RESEARCH ARTICLE
Mic oglial angio ensin ype 2 ecep o s media e sex-speci ic
exp ession o in lamma o y cy okines independen ly
o ci cula ing es ogen
Pablo Ga ido-Gil
1,2
| Ma ia A. Ped osa
1,2
| Ma ia Ga cia-Ga o e
1,2
|
Ana Pequeño-Val ie a
3
| Jo ge Rod íguez-Cas o
3
| Daniel Ga cía-Sou o
3
|
Ana I. Rod íguez-Pé ez
1,2
| Jose L. Labandei a-Ga cia
1,2
1
Labo a o y o Cellula and Molecula
Neu obiology o Pa kinson's disease, Resea ch
Cen e o Resea ch in Molecula Medicine
and Ch onic Diseases (CIMUS), Depa men o
Mo phological Sciences, IDIS, Uni e si y o
San iago de Compos ela, San iago de
Compos ela, Spain
2
Ne wo king Resea ch Cen e on
Neu odegene a i e Diseases (Cibe Ned),
Mad id, Spain
3
Labo a o y o Genomes and Disease, Cen e
o Resea ch in Molecula Medicine and
Ch onic Diseases (CIMUS), Uni e si y o
San iago de Compos ela, San iago de
Compos ela, Spain
Co espondence
Jose L. Labandei a-Ga cia and Ana
I. Rod íguez-Pé ez, Depa men o
Mo phological Sciences, CIMUS, Uni e si y o
San iago de Compos ela, 15782 San iago de
Compos ela, Spain.
Email: joseluis.labandei [email protected] and anai.
[email p o ec ed]
Funding in o ma ion
Conselle ía de Cul u a, Educaci
on e
O denaci
on Uni e si a ia, Xun a de Galicia,
G an /Awa d Numbe s: XUGA, ED431C
2018/10, ED431G/05; Ins i u o de Salud
Ca los III, G an /Awa d Numbe s: PI20/00385,
RD16/0011/0016, CIBERNED; Sec e a ía de
Es ado de In es igaci
on, Desa ollo e
Inno aci
on, G an /Awa d Numbe :
RTI2018-098830-B-I00; Regional Eu opean
De elopmen Fund (FEDER)
Abs ac
The e a e sex di e ences in mic oglia, which can main ain sex- ela ed gene
exp ession and unc ional di e ences in he absence o ci cula ing sex s e oids. The
angio ensin ype 2 (AT2) ecep o s media e an i-in lamma o y ac ions in di e en is-
sues, including b ain. In mice, we pe o med RT-PCR analysis o mic oglia isola ed
om adul b ains and RNA scope in si u hyb idiza ion om males, emales, o a iec o-
mized emales, o chiec omized males and b ain masculinized emales. We also com-
pa ed wild ype and AT2 knockou mice. The exp ession o AT2 ecep o s in
mic oglial cells showed sex di e ences wi h much highe AT2 mRNA exp ession in
emales han in males, and his was no dependen on ci cula ing gonadal ho mones,
as obse ed using o a iec omized emales, b ain masculinized emales and o chiec o-
mized males. These esul s sugges genomic easons, possibly ela ed o sex ch omo-
some complemen , o sex di e ences in AT2 exp ession in mic oglia, as he AT2
ecep o gene is loca ed in he X ch omosome. Fu he mo e, sex di e ences in
exp ession o AT2 ecep o s we e associa ed o sex di e ences in mic oglial exp es-
sion o key an i-in lamma o y cy okines such as in e leukin-10 and p o-in lamma o y
cy okines such as in e leukin-1βand in e leukin-6. In conclusion, sex di e ences in
mic oglial AT2 ecep o exp ession appea as a majo ac o con ibu ing o sex di -
e ences in he neu oin lamma o y esponses beyond he e ec s o ci cula ing
s e oids.
KEYWORDS
AT2 ecep o , gende , gonadal ho mones, in e leukins, neu oin lamma ion, sex ch omosome
complemen , sex dimo phism
Pablo Ga ido-Gil and Ma ia A. Ped osa con ibu ed equally o expe imen s.
Recei ed: 2 Ap il 2022 Re ised: 17 July 2022 Accep ed: 25 July 2022
DOI: 10.1002/glia.24255
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-NoDe i s License, which pe mi s use and dis ibu ion in any
medium, p o ided he o iginal wo k is p ope ly ci ed, he use is non-comme cial and no modi ica ions o adap a ions a e made.
© 2022 The Au ho s. GLIA published by Wiley Pe iodicals LLC.
2348 Glia. 2022;70:2348–2360.
wileyonlinelib a y.com/jou nal/glia
10981136, 2022, 12, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/glia.24255 by Conso cio In e uni e si a io Do Sis ema Uni e si a io De Galicia (Cisug), Wiley Online Lib a y on [03/01/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
1|INTRODUCTION
I is now known ha sex modi ies he ou come o many neu ological dis-
eases and pa icula ly hose ela ed o neu oin lamma ion (Mazu e &
Swendsen, 2016; Villapol e al., 2017). Se e al s udies ha e shown ha
mic oglial cells show sex di e ences, and his may play a majo ole in sex
di e ences in disease ou come (Han e al., 2021;Ke e al.,2019). Ini ially,
sex di e ences we e ela ed o e ec s o ci cula ing gonadal ho mones,
pa icula ly on mic oglial in lammasome (Habib & Beye , 2015). Mic oglial
cells espond o in i o ea men s wi h gonadal s e oids and changes in
neu oin lamma o y o neu odegene a i e esponses we e obse ed a e
o a iec omies (Acos a-Ma inez, 2020; Rod iguez-Pe ez e al., 2011,
2012; VanRyzin e al., 2019). Howe e , a se ies o ecen s udies ha e
e ealed ha o he ac o s, mo e di icul o iden i y in classic in i o
s udies, may also de e mine sex di e ences in mic oglia. Mic oglia can
main ain sex- ela ed gene exp ession and unc ional di e ences in he
absence o ci cula ing sex s e oids (Bo d e al., 2020; Villa e al., 2018). I
has been sugges ed ha ho mones mayac du ing e yea lys ageso
b ain de elopmen , leading o pe manen sex- ela ed changes in mic oglia
(Hanamsaga e al., 2017; Villa e al., 2016). Recen s udies also sugges
he p ima y ac ion o genes loca ed in he sex ch omosomes, so ha ho -
mones may be conside ed as he mos impo an seconda y ac o down-
s eam o p ima y e ec s o sex ch omosomes (A nold, 2020). Incomple e
X ch omosome inac i a ion may lead o double exp ession o some genes
leading o sex di e ences in he ela ed unc ions (Sy e &
Angue a, 2019; Tukiainen e al., 2017), including in lamma o y esponses
(Qi e al., 2021). Mos o he escape genes lie in he Xq egion, which is he
olde e olu iona y egion o he ch omosome (Ross e al., 2005).
