ped-10-793189 Ap il 29, 2022 Time: 13:42 # 1
ORIGINAL RESEARCH
published: 28 Ap il 2022
doi: 10.3389/ ped.2022.793189
Edi ed by:
G aeme R. Polglase,
Monash Uni e si y, Aus alia
Re iewed by:
Vladimi Riljak,
Cha les Uni e si y, Czechia
Robe Galinsky,
The Ri chie Cen e, Hudson Ins i u e
o Medical Resea ch, Aus alia
*Co espondence:
Daniel Alonso-Alconada
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Neona ology,
a sec ion o he jou nal
F on ie s in Pedia ics
Recei ed: 11 Oc obe 2021
Accep ed: 04 Ap il 2022
Published: 28 Ap il 2022
Ci a ion:
Alonso-Alconada D, G essens P,
Golay X and Robe son NJ (2022)
Neu ogenesis Is Reduced a 48 h
in he Sub en icula Zone
Independen o Cell Dea h in a Pigle
Model o Pe ina al Hypoxia-Ischemia.
F on . Pedia . 10:793189.
doi: 10.3389/ ped.2022.793189
Neu ogenesis Is Reduced a 48 h in
he Sub en icula Zone Independen
o Cell Dea h in a Pigle Model o
Pe ina al Hypoxia-Ischemia
Daniel Alonso-Alconada1*, Pie e G essens2, Xa ie Golay3and Nicola J. Robe son4,5
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),
Leioa, Spain, 2Neu oDide o , Inse m, Uni e si é de Pa is, Pa is, F ance, 3Depa men o B ain Repai and Rehabili a ion,
Ins i u e o Neu ology, Uni e si y College London, London, Uni ed Kingdom, 4Ins i u e o Women’s Heal h, Uni e si y
College London, London, Uni ed Kingdom, 5Edinbu gh Neu oscience, Cen e o Clinical B ain Sciences, The Uni e si y
o Edinbu gh, Edinbu gh, Uni ed Kingdom
Cellula and issue damage igge ed a e hypoxia-ischemia (HI) can be gene alized
and a ec he neu ogenic niches p esen in he cen al ne ous sys em. As
neu o egene a ion may be c i ical o op imizing unc ional eco e y in neona al
encephalopa hy, he goal o he p esen wo k was o in es iga e he neu ogenic
esponse o HI in he neu ogenic niche o he sub en icula zone (SVZ) in he neona al
pigle . A o al o 13 la ge whi e male pigle s aged <24 h we e andomized in o wo
g oups: i) HI g oup (n= 7), animals submi ed o ansien ce eb al HI and esusci a ion;
and ii) Con ol g oup (n= 6), non-HI animals. A 48 h, pigle s we e eu hanized, and he
SVZ and i s su ounding egions, such as cauda e and pe i en icula whi e ma e , we e
analyzed o his ology using hema oxylin-eosin s aining and immunohis ochemis y by
e alua ing he p esence o clea ed caspase 3 and TUNEL posi i e cells, oge he wi h
he cell p oli e a ion/neu ogenesis ma ke s Ki67 (cell p oli e a ion), GFAP (neu al s em
cells p ocesses), Sox2 (neu al s em/p ogeni o cells), and doubleco in (DCX, a ma ke
o imma u e mig a ing neu oblas s). Hypoxic-ischemic pigle s showed a dec ease in
cellula i y in he SVZ independen o cell dea h, oge he wi h dec eased leng h o
neu al s em cells p ocesses, neu oblas chains a ea, DCX immuno eac i i y, and lowe
numbe o Ki67 + and Ki67 + Sox2 + cells. These da a sugges a educ ion in bo h
cell p oli e a ion and neu ogenesis in he SVZ o he neona al pigle , which could in u n
comp omise he eplacemen o he los neu ons and he achie emen o global epai .
Keywo ds: newbo n, neona al b ain, hypoxia-ischemia, neu ogenesis, sub en icula zone
INTRODUCTION
Du ing emb yonic de elopmen , he o ma ion o he cen al ne ous sys em (CNS) esul s om
a igh ly egula ed balance be ween he p ocesses o apop osis and neu ogenesis, which occu s
accomplished in ime and space (1,2). A he ime o bi h, howe e , he human b ain is no ye
ully de eloped [almos 2/3 o he cells a e p oduced a e bi h; (3)], so in apa um- ela ed insul s
like hypoxia-ischemia (HI) may disbalance he apop osis-neu ogenesis equilib ium, obs uc ing he
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Alonso-Alconada e al. Hypoxia-Ischemia Reduces SVZ Neu ogenesis
p ope ma u a ional p ocess and leading o li e-long sequelae (4).
Neona al HI igge s a se ies o complex and ha m ul me abolic
cascades ha lead o gene alized cellula and issue damage and
a ec nume ous g ay and whi e ma e b ain egions, bu less is
known abou he e ec o HI on he neu ogenic niches.
