URB447-I)
hippocampi o emales. HI signi ican ly educed cell p oli e a ion compa ed o sham
(Sham-I: 469
±
91 s. HI-I: 0
±
0 cells/mm
2
;p< 0.01), as i did in males. Howe e , his
educ ion in cell p oli e a ion was e e ed in URB447- ea ed emale a s, wi h Ki67+ cell
coun s (HI+URB447: 469
±
106) signi ican ly highe han non- ea ed HI pups (p< 0.001),
and simila o sham.
2.4. Quan i ica ion o Neu oblas s
We u he wan ed o explo e he possible e ec o HI and URB447 on neu ogenesis,
so we analyzed doubleco in (DCX) as a speci ic ma ke o neu oblas s in he neu ogenic
niche o he hippocampal subg anula zone (SGZ). Resul s a e shown in Figu e 4.
In . J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 8 o 15
Figu e 4. Effec o HI and URB447 on neu ogenesis e ealed by DCX+ cell coun s. Da a analysis was
i s pe o med in bo h sexes oge he and hen in males and emales sepa a ely. G aphs show DCX+
cells pe mm
2
in bo h sexes (A), males (B) and emales (C). ** p < 0.01; *** p < 0.001; **** p < 0.0001 s.
HI. Images show ep esen a i e pho og aphs o he DCX exp ession in he SGZ o he ipsila e al
hippocampi om sham (D), HI- (E) and HI+URB447- ea ed (F) animals. DCX+ cells appea ma ked
in da k b own. All images we e aken a 40× magni ica ion. Scale ba : 50 µm.
3. Discussion
In he p esen wo k, he adminis a ion o he cannabinoid URB447 a e HI in neo-
na al a s e ealed s ong neu op o ec i e and neu o es o a i e effec s, as i educed b ain
in a c and global and egional neu opa hological damage, oge he wi h inc eased cell
p oli e a ion and neu oblas numbe in he den a e gy us. We also assessed whe he sex
would in luence he he apeu ic effec o he ea men : ou esul s sugges ha he can-
nabinoid was neu op o ec i e o bo h sexes, wi h s onge neu ogenic bene i in emales.
In he las ew yea s, se e al au ho s ha e no ed ha HI migh affec males and e-
males diffe en ly [6,31–34], which p omp ed us o sepa a e ou da a by sex o e alua e a
possible dimo phic esponse in b ain damage a e HI and subsequen URB447 ea men .
Clinically, male sex is nowadays conside ed a isk ac o in pe ina al b ain inju y [23].
He e, non- ea ed HI males and emales e ealed simila pos mo em neu opa hology,
despi e demons a ing a end owa ds mo e his ological damage in males. In p eclinical
models, o he au ho s ha e epo ed simila esul s o ou s, wi h no sex diffe ences in
lesion size o issue a ophy, whe e his ological b ain damage associa ed wi h HI was
compa able ac oss sex [20,35–37]. The pa hophysiology o neona al HI is complex and no
ully unde s ood ye , and so a e he mechanisms leading o po en ial sexual diffe ences;
u he in es iga ion is needed o unde s and he basis o sexual dimo phism a e HI.
The need o no el and effec i e he apies o ea neona al hypoxic–ischemic enceph-
alopa hy is u gen . Howe e , esea ch wo ks do no usually ake in o conside a ion sex
dimo phism in hei expe imen al designing, despi e sex possibly being a c ucial ac o
when e alua ing he he apeu ic effec i eness o neu op o ec an s. We p e iously e-
po ed ha he cannabinoid URB447 helped mi iga e b ain inju y and whi e ma e demy-
elina ion ollowing neona al HI [19], so we u he wan ed o cha ac e ize sex-speci ic ou -
comes in esponse o URB447 ea men . The neu op o ec ion pa ame e s (hemisphe ic
and hippocampal a ios and global and egional neu opa hological sco es) e ealed in-
c eased bene i in emales, bu bo h ea ed males and emales eco e ed he his ological
alues obse ed o sham. Ve y ew s udies ha e e alua ed he he apeu ic effec o can-
nabinoids based on sex a e acu e b ain damage. In a auma ic b ain inju y model, ∆9-
e ahyd ocannabinol ea men impai ed mo o unc ion and inc eased le els o cy okine
in e leukin-6 in males only [38], hus sugges ing sex-speci ic cannabinoid effec s. Toge he
Figu e 4. E ec o HI and URB447 on neu ogenesis e ealed by DCX+ cell coun s. Da a analysis was
i s pe o med in bo h sexes oge he and hen in males and emales sepa a ely. G aphs show DCX+
cells pe mm
2
in bo h sexes (A), males (B) and emales (C). ** p< 0.01; *** p< 0.001;