The angio ensin ype 2 (AT2) ecep o s media e an i-in lamma o y
ac ions in di e en issues, including b ain (Bha e al., 2019; Fa ima
e al., 2021). In he b ain, AT2 ecep o s ha e been obse ed in neu ons
and glial cells o oden s, monkeys and humans (Ga ido-Gil e al., 2013,
2017), and p omo e neu op o ec i e and an i-in lamma o y e ec s,
inducing a shi om p o-in lamma o y o an i-in lamma o y mic oglial
pheno ypes (Jackson e al., 2020;Rod iguez-Pe eze al.,2020). In e -
es ingly, AT2 ecep o s we e ound o e exp essed in se e al mu ine
emale issues ela i e o males (Ga ido-Gil e al., 2021;Okumu a
e al., 2005; Rod iguez-Pe ez e al., 2011), and he AT2 ecep o gene is
in he X ch omosome, and pa icula ly in he Xq23 egion (de Gaspa o
e al., 2000). The e o e, bo h ho monal and sex ch omosome comple-
men e ec s may be esponsible o p ima y sex di e ences in AT2
exp ession and di e ences in mic oglial esponses.
In he p esen s udy, we isola ed mic oglia om adul heal hy
mouse b ains o males, emales, o a iec omized (OVX) emales, mas-
culinized emales and o chiec omized males, and om bo h wild ype
(WT) and AT2 knockou (KO) mice. In mic oglial cells om he di e -
en ho monal condi ions, we s udied he AT2 ecep o exp ession and
he exp ession o se e al in e leukins (IL) ha ha e p e iously been
shown o be ela ed wi h he AT2 ecep o ac i i y (IL-10, IL-6, and
IL-1β). The subs an ia nig a has he highes le els o mic oglia in
heal hy mouse b ains (Lawson e al., 1990), and was used o u he
con i m he p esence o AT2 ecep o s in mic oglial cells using RNA
scope in si u hyb idiza ion.
2|METHODS
2.1 |Expe imen al design
Young adul (2–4-mon h-old) male and emale wild- ype (WT) and
AT2 ecep o knockou (AT2 KO; gene ous gi o D . Daniel Hen ion)
C57/BL6 mice we e used. Animals we e housed in condi ions o con-
s an oom empe a u e (21–22C) and a 12:12 h ligh –da k cycle and
gi en ee access o ood and wa e . All expe imen s we e ca ied ou
in acco dance wi h he Eu opean Di ec i e 2010/63/EU and he
Spanish RD/53/2013 and we e app o ed by he co esponding E hics
Commi ee a he Uni e si y o San iago de Compos ela.
A i s se ies o expe imen s we e pe o med in WT mice. In a
g oup o mice, adul mic oglial cells we e isola ed om b ains o male
(n=14), emale (n=14) and o a iec omized (OVX)- emale (n=14)
WT mice and we e used o s udy he e ec o sex and es ogen on he
AT2 ecep o and an i-in lamma o y (IL-10) and p o-in lamma o y (IL-
1β, IL-6) cy okine mRNA exp ession. Two mouse b ains we e used o
ob ain one sample o mic oglial cells. A second g oup o male (n=8)
and emale (n=8) WT mice we e quickly pe used wi h ice-cold saline
and b ains ex ac ed and p ocessed o mic oglial isola ion, as con ols
o exclude any con amina ion by ci cula ing blood cells. An addi ional
g oup o male (n=10) and emale (n=10) WT mice we e included o
obse e he mRNA exp ession o AT2 ecep o s in mic oglial cells in
b ain issue sec ions con aining he subs an ia nig a by RNA scope in
si u hyb idiza ion (bo h double in si u o immuno/in si u labeling).
In a second se ies o expe imen s, adul mic oglia we e isola ed
om male AT2 KO mice (n=14), emale AT2 KO mice (n=14) and
o a iec omized emale AT2 KO mice (n=14) o e alua e he e ec o
AT2 ecep o on sex- and es ogen- ela ed di e ences in p o- and
an i-in lamma o y cy okine exp ession.
A hi d se ies o mice we e used o obse e he esponse o
abo e men ioned IL o a p o-in lamma o y s imulus in he p esence
and absence o AT2 ecep o s: WT emale mice (n=4) and AT2 KO
emale mice (n=4) we e ea ed wi h LPS o ehicle (n=4) and sac i-
iced 24 h la e o mic oglia ex ac ion.
In addi ional se ies o mice, o he possible mechanisms in ol ed
in sex di e ences obse ed in mic oglial AT2 ecep o exp ession
we e in es iga ed. Fi s , male mice we e bila e ally o chiec omized
(n=6) o analyze possible e ec s o male gonadal ho mones. Second,
emale pups (n=11) we e subjec ed o b ain masculiniza ion using
subcu aneous injec ions o es adiol benzoa e. Finally, mic oglial cells
isola ed om wild ype emales (n=3) and males (n=3) we e used o
s udy he possibili y o obse ing sex di e ences in he me hyla ion
s a us in he p omo e o he AT2 gene (Ag 2).