In he CNS, he wo neu ogenic niches ha e ain neu al
s em cells and p ogeni o s wi h egene a i e po en ial a e he
subg anula zone o he hippocampal den a e gy us and he
sub en icula zone (SVZ) o he la e al en icle (5). To da e,
con o e sy exis s a ound how HI modi ies he neu ogenic
esponse o he newbo n b ain. While some epo s indica e
ha HI supp esses he endogenous genesis o neu al s em cells
and p ogeni o s (6,7), o he s udies poin ou in he opposi e
di ec ion, sugges ing ha neu ogenesis is inc eased in he SVZ
a e HI (8,9).
Pos -inju y neu ogenesis is a complex p ocess ha can be
a ec ed by a numbe o ac o s, including he du a ion, ype,
loca ion, and in ensi y o damage (10). Fo ins ance, he SVZ has
shown o be s icken a e se e e HI (6), whe eas in mode a e
b ain inju y, neu ogenesis was s imula ed (9,11). The pe iod
o ime since he insul also modula es he neu ogenic esponse
o damage: a e an ini ial phase o dec eased cell p oli e a ion
accompanied by ex ensi e cell dea h in he oden SVZ (6,12),
he mo phology o he ipsila e al SVZ has shown o inc ease i s
size, a phenomenon a ibu ed o augmen ed cell p oli e a ion
(8,9,11). Since HI ypically occu s a a ime when hese niches
a e ac i ely gene a ing new b ain cells, we hypo hesize ha he
endogenous neu ogenic capabili y o he SVZ con ibu ing o he
plas ici y o he newbo n b ain and/o o issue emodeling could
be comp omised i his a ea is a ec ed.
As desc ibed, mos o he wo k on neu al s em cells and
p ogeni o s o he SVZ has been conduc ed in oden s. The
newbo n pigle SVZ sha es many ana omical simila i ies wi h
he SVZ in he human in an , and he SVZ pe sis s beyond
e al de elopmen se ing as a sou ce o pigle new cells (13).
The aim o his s udy was o in es iga e i HI a ec s he SVZ
o he neona al pigle by e alua ing i s possible changes in
cellula i y, cell dea h, cell p oli e a ion, and neu ogenesis ea ly
a e a quan i ied global ce eb al hypoxic-ischemic insul .
MATERIALS AND METHODS
All expe imen a ion was in acco dance wi h UK Home O ice
Guidelines [Animals (Scien i ic P ocedu es) Ac 1986] and
app o ed by he Animal Ca e and Use Commi ee o Uni e si y
College London Biological Se ices and Ins i u e o Neu ology.
Animal Expe imen s and Su gical
P epa a ion
Thi een la ge whi e male pigle s aged <24 h we e included
in his s udy. B ie ly, pigle s we e seda ed wi h in amuscula
midazolam (0.2 mg/kg), and a e ial O2sa u a ion was
moni o ed (Nonin Medical). Iso lu ane anes hesia (4% ol/ ol)
was applied ia a acemask du ing acheos omy and in uba ion
and was main ained (3% du ing su ge y, 2% o he wise).
Pigle s we e mechanically en ila ed o main ain a e ial pa ial
p essu es o O2(PaO2; 8–13 kPa) and CO2(PaCO2; 4.5–6.5 kPa)
allowing o empe a u e co ec ion o he a e ial blood sample.
An umbilical enous ca he e was inse ed o in use
main enance luids (10% dex ose, 60 ml/kg/day), en anyl (3–
6µg/kg/h), and an ibio ics (benzylpenicillin 50 mg/kg and
gen amicin 2.5 mg/kg, e e y 12 h). An umbilical a e ial ca he e
was inse ed o con inuous hea a e (HR) and mean a e ial
blood p essu e (MABP) moni o ing and 6-h blood sampling o
measu e PaO2, PaCO2, pH, elec oly es, glucose (3–10 mmol/L),
and lac a e (Abbo Labo a o ies). Bolus in usions o colloid
(Gelo usin, B B aun Medical L d.) and ino opes main ained
MABP >40 mmHg. A e ial lines we e main ained by in using
0.9% saline solu ion (Bax e , 1 ml/h) wi h hepa in sodium (1
IU/ml) o p e en line blockage. Bo h common ca o id a e ies
we e su gically isola ed a he le el o he ou h ce ical e eb a
and enci cled by emo ely con olled ascula occlude s (OC2A,
In Vi o Me ic). A e su ge y, pigle s we e posi ioned p one in a
plas ic pod wi h hei heads immobilized.
Ce eb al Hypoxia-Ischemia
Be o e insul , pigle s we e andomized in o wo g oups, using
a compu e -gene a ed andomiza ion sequence and opaque
sequen ially numbe ed en elopes. The hypoxic-ischemic (HI,
n= 7) insul was pe o med acco ding o he o iginal hypoxia-
ischemia p o ocol wi h ansien ca o id a e y occlusion and
con empo aneous hypoxia (14–16). Non-HI animals we e no
posi ioned in he scanne no we e subjec ed o hypoxia o
ischemia, se ing as con ols (Con ol, n= 6).