**** p< 0.0001 s.
HI. Images show ep esen a i e pho og aphs o he DCX exp ession in he SGZ o he ipsila e al
hippocampi om sham (D), HI- (E) and HI+URB447- ea ed (F) animals. DCX+ cells appea ma ked
in da k b own. All images we e aken a 40×magni ica ion. Scale ba : 50 µm.
Fi s , we coun ed he numbe o DCX+ neu oblas s in he con ala e al and ipsila -
e al hippocampi o each g oup (i.e., Sham-C s. Sham-I; HI-C s. HI-I; HI+URB447-C s.
HI+URB447-I) o bo h sexes oge he (Figu e 4A). In sham animals, he e we e no di e ences
be ween he con ala e al (2148
±
247 cells/mm
2
) and ipsila e al (
2142 ±267 cells/mm2)
sides. The HI g oup, in con as , showed educed DCX+ cells on he ipsila e al hippocampi
(
690 ±1077 cells/mm2)
, signi ican ly lowe han he HI con ala e al (
2137 ±398 cells/mm2
;
p< 0.001). In URB447- ea ed animals, he e we e no di e ences in DCX+ cell coun s
be ween he con ala e al (2220
±
280 cells/mm
2
) and ipsila e al (2108
±
583 cells/mm
2
)
hippocampi.
We hen compa ed he esul s o he di e en expe imen al g oups o assess he e ec
o HI and he ea men o URB447 in DCX cell coun s om con ala e al (i.e., Sham-C s.
In . J. Mol. Sci. 2024,25, 1607 8 o 15
HI-C s. HI+URB447-C) and ipsila e al (Sham-I s. HI-I s. HI+URB447-I) hippocampi o
bo h sexes oge he . In he con ala e al hippocampi, we ound no signi ican di e ences
be ween any o he g oups. When e alua ing he ipsila e al hippocampi, DCX+ cells
we e only educed in HI animals (Sham: 2142
±
267 s. HI: 690
±
1077 cells/mm
2
;
p< 0.0001
). This educ ion in neu oblas s was es o ed when ea ing animals wi h URB447
(HI+URB447: 2108
±
583 cells/mm
2
), showing signi ican ly highe DCX+ cells compa ed
o HI (p< 0.0001), and simila o sham.
Nex , we compa ed he e ec s o HI and he cannabinoid ea men , sepa a ing animals
by sex.
In males (Figu e 4B), he numbe o neu oblas s in he sham g oup was simila in
bo h hemisphe es (con ala e al: 2160
±
310; ipsila e al: 2215
±
311). In he HI g oup,
ipsila e al cell coun s (HI-C: 1953
±
267) e ealed a no s a is ically signi ican educ ion
in neu oblas s compa ed o con ala e al (HI-I: 778
±
1256 cells/mm
2
). URB447- ea ed
pups e ealed no signi ican di e ences in con ala e al (2169
±
293 cells/mm
2
) and
ipsila e al (
2092 ±773 cells/mm2)
DCX+ cells. URB447- ea ed male a s did no show
highe numbe s o neu oblas s in he ipsila e al SGZ compa ed wi h HI a s.
In emales (Figu e 4C), all g oups showed simila alues o DCX+ cell coun s excep
he HI ipsila e al hippocampi, whose coun s (580
±
873 cells/mm
2
) we e signi ican ly
lowe (p< 0.01) han in he con ala e al ones (2341
±
434 cells/mm
2
). Such a educ-
ion was e e ed by URB447 ea men , showing highe DCX cell coun s (HI+URB447-I:
2126 ±304 cells/mm2) han HI animals (p< 0.01), and simila ones o sham.