2.2 |Es ogen deple ion by o a iec omy and ELISA
analysis
Female mice we e bila e ally o a iec omized h ough a do sal incision
as p e iously desc ibed (Ga ido-Gil e al., 2021). The anes he ized
animal was placed in he p one posi ion wi h he ail acing he
GARRIDO-GIL ET AL.2349
10981136, 2022, 12, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/glia.24255 by Conso cio In e uni e si a io Do Sis ema Uni e si a io De Galicia (Cisug), Wiley Online Lib a y on [03/01/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
su geon and he do sal su gical a ea was sha ed and cleaned wi h
po idone iodine. A skin incision was made a he le el o he midline
be ween he caudal edge o he ib cage and he base o he ail. Then,
an incision (bila e al o he spinal column) was made in he muscle
wall. The o a ies we e hen loca ed and emo ed ou side. A s e ile silk
liga ion was placed a ound he o a y, and i was emo ed. Finally, he
muscle wall was closed wi h s i ches, and he skin wi h s aples. The
animals we e injec ed wi h a ipamezole o p omo e hei eco e y,
and wi h bup eno phine as an analgesic. Mic oglia was isola ed
4 weeks a e he ope a ion.
17β-es adiol le els we e quan i ied (pg/ml) in emale (OVX, and
non-OVX) and male mouse se um using a speci ic enzyme-linked
immunoso ben assay (ab108667) om Abcam, ollowing he manu-
ac u e 's ins uc ions. B ie ly, 17β-es adiol was added o s anda d,
con ols, and samples in he espec i e 96-well mic o i e pla es sup-
plied by manu ac u e and incuba ed o 2 h a 37C. When incuba-
ion was comple ed, he wells we e washed, and TMB subs a e
solu ion was added and incuba ed a oom empe a u e in he da k.
A e ha , s op solu ion was added and he abso bance o he sam-
ples was ead a 450 nm in an In ini e M200 mul iwell pla e eade
(TECAN).
2.3 |B ain masculiniza ion, male o chiec omy, and
LPS ea men
B ain masculiniza ion was ca ied ou by subcu aneous injec ions o
5mgo 17β-es adiol benzoa e (E8515, Sigma) dissol ed in 50 ml o
s e ile sun lowe oil. Male and emale pups (n=11) we e injec ed a
days P2, P5, and P8 (Villa e al., 2018). The e ec i eness o he in e -
en ion was es ed by mic oscopic e alua ion o he ypes o cells
p esen in aginal smea s, a common p ocedu e used o documen he
s ages o he es ous cycle (Co a e al., 2015). The masculinized
emales we e unable o cycle a 8 weeks o age.
Male mice we e bila e ally o chiec omized (n=6) h ough a small
incision a he ip o he sc o um. Anes he ized animals we e immobilized,
sha ed, and disin ec ed. The unic was opened, and he es is and epidid-
ymis we e ex e io ized. A e placing a s e ile silk liga ion a ound he cau-
dal zone o he spe ma ic co d, he es is and epididymis we e emo ed,
and he emaining issue e u ned in o he sac. The p ocedu e was
epea ed wi h he o he es is. Finally, he muscle wall was closed wi h
s i ches, and he skin wi h s aples. In sham o chiec omized animals (n=6)
sc o al sac was opened wi hou emo ing he es is. Finally, he incision
was closed, and su u ed. Animals we e allowed o eco e o 2 weeks
be o e s udy. Tes os e one le els we e quan i ied in sham (i.e., con ols,
n=6) and in o chiec omized mice (n=6) se um using a speci ic compe i-
i e enzyme-linked immunoso ben assay (ADI-900-065 om Enzo), ol-
lowing s ic ly he manu ac u e 's ins uc ions. Op ical densi y was
measu ed a 405 nm (wi h co ec ion a 570 nm) using an In ini e M200
mul iwell pla e eade (TECAN; 2.0 so wa e) and es os e one concen-
a ions we e quan i ied using a speci ic s anda d cu e (4PL cu e i ).
Tes os e one le els we e 4.393 ng/ml ± 0.404 (SEM) in sham-ope a ed
mice and 0.513 ng/ml ± 0.0289 (SEM) in o chiec omized mice.
To obse e he esponse o he LPS p o-in lamma o y s imulus in
he p esence and absence o AT2 ecep o s, WT emale mice (n=4)
and AT2 KO emale mice (n=4) we e ea ed wi h LPS (lipopolysac-
cha ide; 5 mg/kg i.p.; Sigma) and sac i iced 24 h la e , as p e iously
desc ibed (Rod iguez-Pe ez e al., 2020). The con ol g oup we e WT
emale mice ea ed wi h s e ile saline solu ion (n=4).
Finally, mic oglial cells isola ed om a g oup o male (n=3) and
emale (n=3) mice we e used o s udy possible sex di e ences in he
me hyla ion s a us in he p omo e o he AT2 gene (Ag 2).
2.4 |Adul mic oglia isola ion
Isola ion o adul mic oglia om mouse b ains was pe o med as p e-
iously desc ibed (Rod iguez-Pe ez e al., 2020) wi h sligh modi ica-
ions. Two mouse b ains we e used o ob ain one sample o mic oglial
cells. B ains om pe used (ice-cold saline; 0.9% in DEPC wa e ) o
non-pe used mice we e emo ed and washed in ee-se um DMEM/
F12 wi h penicillin/s ep omycin and glucose. Then, each b ain hemi-
sphe e was ully minced wi h a scalpel in o small pieces (app oxima e
1mm
2
), epea edly washed o emo e blood cells om essels, ans-
e ed o a Dounce homogenize con aining 5 ml o he enzyma ic mix
(1 mg/ml papain, 6 U/ml dispase and 20 U/ml DNase in a DMEM/F12
solu ion wi h 12.5 mM HEPES and 37.5 mM NaCl) and dissocia ed on
ice wi h 10–12 passes using he loose- i ing pes le. Dissocia ed is-
sue was ans e ed o a 50 ml conical ube and he enzyma ic diges-
ion was pe o med a 37C o 30 min. Then, cell suspension was
washed wi h 10 ml o ice-cold Hanks' balanced sal solu ion (HBSS),
il e ed (100 μm cell s aine ) and cen i uged (300 g a 4C o 5 min).