A magne ic esonance spec oscopy (MRS) su ace coil was
secu ed o he c anium, and he animal was posi ioned in a 9.4
Tesla Agilen MRI scanne , while in he MRI scanne , ansien
HI was induced by emo e occlusion o bo h common ca o id
a e ies, using in la able ascula occlude s, and educing he
ac ion o inspi ed oxygen (FiO2) o 12% ( ol/ ol).
Du ing HI (n= 7), ce eb al ene ge ics we e moni o ed
e e y 2 min by phospho us (31P) MRS, and he β-
nucleo ide iphospha e (β-NTP; mainly ATP) peak heigh
was au oma ically measu ed. When β-NTP peak heigh had
allen o 40% o baseline, FiO2was adjus ed in o de o
s abilize β-NTP a ha le el o 12.5 min. A he end o his
12.5-min pe iod, he occlude s we e de la ed and FiO2was
no malized; 31P spec a we e acqui ed o a u he 1 h o
moni o eco e y om HI. The ime in eg al o he dec emen
o β-NTP/EPP [EPP = exchangeable phospha e pool = ino ganic
phospha e + phosphoc ea ine + (2γ+β)-NTP] du ing HI and he
i s 1 h o esusci a ion quan i ied he acu e ene gy deple ion.
Pigle s we e main ained no mo he mic ( ec al empe a u e
38.5◦C) h oughou he en i e expe imen by using a wa med
wa e ma ess (Teco he m) abo e and below he animal.
All animals ecei ed con inuous physiological moni o ing
(SA ins umen s) and in ensi e li e suppo h oughou
expe imen a ion.
His ology and Immunohis ochemis y
A 48 h, pigle s we e eu hanized wi h pen oba bi al, and he b ain
was ixed by ca diac pe usion wi h cold 4% pa a o maldehyde,
dissec ed ou , and pos - ixed a 4◦C in 2% pa a o maldehyde
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Alonso-Alconada e al. Hypoxia-Ischemia Reduces SVZ Neu ogenesis
FIGURE 1 | Magni ied image o co onal sec ion o he SVZ a b egma –2.0 (A) and –4.0 (B) b ain le els sampled in his s udy. H&E s aining. LV, la e al en icle; SVZ,
sub en icula zone; DL-SVZ, do sola e al-SVZ; L-SVZ, la e al-SVZ. O iginal magni ica ion 40×. Scale ba : 500 µm.
o 7 days; 5-mm- hick co onal slices o he igh hemisphe e,
s a ing om an e io o he op ic chiasma, we e embedded in
pa a in, sec ioned o 5 µm hickness, and s ained wi h H&E o
alida e he b egma o analysis.
Fo each animal, 2 le els (b egma −2.0 and −4.0) we e
e alua ed. The e we e i e egions o in e es , namely, he
SVZ, which was in u n di ided in o 3 suba eas (Figu e 1):
oo , do sola e al-SVZ (DL-SVZ, close o he pe i en icula
whi e ma e ), and la e al-SVZ (L-SVZ, apposed o he cauda e
nucleus); and he cauda e nucleus and he pe i en icula whi e
ma e , bo h being SVZ-su ounding egions (Figu e 1).
Hema oxylin-Eosin S aining
Pa a in-embedded b ain samples we e s ained ollowing he
au oma ed p ocedu e co esponding o Hema oxylin-Eosin
(H&E) s aining by a Shandon Va is ain V24-4 (The mo Elec on
Co po a ion, Uni ed S a es) using Ha is hema oxylin (Shandon
Gill 2 Hema oxylin, The mo Scien i ic, Uni ed S a es) and eosin Y
(Shandon Eosin-Y Alcoholic, The mo Scien i ic, Uni ed S a es).
His ological Examina ion o Cell Dea h in
Hema oxylin-Eosin Samples
His ological changes we e e alua ed in he SVZ ( oo , DL-
SVZ, and L-SVZ), cauda e nucleus, and pe i en icula whi e
ma e b ain egions a bo h b egma −2.0 and −4.0 le els
(Supplemen a y Figu es 1, 2) by using H&E s aining and
analyzed wi h Fiji/ImageJ image so wa e. Fo each animal, le el,
and sec ion, a o al o i een non-o e lapping mic opho og aphs
(3 om each a ea) we e aken a 400 ×magni ica ion in
a ligh ield op ical mic oscope (Olympus BX50F4, Japan).
A blinded his ologis coun ed mo phologically well-p ese ed
cells (undamaged), oge he wi h cells wi h apop o ic o nec o ic
ea u es. Apop o ic-like cells we e cha ac e ized by he p esence
o nuclea ka yo hexis and low cy oplasmic change, whe eas
nec o ic cells we e iden i ied by a pykno ic nucleus o no nucleus,
along wi h a swollen, eosinophilic cy oplasm (17). We did no
coun apop o ic no nec o ic p o iles ha we e wi hin o adjacen
o blood essels o a oid including apop o ic/nec o ic endo helial
and whi e blood cells. The undamaged, apop o ic and nec o ic
cell coun was a e aged om 3 high-powe ields om 3 slides
om he same egion in each animal, and alues a e gi en as
cells pe mm2.