3. Discussion
In he p esen wo k, he adminis a ion o he cannabinoid URB447 a e HI in neona al
a s e ealed s ong neu op o ec i e and neu o es o a i e e ec s, as i educed b ain in a c
and global and egional neu opa hological damage, oge he wi h inc eased cell p oli e -
a ion and neu oblas numbe in he den a e gy us. We also assessed whe he sex would
in luence he he apeu ic e ec o he ea men : ou esul s sugges ha he cannabinoid
was neu op o ec i e o bo h sexes, wi h s onge neu ogenic bene i in emales.
In he las ew yea s, se e al au ho s ha e no ed ha HI migh a ec males and
emales di e en ly [
6
,
31
–
34
], which p omp ed us o sepa a e ou da a by sex o e alua e a
possible dimo phic esponse in b ain damage a e HI and subsequen URB447 ea men .
Clinically, male sex is nowadays conside ed a isk ac o in pe ina al b ain inju y [
23
]. He e,
non- ea ed HI males and emales e ealed simila pos mo em neu opa hology, despi e
demons a ing a end owa ds mo e his ological damage in males. In p eclinical models,
o he au ho s ha e epo ed simila esul s o ou s, wi h no sex di e ences in lesion size o
issue a ophy, whe e his ological b ain damage associa ed wi h HI was compa able ac oss
sex [
20
,
35
–
37
]. The pa hophysiology o neona al HI is complex and no ully unde s ood
ye , and so a e he mechanisms leading o po en ial sexual di e ences; u he in es iga ion
is needed o unde s and he basis o sexual dimo phism a e HI.
The need o no el and e ec i e he apies o ea neona al hypoxic–ischemic en-
cephalopa hy is u gen . Howe e , esea ch wo ks do no usually ake in o conside a ion
sex dimo phism in hei expe imen al designing, despi e sex possibly being a c ucial ac o
when e alua ing he he apeu ic e ec i eness o neu op o ec an s. We p e iously epo ed
ha he cannabinoid URB447 helped mi iga e b ain inju y and whi e ma e demyelina ion
ollowing neona al HI [
19
], so we u he wan ed o cha ac e ize sex-speci ic ou comes in e-
sponse o URB447 ea men . The neu op o ec ion pa ame e s (hemisphe ic and hippocam-
pal a ios and global and egional neu opa hological sco es) e ealed inc eased bene i in
emales, bu bo h ea ed males and emales eco e ed he his ological alues obse ed o
sham. Ve y ew s udies ha e e alua ed he he apeu ic e ec o cannabinoids based on
sex a e acu e b ain damage. In a auma ic b ain inju y model,
∆
9- e ahyd ocannabinol
ea men impai ed mo o unc ion and inc eased le els o cy okine in e leukin-6 in males
only [
38
], hus sugges ing sex-speci ic cannabinoid e ec s. Toge he wi h he ho monal
modula ion o he ECS [
39
], di e en esponses o cannabinoid adminis a ion on males
In . J. Mol. Sci. 2024,25, 1607 9 o 15
and emales ha e been desc ibed [
40
], and also sex di e ences in CB1 and CB2 ecep o
exp ession: emale a s showed highe le els o CB1 and CB2 mRNA in he amygdala,
hypo halamus, hippocampus and co ex compa ed o males unde con ol condi ions [
41
],
he las wo being egions a ec ed by HI in ou model and e alua ed he e.
Mos o he s udies assessing he he apeu ic e ec o he ECS a e neona al b ain
inju y ha e ocused on neu op o ec ion [
19
,
42
,
43
], bu li le is known abou he po en ial
o cannabinoids as b ain- issue- epai ing he apies a e HI. Mo eo e , he neu ogenic e-
sponse o he neona al b ain a e damage is s ill no ully clea , wi h wo ks sugges ing ha
HI can enhance neu ogenesis [
44
,
45
], while o he s epo educed cell
p oli e a ion [46,47]
.
He e, we wan ed o explo e he po en ial o URB447 as a neu ogenic he apy, and
showed bo h enhanced cell p oli e a ion and an inc eased numbe o neu oblas s, hus
sugges ing a double neu op o ec i e/neu o es o a i e e ec o he cannabinoid a e HI.