The pelle was esuspended in 8 ml o 35% Pe coll a RT and ans-
e ed o a clean conical ube. A e o e laying wi h 5 ml o HBSS,
each sample was incuba ed o 5 min on ice, and cen i uged (800 g a
4C o 20 min) wi hou b aking. Then, he cellula pelle was washed
wi h HBSS, il e (30 μm), cen i uged (400 g a 4C o 5 min), and
subsequen ly, esuspended in 90 μl o sepa a ion bu e (PBS wi h
0.5% bo ine se um albumin and 2 mM EDTA), and was ans e ed o
a 15 ml conical ube. Fo mic oglia isola ion, 10 μl o CD11b
(Mic oglia) Mic oBeads (130-093-634, MACS Mil enyi Bio ec) we e
added, and hen, he cell-bead suspension was incuba ed (15 min a
4C) unde agi a ion, washed, esuspended in 500 μl o sepa a ion
bu e and applied on o MS columns (130-042-210, MACS Mil enyi
Bio ec) acco ding o manu ac u e ins uc ions.
2.5 |Flow cy ome y analysis
Mic oglial cells isola ed by magne ic so ing we e incuba ed (15 min
a 4C) unde agi a ion wi h monoclonal an ibodies agains CD11b
(PE-conjuga ed: eBioscience 12-0112, 1:170), CD45 (FITC-conju-
ga ed: eBioscience 11-0451, 1:200) o iso ype con ols (eBioscience
12-4331, 1:50, and 11-4331, 1:100). Then, cells we e insed in 2 ml
ice-cold PBS, pelle ed a 300 g o 10 min a 4C and esuspended in
300 μl o sepa a ion bu e . To disc imina e be ween li ing and dead
2350 GARRIDO-GIL ET AL.
10981136, 2022, 12, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/glia.24255 by Conso cio In e uni e si a io Do Sis ema Uni e si a io De Galicia (Cisug), Wiley Online Lib a y on [03/01/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
cells, cells we e incuba ed wi h DAPI 10 min be o e low cy ome y.
Cells we e analyzed on an Accu i C6 low cy ome e (BD Biosciences).
Fo each s aining condi ion (cells only, iso ype con ol, and an ibody
o in e es ), a leas 1 10
4
e en s we e collec ed and hen analyzed
using a FlowJo 10 so wa e (BD Biosciences). Mic oglia pu i y was
de ined as he pe cen age o all li ing CD11b+cells ha showed
CD45 low exp ession. Posi i e labeled cells we e de e mined by se -
ing h eshold on backg ound s aining o iso ype con ol.
2.6 |Real ime quan i a i e PCR
Fi s ly, o al RNA ex ac ion was pe o med wi h he RNeasy miniki
(Qiagen) p o ocol and hen, he RNA was e e se ansc ibed o com-
plemen a y DNA using he high-capaci y RNA- o-cDNA ki (4387406,
Applied Biosys ems). Subsequen ly, he eal ime qPCR analysis was
pe o med wi h a Quan S udio3 pla o m (Applied Biosys ems) and
he E aG een qPCR Mas e Mix (Applied Biological Ma e ials Inc.).
β-ac in was used as housekeeping gene and was ampli ied in pa allel
wi h he genes o in e es . The ela i e le els o mRNA we e e alu-
a ed by he del a C me hod (2
ΔΔC
), whe e C is he cycle h eshold.
Exp ession o each gene was ob ained as ela i e o ha o he house-
keeping ansc ip s. Fo wa d (F) and e e se (R) p ime s sequences,
designed using Beacon Designe so wa e (P emie Bioso ), we e as
ollows: o β-ac in, F 50-TCGTGCGTGACATTAAAGAG-30,R5
0-TGC
CACAGGATTCCATACC-30; o AT2, F 50-TGTGGTCTCACTGTTTT
GTTGTCA-30,R5
0-TTGCCCAGAGAGGGAGGGTA-30; o IL-1β,
F5
0- CCGTGTCTTCCTAAAGTATGG-30,R5
0-GTTTCTTGTGACCCTG
AGC-30; o IL-6, F 50-GACTGATGCTGGTGACAAC-30,R5
0-GAGT
GGTATCCTCTGTGAAGT-30; and o IL-10, F 50-CATACTGCTAACCG
ACTC-30,R5
0-AATGCTCCTTGATTTCTGG-30.
2.7 |RNA scope in si u hyb idiza ion
B ains om adul mice we e apidly emo ed, embedded in OCT,
ozen wi h liquid ni ogen cooled-isopen ane, and s o ed a 80C.
Then, co onal sec ions (15 μm hick) con aining he subs an ia nig a
we e cu a 20C wi h a c yos a (The mo Scien i ic), moun ed on
glass slides (Supe os Plus, The mo Fishe Scien i ic), and s o ed a
80C un il use. Tissue collec ion and sec ioning we e pe o med in
RNase ee condi ions.
Fo in si u hyb idiza ion, slides con aining nig al issue sec ions
we e p ocessed wi h he RNAscope Fluo escen Mul iplex Assay using
he RNAscope
®
Fluo escen Mul iplex Reagen Ki (320293-USM;
Ad anced Cell Diagnos ics, ACD, Newa k, CA), acco ding o he manu-
ac u e 's ins uc ions. Tissue sec ions we e ixed wi h 4% pa a o mal-
dehyde o 30 min a 4C, dehyd a ed in g aded e hanol (50%, 70%,
and 100% o 5 min each s ep) and incuba ed wi h P o ease IV
(322340, ACD) o 30 min a RT. Then, hyb idiza ion was pe o med
in o he ACD HybEZ™II o en (321720, AC) a 40C o 2 h using he
ollowing a ge p obes: Mus musculus angio ensin II ecep o ype
2mRNA(Ag 2 p obe; accession numbe NM_007429.5, a ge egion
397-1697; 406471-C2, ACD) and Mus musculus in eg in alpha M (I gam),
ansc ip a ian 1, mRNA (accession numbe NM_001082960.1, a ge
egion 538-1528; 311491 -C3, ACD), as a mic oglial ma ke . In addi ion,
RNAscope 3-plex Nega i e Con ol P obe_Mm (320871, ACD) and RNA-
scope 3plex Posi i e Con ol P obe_Mm (320881, ACD) we e used as
con ol p obes. A e p obes hyb idiza ion, he issue sec ions we e
sequen ially incuba ed in o he ACD HybEZ™II o en a 40C wi h ampli-
ica ion eagen s (De ec ion Reagen s ki , 320851, ACD): AMP-1 o
30 min, AMP-2 o 15 min, AMP-3 o 30 min and AMP-4 C o 15 min,
which assigned he di e en luo opho es o each channel: Ag 2 p obe
(channel 2; luo opho e: A o 647), I gam (channel 3; Alexa 488). Then, is-
sue sec ions we e incuba ed wi h DAPI (320858, ACD) o 30 s a RT and
immedia ely co e slipped wi h Fluo omoun -G™Moun ing Medium
(00-4958-02, In i ogen).