T ans e ase-Media ed Inco po a ion o
Digoxigenin-Labeled Nucleo ide
DNA agmen a ion was e ealed by using he e minal
deoxynucleo idyl ans e ase-media ed inco po a ion o
digoxigenin-labeled nucleo ide (TUNEL) assay (Roche, Bu gess
Hill, Uni ed Kingdom). B ie ly, in si u end-labeling o agmen ed
DNA was ca ied ou on b ain slices ha we e i s depa a ina ed,
hyd a ed, p e ea ed in 3% H2O2, and subjec ed o a p o ease-K
diges ion (P omega, Sou hamp on, Uni ed Kingdom). TUNEL
was isualized using a idin-bio inyla ed ho se adish complex
(ABC, Vec o Labo a o ies, Pe e bo ough, Uni ed Kingdom) and
diaminobenzidine/H2O2(DAB, Sigma, Poole, Uni ed Kingdom)
enhanced wi h CoSO4and NiCl2. Finally, TUNEL sec ions
we e dehyd a ed and co e -slipped wi h DPX (VWR, Leigh on
Buzza d, Uni ed Kingdom).
Caspase 3 Immunohis ochemis y
A e depa a ina ion, an igen e ie al was pe o med using a
pH 6 solu ion o 10 mM sodium ci a e + 0.05% Tween20 in
dis illed wa e whe e samples we e boiled 3 imes be o e being
kep o 20 min a 95–98◦C. A e cooling a oom empe a u e,
he endogenous pe oxidase was blocked, and samples we e
incuba ed in 5% bo ine se um albumin blocking bu e . B ain
slices we e hen incuba ed wi h p ima y an ibody abbi an i-
Caspase 3 (1:100, 9661 L, Cell Signaling, Uni ed S a es)
o e nigh . The nex day, samples we e incuba ed wi h a bio in-
conjuga ed seconda y an ibody (1:500, goa an i- abbi , 65-6140,
In i ogen, Uni ed S a es) o 1 h a oom empe a u e ollowed
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Alonso-Alconada e al. Hypoxia-Ischemia Reduces SVZ Neu ogenesis
by ho se adish pe oxidase-s ep a idin conjuga e (1:500, 43-
4323, The mo Fishe , Uni ed S a es) plus diaminobenzidine.
Righ a e , sec ions we e coun e s ained wi h hema oxylin and
moun ed wi h DPX.
An in es iga o blind o he ea men g oup pe o med he
quan i a i e analyses o caspase 3 exp ession. Fo each le el,
sec ion, and b ain egion, caspase-3 posi i e cells we e coun ed
in h ee ields (a 40 ×magni ica ion, wi h an a ea o 0.077 mm2)
and he a e age was con e ed in o coun s pe mm2.
Fluo escen Immunohis ochemis y
B ain slices o luo escence immunohis ochemis y we e
managed as de ailed in he “Immunohis ochemis y” sec ion
un il incuba ion wi h p ima y an ibody. Fo single labeling,
adial-glia/neu al s em cells we e iden i ied using an an i-glial
ib illa y acidic p o ein (GFAP) an ibody o assess neu ogenic
ac i i y close o he en icula wall (mouse an i-GFAP, 1:100,
MA5-12023, The mo Fishe , Uni ed S a es); an an i-doubleco in
(DCX) an ibody was used o iden i y young neu ons/neu oblas s
(mouse an i-DCX, 1:50, sc-271390, San a C uz Bio echnology,
Uni ed S a es); cell p oli e a ion was iden i ied by an an i-Ki67
an ibody (mouse an i-Ki67; 1:50, STJ96966, S Johns Labs,
Uni ed Kingdom). Double immunohis ochemical s aining was
used o de ec he cellula co-localiza ion o Ki67 wi h Sox2
(neu al s em/p ogeni o cells; 1:100, San a C uz Bio echnology,
Uni ed S a es). Immuno eac i i y was e ealed using Alexa
Fluo 488 and Texas Red (1:300, The mo Fishe , Uni ed S a es)
seconda y an ibodies incuba ed in he da k o 1 h a oom
empe a u e. A e inal washes, luo omoun aqueous moun ing
medium (F4680, Sigma) was added, and each sec ion was co e ed
by a co e slip. Nega i e con ols ecei ed iden ical ea men
excep o he omission o p ima y an ibodies and showed no
speci ic s aining.
His ological E alua ion o Ma ke s o
Neu ogenesis
Sec ions we e examined using Fiji/ImageJ image so wa e, and
analyses and quan i ica ions we e pe o med by wo independen
in es iga o s blinded o he ea men g oup. We ex ended he
analysis including he cell-dense band o DCX + young neu ons
along he walls o he la e al en icle le els (Supplemen a y
Figu e 3). The SVZ is u he subdi ided in o oo , do sola e al-
SVZ (DL-SVZ, close o he pe i en icula whi e ma e ), and
la e al-SVZ (L-SVZ, apposed o he cauda e nucleus). Fo each
animal, le el, and sec ion, a o al o nine non-o e lapping
mic opho og aphs (3 om each a ea) we e aken a 200 ×o
400 ×magni ica ion.