Di e en in e ac ions wi h CB1 and CB2 ecep o s ha e p e iously been epo ed o mod-
ula e neu ogenesis, including he inc eased p oli e a ion o neu al p ogeni o s [
48
] and
mig a ion [
49
]. In he con ex o neona al HI, we and o he s ha e desc ibed inc eased cell
p oli e a ion in he hippocampal den a e gy us [
46
] and sub en icula zone [
50
] by CB1
agonis s 2-AG and WIN55,212-2, espec i ely. Howe e , CB1 agonism may no be neu o-
p o ec i e in ce ain ypes o neu ons (like GABAe gic) [
51
], so cau ion mus be aken when
choosing he cannabinoid o es . In his wo k, we op ed o a CB1 an agonis o disca d
possible non-desi able neu o oxic o side e ec s. Fu he , CB2 ecep o ac i a ion is also
ela ed o he enhancemen o neu ogenesis [
48
,
49
], as CB2-de icien mice showed impai ed
neu al p ogeni o p oli e a ion [
48
]. URB447 is also a CB2 agonis , so his cannabinoid may
be a p omising he apy a ge ing bo h neu op o ec ion and neu o egene a ion a e HI
wi h a single molecule. In line wi h ou esul s, A aham and collabo a o s obse ed ha
he inhibi ion o neu ogenesis was e e ed in oden s a e using a CB2 agonis [52]. CB2
exp ession le els a e highe in less-di e en ia ed cells [
53
,
54
], and CB2 ecep o inhibi ion
impai ed neu oblas mig a ion a e s oke in mice [55].
We also wan ed o es whe he sex could a ec neu ogenesis a e inju y and subse-
quen URB447 adminis a ion. In non-pa hological si ua ions, some au ho s ha e epo ed
ha males gene a e mo e new cells in he hippocampus in he i s pos na al week [
56
,
57
],
whe eas o he s ha e no ed highe p oli e a ion le els in emales [
58
,
59
]. He e, we i s
showed ha HI gene a ed a simila educ ion in cell p oli e a ion and neu oblas coun s
o bo h sexes. When e alua ing he neu ogenic e ec o he cannabinoid, emales showed
inc eased cell p oli e a ion and neu oblas coun s when compa ing o non- ea ed pups, a
esponse no obse ed o males. Du ing b ain de elopmen , sex may accoun o b ain
dimo phism [
60
–
62
], hus becoming a po en ial ac o in luencing di e en esponses o
damage and ul e io neu o es o a i e ou comes o cannabinoids o o o he ea men s.
In ou wo k, we obse ed augmen ed Ki67+ p oli e a ing cells in he con ala e al
hemisphe e o non- ea ed HI animals, a possible compensa o y mechanism o he non-
damaged hemisphe e in esponse o inju y, as p e iously epo ed [
63
]. In ou model,
animals a e exposed o sys emic hypoxia, which also a ec s he con ala e al hemisphe e.
A low oxygen concen a ions, hypoxia-inducible ac o -1
α
(HIF-1
α
) may p omo e neu al
s em cell p oli e a ion [64], which may accoun o he obse ed inc ease in Ki67+ cells.
Based on he esul s p esen ed, some s udy limi a ions should be conside ed. The
p esen wo k e ealed impo an di e ences in he esponses o males and emales o HI
and URB447 ea men , bu he pa hways in ol ed could no be epo ed by he his ological
me hods used. To be e unde s and he mechanisms ha ollow HI and he ea men wi h
he cannabinoid, u he expe imen s should be pe o med in u u e wo ks, which may
include he analysis o HIF-1
α
exp ession. Also, long- e m s udies e alua ing he e ec
o URB447 on b ain neu op o ec ion/neu o- egene a ion a e HI mus be conside ed, as
simila ly epo ed o 2-AG [46].
In conclusion, URB447 showed obus neu op o ec i e e ec s in male and emale a s
subjec ed o HI, as bo h sexes showed educed b ain in a c a eas and imp o ed o al and
egional neu opa hological sco es. No only ha , URB447 also enhanced neu ogenesis in