2.8 |RNA scope in si u hyb idiza ion combined
wi h immunohis ochemis y
The combina ion o he RNA scope in si u hyb idiza ion echnique
and he immuno luo escence echnique was pe o med as p e iously
desc ibed (Lan anco e al., 2017; Villapol e al., 2017), wi h sligh
modi ica ions. Fo issue p epa a ion, mice we e i s ansca dially
pe used wi h 0.9% saline and hen wi h cold 4% pa a o maldehyde in
0.1 M PBS (pH 7.4). The b ains we e emo ed, washed and c yop o-
ec ed in PBS con aining 20% suc ose (24 h a 4C). B ains we e
embedded in OCT and ozen wi h liquid ni ogen cooled-isopen ane.
Then, co onal issue sec ions (15 μm hick) we e cu wi h a c yos a
(The mo Scien i ic), moun ed on glass slides (Ep edia™Polysine Adhe-
sion Slides, The mo Fishe Scien i ic), and s o ed a 80C un il use.
Fluo escen in si u hyb idiza ion was pe o med using RNA
scope
®
Technology 2.5 Red luo escen ki o ixed issue (Ad anced
Cell Diagnos ics, ACD; Haywa d, CA), acco ding o he manu ac u e 's
ins uc ions. B ie ly, issue sec ions we e incuba ed (30 min a 60C)
in a HybEZTM II o en (321720, AC), p e ea ed (10 min a RT) wi h a
H
2
O
2
solu ion (322335, ACD) and boiled (5 min) wi h RNA scope Ta -
ge Re ie al (322001, ACD). Then issue sec ions we e washed in
100% e hanol and incuba ed wi h P o ease Plus (322331, ACD) o
30 min a 40C. In si u hyb idiza ion was pe o med using a Mus mus-
culus angio ensin II ecep o ype 2 mRNA p obe (Ag 2; accession
numbe NM_007429.5, a ge egion 397-1697; 403991-C1, ACD)
o 2 h a 40C. Then, sequen ial ampli ica ion s eps we e pe o med
a 40C (De ec ion Reagen -RED ki , 322360, ACD). Finally, issue
sec ions we e incuba ed wi h a de ec ion solu ion con aining a mix-
u e a io o Red-A o Red-B o 1:60. Posi i e hyb idiza ion consis ed
o a punc a e signal in he a - ed spec um (Exci a ion: 647 nm; Emis-
sion: 657–727 nm) ep esen ing a single mRNA a ge molecule.
Following in si u hyb idiza ion, issue sec ions we e p ocessed o
immuno luo escence o he mic oglial ma ke Iba1. In sho , pos -
hyb idized sec ions we e i s blocked wi h 5% no mal donkey se um
(Sigma) in PBS- ween o 1 h a RT and hen incuba ed o e nigh a
4C wi h a abbi polyclonal an ibody agains Iba-1 (1:100;
019-19741, Wako). The immuno eac ion was isualized wi h an Alexa
GARRIDO-GIL ET AL.2351
10981136, 2022, 12, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/glia.24255 by Conso cio In e uni e si a io Do Sis ema Uni e si a io De Galicia (Cisug), Wiley Online Lib a y on [03/01/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
luo 488-conjuga ed donkey an i- abbi IgG (1:200, 2 h a RT; Molec-
ula P obes, Eugene, OR). Finally, issue sec ions we e incuba ed wi h
DAPI (320858, ACD) o 30 s a RT and immedia ely co e slipped
wi h Fluo omoun -G™Moun ing Medium (00-4958-02, In i ogen).
2.9 |Images acquisi ion and quan i a i e analysis
Tissue sec ions p ocessed o RNA scope in si u hyb idiza ion alone
o in combina ion wi h immuno luo escence echnique we e isual-
ized using a con ocal lase scanning mic oscope (AOBS-SP5X; Leica
Mic osys ems Heidelbe g GmbH, Mannheim, Ge many). Fluo escen
signals o AT2 mRNA and CD11b mRNA hyb idiza ion and immuno-
luo escence o Iba1 we e imaged wi h a 40and 63
objec i e lens.
Fo quan i a i e analysis, h ee subs an ia nig a sec ions om
each animal (n=5 pe g oup), selec ed h oughou i s an e opos e io
axis (an e io , cen al, and pos e io le els) we e analyzed by in es i-
ga o s blinded o expe imen al condi ions. Images om six mic o-
scopic ields (400.15 mm
2
by ield) pe each issue sec ion we e
ob ained wi hin he subs an ia nig a by lase scanning mic oscopy and
using cons an mic oscope pa ame e s and simila lase in ensi y. The
o al numbe o mic oglial cells and he o al numbe o mic oglial cells
ha exp ess AT2 mRNAs we e quan i ied using he ImageJ64 so -
wa e (Na ional Ins i u e o Heal h. Be hesda, MD, USA). Only cell p o-
iles showing a labeled nucleus and mic oglial mo phology we e
included. The nega i e p obe used as a con ol did no con ain any
s ained cells. Da a we e p esen ed as he mean numbe o cells pe
mm
2
. The pe cen age o mic oglial cells exp essing AT2 mRNA was
calcula ed om he co esponding absolu e alues. In addi ion, he
numbe o AT2 mRNA posi i e do s pe each cell was quan i ied in
hese mic oscopic ields o assess he in acellula mRNA le els o
AT2. A o al o 60 mic oglial cells pe animal we e analyzed using he
ImageJ64 so wa e.