The leng h o he GFAP posi i e cells p ocesses lining he SVZ
was measu ed and a e aged using Fiji/ImageJ so wa e in h ee
sepa a e mic oscopic ields a 40 ×in he oo , DL-SVZ, and
L-SVZ a eas wi h a luo escence lase mic oscope. The so wa e
was p e iously calib a ed, and he mean leng h o he p ocesses
was ob ained a e 6 andom measu emen s (Supplemen a y
Figu e 4) o each pho og aph (18). Values a e gi en as µm.
Quan i ica ion o Ki67 + and Sox2 + Ki67 + cells
was pe o med in h ee non-adjacen ields o iew a
20 ×magni ica ion along he DL-SVZ and L-SVZ edges o
he la e al en icle (18,19). In each case, he mean o Ki67 + and
Sox2 + Ki67 + cells we e di ided by he a ea o ob ain a
measu emen o cells pe mm2.
F om sec ions s ained wi h H&E, pho omic og aphs we e
ob ained, and he a ea o neu oblas chains in he SVZ [high
densi y o cells ha ake up hema oxylin ha co esponds o
DCX, (20)] was aced and measu ed in squa e millime e s.
Fo DCX luo escen immunohis ochemical e alua ion, all
he immuno-posi i e a eas along he SVZ we e digi alized, and
whole luo escence was ob ained because o he con luence o he
exp ession (21). Neu oblas s gene a ed om ype 2 p ogeni o s
exp ess ma ke s o he neu onal lineage like DCX om hei i s
exp ession o neu onal lineage and du ing mig a ion, well be o e
hey ma u e as neu ons, so DCX hus is a good ea ly ma ke in
human e al b ain and also in animal s udies o neu al cell lineage
in neu epi helium (22).
S a is ical Analysis
A wo- ailed, unpai ed S uden ’s - es was pe o med o
compa isons; da a we e conside ed signi ican ly di e en i
p<0.05. Ba g aphs appea as mean wi h 95%CI. S a is ical
analysis was pe o med using he G aphpad P ism 8 so wa e
package (G aphPad So wa e, Inc., La Jolla, CA, Uni ed S a es).
RESULTS
Hypoxia-Ischemia Induced a Dec ease in
Cellula i y in he Neona al Pigle
Sub en icula Zone, Cauda e Nucleus,
and Pe i en icula Whi e Ma e
In he sagi al sec ion, he SVZ can be easily iden i ied using
con en ional his ological s ains as an agg ega ion o small da kly
s ained cells loca ed immedia ely adjacen o he en icles and
descending along he leng h o he do sola e al (DL, close o he
pe i en icula whi e ma e ) and la e al (L-SVZ, close o he
cauda e nucleus) walls o he la e al en icle (Figu e 1).
We i s e alua ed he cellula i y o he SVZ, cauda e nucleus,
and pe i en icula whi e ma e quan i ying he numbe o
mo phologically well-p ese ed (undamaged) cells. A e a HI
e en , he cellula i y o he SVZ o he neona al pigle was
educed. The oo (p<0.01), he L-SVZ (p<0.01), and
he DL-SVZ (p<0.05) suba eas showed signi ican ly lowe
coun s o mo phologically well-p ese ed cells a e neona al
HI (Figu e 2, uppe g aph). Quan i a i e e alua ion o he
cauda e nucleus (p<0.05) and pe i en icula whi e ma e ’s
cellula i y (p<0.001) con i med he dec ease in well-p ese ed
cells in he asphyc ic pigle in bo h SVZ-su ounding egions
(Figu e 2, lowe g aph).
The Neona al Pigle Sub en icula Zone
Is Resis an o
Hypoxia-Ischemia-Induced Cell Dea h
Al hough he SVZ appea ed swollen a 48 h o eco e y om HI,
H&E-s ained samples e ealed less dea h in his a ea, sugges ing
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Alonso-Alconada e al. Hypoxia-Ischemia Reduces SVZ Neu ogenesis
FIGURE 2 | Rep esen a i e mic opho og aphs o he sub en icula zone (SVZ), cauda e nucleus (CDT), and pe i en icula whi e ma e (P WM) b ain egions om
con ol and HI pigle s. The pigle SVZ is a highly cellula a ea wi h small and closely si ua ed o each o he ound cells (A). SVZ appea ed swollen a 48 h a e HI bu
wi h e y less o absen nec o ic o apop o ic ea u es (B). In CDT, well-dema ca ed cells (black a ows) wi h well-de ined nucleolus (seen in he inse a
630 ×magni ica ion), and dense neu opil we e obse ed in con ol pigle s (C). In con as , in HI animals (D) cells wi h nec o ic (black a owheads) and apop o ic
(whi e a ow) ea u es we e p esen (seen in he inse a 630 ×magni ica ion), wi h di use edema ous ma ix and hypodense neu opil. In P WM, black a ows iden i y
examples o mo phologically undamaged oligodend ocy es (E).(F) Shows damaged cells (whi e a ows) oge he wi h di use edema and dis up ed neu opil om an
HI-inju ed pigle . G aphs: HI educed he cellula i y o he oo (**p<0.01), L-SVZ (**p<0.01), and DL-SVZ (*p<0.05) suba eas o he SVZ, an e ec also obse ed
in CDT (*p<0.05) and P WM (***p<0.001) cell coun s. LV, la e al en icle; SVZ, Sub en icula zone; CDT, cauda e nucleus; P WM, pe i en icula whi e ma e .
H&E s aining. O iginal magni ica ion 400×. Scale ba : 100 µm.
ha hese cells a e ela i ely esis an o damage. A quan i a i e
e alua ion la e e ealed ha damaged cells (ei he wi h nec o ic
o apop o ic ea u es) we e a ely obse ed in he SVZ o con ol
(Figu e 2A) o HI g oup (Figu e 2B): alues we e e y low o
nec o ic- (0–0.8 cells pe mm2) o apop o ic-like cells (0.7–1.2
cells pe mm2) o bo h expe imen al g oups, wi h no di e ences
be ween con ol and HI in he h ee SVZ suba eas e alua ed
( oo , DL-SVZ, o L-SVZ). The absence o cell dea h in he SVZ
was u he con i med when s udying TUNEL immune-s ained
slices om bo h con ol and HI pigle s (Figu es 3A,B).
La e , we ocused on he cauda e nucleus and pe i en icula
whi e ma e . In H&E-s ained samples, we obse ed ex ensi e
cell dea h in bo h he cauda e nucleus (Figu e 2D) and
pe i en icula whi e ma e (Figu e 2F) a e HI. Cauda e
nucleus and pe i en icula whi e ma e o HI pigle s showed
inc eased pe i ascula and pe icellula space sugges ing edema.
Addi ional changes in he su ounding neu opil included
swelling o endo helial cells. Damaged cells appea ed nec o ic
in appea ance (nuclea pyknosis wi h a swollen, eosinophilic
cy oplasm) and also apop o ic ( agmen ed, ounded, dense
ch oma in wi h minimal cy oplasmic change) (Figu es 2D,F).
Quan i ica ion o cha ac e is ic mo phologically al e ed cell
nuclei and cy oplasm e ealed no o ious nec o ic and apop o ic
p ocesses in he cauda e nucleus and pe i en icula whi e
ma e a e HI: cauda e nucleus showed high coun s o nec o ic
(Con ol:2.3 ±12.6 s. HI: 99.31 ±77.4 cells pe mm2;p<0.01)
and apop o ic (Con ol:1.9 ±11.2 s. HI: 6.8 ±11.0 cells pe
mm2;p<0.002) cell dea h, and an inc ease was obse ed
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Alonso-Alconada e al. Hypoxia-Ischemia Reduces SVZ Neu ogenesis
FIGURE 3 | Rep esen a i e mic opho og aphs o TUNEL immunos aining in he SVZ, cauda e nucleus (CDT), and pe i en icula whi e ma e (P WM) b ain egions
om con ol (A,C,E) and HI (B,D,F) pigle s. SVZ om bo h expe imen al g oups showed no signs o cell dea h, as no TUNEL posi i e nuclei we e obse ed (A,B). In
CDT and P WM, howe e , immuno eac i e cells a e obse ed a e HI, especially in he o me [black a ows, (D)]. LV, la e al en icle; SVZ, sub en icula zone;
CDT, cauda e nucleus; P WM, pe i en icula whi e ma e . O iginal magni ica ion 400×. Scale ba , 100 µm.
o pe i en icula whi e ma e o nec osis (2.9 ±11.4 s.
99.5 ±52.2 cells pe mm2;p<0.05) o apop osis (2.6 ±10.4
s. 1.2 ±3.5 cells pe mm2;p<0.05). The lack o DNA
agmen a ion desc ibed abo e o he SVZ (Figu es 3C,E)
con as ed wi h he ob ious p esence in he cauda e nucleus
and pe i en icula whi e ma e a e HI (Figu es 3D,F) using
TUNEL immunohis ochemis y.
Caspase 3 Exp ession in he
Sub en icula Zone Is Main ained A e
Hypoxia-Ischemia
Immunohis ochemical s aining showed ha caspase-3 was
p esen in he SVZ o bo h con ol and HI animals (Figu e 4). In
he ependymal laye , mos o he cells appea posi i ely s ained.
Ependymal cells can be dis inguished by hei loca ion, la ge
nuclei, cilia ed apical domain, and o ganiza ion in a simple
epi helium. These cells we e no included in he coun s.
Caspase-3 + cells we e also obse ed in he zone immedia ely
subjacen o he ependymal laye o con ol and HI pigle s
(Figu es 4A,B), bu he quan i a i e analysis did no e eal
di e ences in he coun s pe mm2in he h ee SVZ suba eas
(Figu e 4, g aph). Fu he , caspase-3 + nuclei we e om non-
pykno ic cells. Cauda e nucleus and pe i en icula whi e ma e
om con ol pigle s showed a low p esence o caspase-3,
con as ing wi h ex ensi ely labeled HI samples.