2.10 |Me hyla ion s a us on he Ag 2 gene
p omo o
DNA was isola ed om mic oglia om b ains o male (n=3) and
emale (n=3) wild ype mice and con e ed wi h sodium bisul i e
using he EZ DNA Me hyla ion-Gold Ki (Zymo Resea ch; D5005)
ollowing he manu ac u e ecommenda ions. A se o PCR p ime s
(MMU_AGTR2_me 1L: 50-TTAGAATTTTGTAGGTTGAAGGTTT-30;
MMU_AGTR2_me 1R: 50-CAAAATACAATTAAAATACAAAAAAAA-30)
was designed wi h Me hp ime (Li & Dahiya, 2002) o ampli y a
250 bp agmen encompassing he en i e p omo e o he AT2 ecep-
o gene (Ag 2), as eco e ed om he Mus musculus e e ence
genome (NC_000086.8). Nex , we ca ied ou PCR ollowed by Sange
and single-molecule sequencing o he esul ing amplicons using a
Seqs udio (Applied Biosys ems) and a MinION (Ox o d Nanopo e
Technologies L d, ONT), espec i ely. PCR condi ions included
35 cycles o 95C o 20 s, 50C o 20 s and 72C o 20 s, in a mix
including 40 ng ans o med DNA and 1D eamTaq Ho S a G een
FIGURE 1 Flow cy ome y analysis o he CD11b+cells. Cells isola ed om non-pe used [(a)–(e)] and pe used [( )–(j)] b ains did no show
signi ican di e ences. [(a), ( )] His og ams showing wo CD45-exp essing cell popula ions: CD45 low (mic oglia) and CD 45 high (possible
mac ophages). [(b), (g)] Mic oglia, iden i ied as CD11b+and CD45 low (Q3), comp ised he highes p opo ion o cells (91.7%–94.5%), whe eas a
low pe cen age o po en ial mac ophages (CD11b+/CD45 high, Q2) we e obse ed. [(c), (h)] Fo wa d sca e (FSC) analysis showing a sligh
smalle size o he CD45 low cells ( ed) espec o CD45 high (blue) cells. [(d), (i)] CD11b analysis showed a sligh lowe exp ession wi hin CD45
low cells ( ed) espec o CD45 high (blue) cells. [(e), (j)] Fo wa d sca e (FSC) and side sca e (SSC) cha ac e is ics o CD11b+/CD45 low ( ed)
and CD11b+/CD45 high (blue) a e shown
2352 GARRIDO-GIL ET AL.
10981136, 2022, 12, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/glia.24255 by Conso cio In e uni e si a io Do Sis ema Uni e si a io De Galicia (Cisug), Wiley Online Lib a y on [03/01/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
PCR Mas e Mix (The mo Scien i ic; MAN0015977). PCR amplicons
we e pu i ied wi h ExoSAP-IT Exp ess (Applied Biosys ems; 75001.1.
ML), ampli ied using he BigDye Te mina o 3.1 ki (The moFishe ;
4337455) and cleaned wi h BigDye XTe mina o ki (The moFishe ;
4376486) p io o Sange sequencing in a SeqS udio gene ic analyze
con olled by he Sequencing Analysis So wa e 6.0 (Applied Biosys-
ems; 4474950). Elec ophe og ams we e immed, isualized and
mapped o he e e ence sequence wi h Geneious. ONT lib a ies we e
cons uc ed wi h he Sequencing 1D liga ion ki (Ox o d Nanopo e
Technologies L d; SQK-LSK109), including DNA epai wi h NEBNex
FFPE DNA Repai Mix (New England BioLabs) and NEBNex Ul a II
Liga ion Module (New England BioLabs) and loaded on o MinION
R9.4 lowcells (Ox o d Nanopo e Technologies L d; FLO-MIN106 e -
D), con olled by MinKNOW 19.12.5 and sequenced o 1000co -
e age. High accu acy base-calling was pe o med wi h Guppy 2.3.1
o gene a e as q iles and hese we e u he mapped o he Mus
musculus e e ence genome (NC_000086.8) wi h minimap2
2.14- 88344. The esul ing SAM iles we e p ocessed wi h Sam ools
1.744 and isualized wi h he In eg a i e Genome Viewe (IGV) o
assess cy osine me hyla ion s a us in he p omo o and adjacen
egions.
2.11 |S a is ical analysis
All da a we e ob ained om a leas h ee independen expe i-
men s and we e exp essed as means ± SEM. Two g oup compa i-
sons we e pe o med wi h S uden 's - es . Mul iple compa isons
we e analyzed by one-way ANOVA (when one ac o was s ud-
ied) o wo-way ANOVA (when wo ac o s we e s udied), ol-
lowed by S uden –Newman–Keuls pos -hoc es o he
co esponding nonpa ame ic es . The no mali y o popula ions
and homogenei y o a iances we e es ed be o e each ANOVA.
Di e ences we e conside ed s a is ically signi ican a p≤.05.
S a is ical analyses we e ca ied ou wi h SigmaPlo 11.0 (Sys a
So wa e, Inc., San José, CA/USA). S a is ics a e de ailed in Sup-
plemen al ile 2: Table S1.
3|RESULTS
3.1 |Cha ac e iza ion o models
Fi s , we cha ac e ized ou model o mic oglial isola ion o con i m
ha he isola ed CD11b+cells we e mic oglial cells and ha he ana-
lyzed popula ion was no cons i u ed by o he ypes o b ain cells o
blood cells. The e icacy o mic oglial isola ion, con i med by low
cy ome y analysis o hese CD11b+cells, showed simila esul s
when pe used (Figu e 1a–e) o non-pe used (Figu e 1 –j) b ains
we e used o isola ion.