Hypoxia-Ischemia Reduced he Leng h
o Glial Fib illa y Acidic P o ein + Cells
P ocesses
The p ocess leng h o GFAP + cells ( adial-glia like cells o
ype 1 o neu al s em cells) in he SVZ was measu ed in he
h ee SVZ suba eas (i.e., oo , DL-SVZ, and L-SVZ) o assess he
neu ogenic ac i i y close o he en icula wall. GFAP + cells
showed a la ge cy oplasm wi h hin p ocesses ha can ex end
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Alonso-Alconada e al. Hypoxia-Ischemia Reduces SVZ Neu ogenesis
FIGURE 4 | Rep esen a i e mic opho og aphs o Caspase-3 immunos aining in SVZ, cauda e nucleus (CDT), and pe i en icula whi e ma e (P WM) b ain egions
om con ol (A,C,E) and HI (B,D,F) pigle s. Caspase 3 posi i e cells can be obse ed in he SVZ o bo h expe imen al g oups (black a ows), including he
ependymal laye and he unde nea h SVZ (A,B). Caspase-3 immuno eac i i y was also e iden in CDT (D) and P WM (F) om HI animals. G aph: no di e ences
we e obse ed in caspase-3 posi i e cell coun s in he oo , L-SVZ, o DL-SVZ suba eas o he SVZ be ween con ol and HI pigle s. LV, la e al en icle; SVZ,
sub en icula zone; CDT, cauda e nucleus; P WM, pe i en icula whi e ma e . O iginal magni ica ion 400×. Scale ba : 100 µm.
se e al mic ome e s away om he ependyma lining he la e al
en icle owa d he inne b ain pa enchyma (Figu e 5). The
leng h o GFAP + p ocesses di e ed om each SVZ suba ea being
longe in he oo and dec easing i s size when ge ing away om
his a ea along he DL- and L-SVZs. When compa ing con ol
and HI g oups, HI signi ican ly (p<0.01 o oo and DL-SVZ;
p<0.05 o L-SVZ) educed he leng h o GFAP + p ocesses in
he h ee suba eas o he SVZ (Figu e 5, g aph).
Hypoxia-Ischemia Diminished Cell
P oli e a ion in he Sub en icula Zone
Ki67-posi i e cells we e p esen as indi idual o small clus e s
o cells in he SVZ o bo h con ol and HI pigle s, ex ending
se e al cells hick om he ependymal laye along he DL-SVZ
and L-SVZ edges o he la e al en icle (Figu e 6A). Con ol
animals showed sligh ly highe alues o Ki67 + cells in he L-SVZ
(63.89 ±19.01 cells pe mm2) han in he DL-SVZ (44.13 ±9.33
cells pe mm2). HI educed cell p oli e a ion by hal in bo h
L-SVZ (31.94 ±6.98 cells pe mm2;p<0.001 s. con ol) and
DL-SVZ (19.44 ±8.65 cells pe mm2;p<0.001 s. Con ol)
egions (Figu e 6, uppe g aph).
Consis en wi h his inding, we also obse ed a signi ican
dec ease in he numbe o Sox2 + Ki67 + cells in he same
egions a e HI. Neu al s em/p ogeni o cells (Sox2 +) appea ed
in con ol (Figu e 6C) and HI (Figu e 6D) pigle s, wi h mo e
p esence in he L-SVZ. I s p oli e a ion, de e mined by Sox2 and
Ki67 double labeling, was also a ec ed a e HI, wi h a signi ican
dec ease in he numbe o Sox2 + Ki67 + cells in bo h L-SVZ
(p<0.0001 s. con ol) and DL-SVZ (p<0.0001 s. con ol)
suba eas (Figu e 6, lowe g aph).
Neu oblas Chain A ea and Doubleco in
S aining A e Reduced A e
Hypoxia-Ischemia
Con ol animals showed an abundance o neu oblas chains
(assessed by H&E s aining; Figu es 7A,B) and DCX + neu oblas s
(immuno luo escence; Figu es 7C,D). Bo h echniques ga e us a
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Alonso-Alconada e al. Hypoxia-Ischemia Reduces SVZ Neu ogenesis
FIGURE 5 | Fluo escen mic opho og aphs o GFAP immune-s ained samples om con ol and HI pigle s. GFAP e eals he leng h o he p ocesses o
adial-glia/neu al s em cells in he SVZ (A). HI [(B) and g aph] signi ican ly educed he leng h o GFAP + p ocesses in he h ee suba eas o he SVZ: oo
(∗∗p<0.01), DL-SVZ (∗∗p<0.01), and L-SVZ (∗p<0.05). LV, la e al en icle; SVZ, sub en icula zone; CDT, cauda e nucleus; P WM, pe i en icula whi e ma e .
O iginal magni ica ion 400×. Scale ba : 100 µm.
s ong posi i e co ela ion be ween he a ea o neu oblas chains
and DCX immuno luo escence (R2= 0.6447, p<0.0001). HI
displayed a signi ican educ ion in he a ea o neu oblas chains
(H&E, p= 0.0003) and DCX immuno luo escence (p= 0.0044)
compa ed wi h con ol animals (Figu e 7, g aph).
DISCUSSION
In his s udy, hypoxic-ischemic pigle s showed a dec ease in
cellula i y in he SVZ independen o cell dea h a 48 h.
Fu he mo e, HI dec eased he leng h o GFAP + p ocesses, he
neu oblas chains a ea, DCX immuno eac i i y, and he numbe
o Ki67 + and Ki67 + Sox2 + cells, hus sugges ing ha , ea ly
a e he HI e en , a educ ion in bo h cell p oli e a ion and
neu ogenesis occu s.
Cellula and issue damage igge ed a e HI can be
gene alized and a ec he neu ogenic niches p esen in he cen al
ne ous sys em. Le ison e al. (6) desc ibed ha mode a e- o-
se e e HI educed 20% o he o al cells om he SVZ in oden s,
so we i s wan ed o explo e i HI could educe he cellula i y in
he SVZ o he newbo n pigle . The po cine SVZ is s uc u ally
simila o i s human coun e pa , made up o a c owd o small
cells closely si ua ed o each o he , adjacen o he ependyma
o he la e al en icles and ex ending owa d he inne b ain
pa enchyma. In ou model, HI educed he numbe o cells in
he pigle SVZ wi hou inc easing cell dea h, as he quan i ica ion
o nec o ic o apop o ic p o iles was minimal o absen , la e
con i med by he a e p esence o TUNEL-posi i e cells. In he
same manne , Mo on e al. (18) ound no di e ences in cell
dea h wi hin he SVZ in hypoxic b ains using a po cine model
o hypoxia-only inju y, hus indica ing ha cell dea h is no
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Alonso-Alconada e al. Hypoxia-Ischemia Reduces SVZ Neu ogenesis
FIGURE 6 | Fluo escen mic opho og aphs o Ki67 [cell p oli e a ion, (A,B)], Sox2/Ki67 [neu al s em/p ogeni o cell p oli e a ion, (C,D)], and DAPI [nuclei, (E,F)]
immune-s ained samples om con ol and HI pigle s. Ki67 posi i e cells (g een) a e abundan in he con ol pigle (A), whe eas HI (B) signi ican ly educed i s coun s
by hal in L-SVZ and DL-SVZ (bo h ∗∗∗p<0.001). Double Sox2 ( ed) + /Ki67 (g een) + cells a e obse ed in con ol animals; again, HI educed neu al
s em/p ogeni o cell coun s in L-SVZ and DL-SVZ (bo h ∗∗∗p<0.001). DAPI immunos aining e eals o al nuclei. LV, la e al en icle; SVZ, sub en icula zone; CDT,
cauda e nucleus; P WM, pe i en icula whi e ma e . O iginal magni ica ion 400×. Scale ba : 100 µm.
a majo unde lying mechanism in he educ ion o cellula i y
in he pigle SVZ.
Simila ly, he oden SVZ became swollen du ing eco e y
om pe ina al HI (as seen he e), bu ew o hose cells died (7,
23), wi h cell dea h alues o app oxima ely 0.3% o he o al
SVZ cells. The same au ho s also e alua ed caspase 3 ac i a ion,
desc ibing no di e ences in he pe cen age o ac i e caspase-3-
posi i e cells in ei he he ipsila e al o con ala e al SVZs a e HI
in oden s (7,23). In ou wo k, con ol animals showed abundan
immuno eac i i y o caspase-3 in he SVZ neu ogenic niche,
bu HI did no a ec he cell coun s. Indeed, caspase-3 posi i e
nuclei we e obse ed in non-pykno ic cells, so we canno ule
ou a possible non-apop o ic ole o his enzyme (24,25) in
he pigle SVZ. Despi e he mos ecognized ole o caspase-3,
i.e., i s capaci y o inducing DNA agmen a ion, i s ac i a ion
modula es a numbe o biological p ocesses ha do no cause cell
dea h, including dend i ic p uning, cell di e en ia ion, and cell
p oli e a ion (26,27).
Whe eas he pigle SVZ could be a b ain egion esilien
o HI-induced cell dea h, SVZ-su ounding egions like he
pe i en icula whi e ma e and cauda e nucleus appea ed
ulne able o HI. As p e iously epo ed by ou g oup in
pigle s (16) and by o he au ho s in oden s (23), in his s udy
we show cellula degene a i e changes in bo h b ain egions
a 48 h a e HI, wi h low coun s o mo phologically well-
p ese ed cells and high alues o apop o ic and nec o ic p o iles,
con i med wi h immunohis ochemical echniques. Unlike
“ma u e issues” like he cauda e nucleus o pe i en icula
whi e ma e , hose con aining neu onal p ecu so s like
he SVZ ha e hei pa icula a chi ec u e, unc ions, and
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