Flow cy ome y analysis showed wo CD45-exp essing cell sub-
popula ions: CD45 low (mic oglia) and CD 45 high (possible mac o-
phages) (Figu e 1a, ). Mic oglia, iden i ied as CD11b+and CD45 low,
comp ised he highes p opo ion o cells (91.7%–94.5%), whe eas a
low pe cen age o po en ial mac ophages (CD11b+/CD45 high) we e
obse ed (Figu e 1b,g). Howe e , b ain- esiden mic oglia can up egu-
la e CD45 exp ession in esponse o insul s (Sedgwick e al., 1991),
indica ing di icul y o s ablish ha high CD45 a e no mic oglia. The
p esence o hese wo cell popula ions was also con i med by analysis
o size (Figu e 1c,h), CD11b exp ession le els (Figu e 1d,i) and in e nal
complexi y (i.e., g anula i y) (Figu e 1e,j). Thus, he mic oglia
(CD11b+/CD45 low) showed a smalle size, a lowe exp ession o
CD11b and a lowe in e nal complexi y han CD11b+/CD45 high
cells.
FIGURE 2 Es adiol concen a ions (pg/ml) in di e en mouse
g oups and absence o AT2 mRNA in AT2 KO mic oglia. Es adiol
(E2) se um concen a ions in wild ype (w ) males, w emales,
o a iec omized (OVX) w emales, AT2 KO emales and OVX AT2 KO
emales a e shown in (a). The lack o exp ession o AT2 mRNA in
adul mic oglia isola ed AT2 KO mice and he p esence in WT mice
was demons a ed by RT-PCR [(b)–(d)]. Speci ic AT2 ampli ica ion by
RT-PCR in adul mic oglia o WT mice was demons a ed by he
p esence o a unique peak a he co esponding mel ing empe a u e
(T
m
) in he mel cu es, and no peak was obse ed in he mel cu e
plo o AT2 KO mice (d). Rep esen a i e bands o AT2 exp ession by
RT-PCR in isola ed mouse WT and AT2 KO adul mic oglia [(b), (c)].
Da a a e mean ± SEMs. *p< .05 compa ed o male g oup,
#
p< .05
compa ed o w emale g oup,
&
p< .05 compa ed o AT2 KO emales.
Mul iple compa isons we e analyzed by K uskal-Wallis one way
analysis o a iance on anks ollowed by Dunn's me hod pos -hoc
es . KO, knockou ; OVX, o a iec omized; w , wild ype
GARRIDO-GIL ET AL.2353
10981136, 2022, 12, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/glia.24255 by Conso cio In e uni e si a io Do Sis ema Uni e si a io De Galicia (Cisug), Wiley Online Lib a y on [03/01/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
The le els o ci cula ing es ogens in he di e en mouse
models used in his s udy and e icacy o o a iec omies we e es i-
ma ed by de e mina ion o le els o Es adiol (E2) concen a ions in w
males (4.658 ± 0.579 pg/ml), w emales (72.123 ± 5.526 pg/ml),
o a iec omized (OVX) w emales (4.869 ± 0.551 pg/ml), AT2
KO emales (72.702 ± 4.866 pg/ml) and OVX AT2 KO emales
(4.385 ± 0.645 pg/ml) (Figu e 2a). These le els o E2 we e simila
o hose ob ained in ou p e ious s udies in mice (Ga ido-Gil
e al., 2021).
Then, we con i med he speci ic AT2 ampli ica ion by RT-PCR
in adul mic oglia. The speci ici y was demons a ed by he p es-
ence o a unique peak a he co esponding mel ing empe a u e
(T
m
) in he mel cu es, while no peak was obse ed in he mel
cu e plo o he nega i e con ols. The lack o AT2 ecep o s in he
mic oglia isola ed om AT2 KO mice was con i med using p ime s
designed agains he mRNA egion (second pu a i e ansmemb ane
domain) ha is codi ied by he dele ed sequence (exon 3) used o
dis up ing he Ag 2 gene in hese KO mice, which clea ly showed
ha AT2 mRNA was exp essed in mic oglia isola ed om WT mice
bu was absen in mic oglia isola ed om AT2 KO mice
(Figu e 2b–d).
3.2 |Le els o AT2 mRNA and in lamma ion-
ela ed cy okine exp ession in mic oglia om WT
males and emales
We analyzed he exp ession o AT2 mRNA and in lamma ion- ela ed
cy okines mRNA in mic oglial cells isola ed om non-pe used
(Figu e 3a–d) and pe used (Figu e 3e–h) b ains o adul WT males,
WT emales and WT OVX emales. AT2 mRNA exp ession was much
highe in mic oglia om emales and OVX emales han in mic oglia
om males (Figu e 3a,e). The e was a dec ease in mic oglial AT2
exp ession in OVX emales as compa ed wi h con ol emales ha
was no s a is ically signi ican .
Mic oglia isola ed om male b ains showed lowe le els o mRNA
exp ession o he an i-in lamma o y cy okine IL-10 han mic oglia
om emales and OVX emales, and no signi ican di e ence was
obse ed be ween emales and OVX emales (Figu e 3b, ). Howe e ,
mRNA exp ession o he p o-in lamma o y cy okines IL-1βand IL-6
was signi ican ly highe in mic oglia isola ed om males han in mic o-
glia isola ed om emales o OVX emales, and no signi ican di e -
ences in IL-1βand IL-6 exp ession we e de ec ed be ween emales
and OVX emales (Figu e 3c,d,g,h). These esul s we e con i med bo h
FIGURE 3 mRNA exp ession o AT2 [(a), (e)], IL-10 [(b), ( )], IL-1β[(c), (g)] and IL-6 [(d), (h)] in mic oglia. Mic oglia was isola ed om pe used
[(a)-(d)] and non-pe used [(e)–(h)] b ains o wild ype (WT) males, WT emales and o a iec omized (OVX) WT emales using RT-PCR. Fo mRNA
he compa a i e cycle h eshold alues me hod (2
ΔΔC
) was used. Da a a e mean ± SEMs. *p< .05 compa ed o wild ype males. One-way
ANOVA ollowed by S uden 's Newman–Keuls pos -hoc es [(a)–(d)] and S uden 's es s [(e)–(h)]
2354 GARRIDO-GIL ET AL.
10981136, 2022, 12, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/glia.24255 by Conso cio In e uni e si a io Do Sis ema Uni e si a io De Galicia (Cisug), Wiley Online Lib a y on [03/01/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
in pe used and non-pe used b ains o WT mice indica ing ha hese
e ec s a e no ela ed o ci cula ing mac ophages. Al oge he sug-
ges s ha di e ences in exp ession o AT2 ecep o s, he an i-
in lamma o y cy okine IL-10 and he p o-in lamma o y cy okines IL-
1βand IL-6 in mic oglia o adul heal hy WT mice a e p ima y ela ed
o sex o mice.
Finally, we con i med he exp ession o AT2 mRNA in mic oglia
om heal hy b ains o WT male and emale mice using in si u RNA
scope in issue sec ions om he nig al egion, as well as in si u
hyb idiza ion combined wi h immunohis ochemis y (Figu e 4). This
again con i med he p esence o AT2 exp ession in cells labeled by
mic oglial ma ke s and showing clea mic oglial mo phology
(i.e., mic oglia) in con ol b ains. As obse ed in isola ed mic oglia,
AT2 mRNA labeling was mo e ma ked in emale han in male
mic oglia (Figu e 4a–l). In si u p ocedu es also showed ha emales
had signi ican ly highe numbe o AT2-labeled mic oglial cells, and
signi ican ly highe numbe o AT2 mRNA do s by cell han males
(Figu e 4m–p).
3.3 |Le els o in lamma ion- ela ed cy okine
exp ession in mic oglia om WT and AT2 KO males
and emales
In a second se ies o expe imen s, we used wild- ype and AT2 KO
males, emales and OVX emales o know whe he di e ences in he
mic oglial exp ession o IL-10, IL-1βand IL-6 be ween males and
emales (con ol o OVX emales) and no dependen o ci cula ing
FIGURE 4 AT2 ecep o mRNA exp ession using in si u RNA scope p obes (Ag 2) and double immune/in si u RNA scope. Rep esen a i e
pho omic og aphs showing mic oglia om he nig al egion in male [(a)–(d)] and emale [(e)–(h)] wild- ype (w ) mice using double in si u RNA
scope (Ag 2 in ed and in eg in alpha M, I gam in blue), and coun e s ained wi h DAPI (whi e). [(i)–(l)] Double immuno/in si u showing Iba-1
(mic oglial ma ke ; g een) and Ag 2 ( ed, a ows). [(m)–(p)] Ag 2 was mo e abundan in mic oglia om emales bo h in numbe (m), pe cen age
(o) o labeled cells and do s by cell (p), wi hou signi ican di e ence in he o al numbe o mic oglial cells (n). Da a a e mean ± SEMs. *p< .05
compa ed o w males. S uden 's es s [(m)–(p)]. Scale ba 12.5 μm
GARRIDO-GIL ET AL.2355
10981136, 2022, 12, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/glia.24255 by Conso cio In e uni e si a io Do Sis ema Uni e si a io De Galicia (Cisug), Wiley Online Lib a y on [03/01/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
gonadal s e oids could be ela ed o he obse ed di e ences in AT2
exp ession be ween males and emales. Changes in IL-10, IL-1β, and
IL-6 exp ession induced by AT2 dele ion we e mo e ma ked in
emales han in males, which is consis en wi h he highe le els o
AT2 exp ession obse ed in emales ela i e o males. In e es ingly,
no signi ican di e ences we e obse ed in mic oglial exp ession
o IL-10, IL-1β, and IL-6 be ween males, emales and OVX
emales o AT2 KO mice, sugges ing ha di e ences in AT2
exp ession play a majo ole in sex di e ences in egula ion o
mic oglial IL-10, IL-1β, and IL-6 exp ession in he con ol b ains
(Figu e 5a–c). Unde in ense p o-in lamma o y s imulus wi h LPS,
he e was a ma ked inc ease in le els o mRNA exp ession o IL-
10 in WT mice ha was much highe han ha obse ed in AT2
KO mice, while mRNA exp ession o he p o-in lamma o y cy o-
kine IL-6 was signi ican ly highe in he AT2 KO mice ela i e o
heWTmice(Figu e6a,b).
FIGURE 5 IL-10 (a), IL-1β(b), and IL-6 (c) mRNA in mic oglia
isola ed om di e en mouse g oups. Wild- ype (w ) o AT2 knockou
(KO) male, emale and o a iec omized (OVX) emale b ains we e
analyzed using RT-PCR. Fo mRNA, he compa a i e cycle h eshold
alues me hod (2
ΔΔC
) was used. Da a a e mean ± SEMs. *p< .05
compa ed o he co esponding w con ol,
$
p< .05 compa ed o w
males. Mul iple compa isons we e analyzed by wo-way ANOVA
ollowed by S uden –Newman–Keuls pos -hoc es [(b), (c)] and wo-
way ANOVA on anks K uskal Wallis, (a)
FIGURE 6 E ec s o LPS injec ion on mic oglial in e leukin
mRNA exp ession in he p esence o he absence o AT2 ecep o s
(a, b), and e ec o emale b ain masculiniza ion (c) and male
o chiec omy (d) on AT2 mRNA exp ession using RT-PCR. In i o
injec ion o LPS induced a ma ked inc ease in le els o IL-10 mRNA
exp ession in mic oglia om WT mice ha was much highe han in
mic oglia om AT2 KO mice (a), while IL-6 mRNA exp ession was
signi ican ly highe in mic oglia om AT2 KO mice han in mic oglia
om WT mice (b). Mic oglia om masculinized emale b ains
showed highe AT2 mRNA exp ession han mic oglia om WT
males (c). Mic oglia om o chiec omized WT males showed AT2
mRNA exp ession simila han mic oglia om con ol WT males (d).
Fo mRNA, he compa a i e cycle h eshold alues me hod (2
ΔΔC
)
was used. Da a a e mean ± SEMs. *p< .05 compa ed o he
co esponding WT con ol,
#
p< .05 compa ed o WT +LPS. One-
way ANOVA ollowed by S uden 's Newman–Keuls pos -hoc es
(a); K uskal-Wallis one way analysis o a iance on anks ollowed
by S uden 's Newman–Keuls pos -hoc es (b) and S uden 's es s
[(c), (d)]
2356 GARRIDO-GIL ET AL.
10981136, 2022, 12, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/glia.24255 by Conso cio In e uni e si a io Do Sis ema Uni e si a io De Galicia (Cisug), Wiley Online Lib a y on [03/01/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
