Expe imen al Neu ology
Delayed changes in he ansc ip omic p o ileo ce eb al a e ies in a a model o
suba achnoid hemo hage
--Manusc ip D a --
Manusc ip Numbe : EXNR-24-735R1
A icle Type: Resea ch Pape
Sec ion/Ca ego y: Cellula and Molecula Neu oscience
Keywo ds: Suba achnoid Hemo hage (SAH); ce eb al a e ies; mic oa ay analysis; unc ional
en ichmen ; immune esponse
Co esponding Au ho : An onio Cas ellano, Ph.D.
Uni e si y o Se ille
SPAIN
Fi s Au ho : Gonzalo Re illa-González
O de o Au ho s: Gonzalo Re illa-González
Ma ía del Ca men González-Mon elongo
El on JR Vasconcelos
Juan U eña
Jian Shi
An onio Cas ellano, Ph.D.
Abs ac : Aneu ismal suba achnoid hemo hage (aSAH) is a neu o ascula disease
cha ac e ized by blood eleased in o he suba achnoid space due o up u e o he
ce eb al a e ies. A e he onse o bleeding, seconda y associa ed asospasm (VSP)
emains a d ama ic side e ec ha causes se e e como bidi ies. We analyzed
al e a ions in he exp ession p o iles o a e ies om a a model o SAH using
mic oa ay and bioin o ma ics app oaches. A single injec ion au ologous blood a
model, p e iously cha ac e ized in ou labo a o y, was used. We pe o med a o al
RNA ex ac ion and a mic oa ay analysis o ce eb al a e ies om animals 7 days a e
su ge y o s udy he delayed ansc ip ional changes induced by SAH. To assess he
unc ional ela ionship be ween di e en ly exp essed genes, we un a combina ion o
gene en ichmen ools: GSEA, ClueGO, and Clus e P o ile . Ou esul s showed ha in
SAH animals, he gene se s ela ed o in lamma ion and immune sys em ac i a ion
we e up- egula ed; genes ela ed o he pa hways in ol ed in he egula ion o muscle
con ac ion had hei exp ession dis u bed; and he gene ca ego ies associa ed wi h
DNA damage and epai we e o e ep esen ed. In conclusion, ou esul s sugges ha ,
a e he SAH insul , mul iple mechanisms, a he han a single cause, a e ac i a ed a
he same ime in he ce eb al essels o igge ascula al e a ions.
Powe ed by Edi o ial Manage ® and P oduXion Manage ® om A ies Sys ems Co po a ion
SAH Animal model
Single injec ion o au ologous blood in Cis e na Magna model.
Ce eb al a e ies isola ion and RNA ex ac ion was done 7 days a e su ge y
(long- e m).
Ou esul s sugges ha , long- e m a e SAH insul , ascula al e a ions and VSP could be igge ed by dis up ing mul iple
mechanisms a he same ime in he ce eb al essels. Ou da a p o ide new clues abou he mechanisms unde lying he
changes induced by SAH and will help pa e he way o new basic and applied esea ch on his pa hology.
T ansc ip ional and en ichmen analysis o a e ies om he SAH animal model
In lamma ion and immune sys em al e a ions we e up- egula ed and en iched long-
e m a e bleeding.
Pa hways ela ed o asospasm (VSP) we e dis u bed and DNA damage-p oducing
p ocesses induced.
G aphical Abs ac
Delayed changes in he ansc ip omic p o ile al e a ions o ce eb al
a e ies in a a model o suba achnoid hemo hage
Gonzalo Re illa-Gonzáleza,b,c,+,*, Ma ía del Ca men González-Mon elongoa,b,e, ,†,*,#,
El on JR Vasconcelosd, Juan U eñaa,b, Jian Shic,&, An onio Cas ellanoa,b,#,&
aIns i u o de Biomedicina de Se illa, IBIS/ Hospi al Uni e si a io Vi gen del Rocío/CSIC/ Uni e sidad de
Se illa. Se illa, Spain.
bDp o. Fisiología Médica y Bio ísica, Facul ad de Medicina, Uni e sidad de Se illa, Spain.
cLeeds Ins i u e o Ca dio ascula and Me abolic Medicine (LICAMM), School o Medicine, Uni e si y o
Leeds. Leeds LS2 9JT. Uni ed Kingdom.
dLeeds Omics, Uni e si y o Leeds. Leeds LS2 9JT. Uni ed Kingdom.
eIns i u o de In es igación e Inno ación Biomédica de Cádiz (INiBICA), Unidad de In es igación, Hospi al
Uni e si a io Pue a del Ma , A . Ana de Viya 21, 11009 Cádiz, Spain.
Á ea de Fisiología, Facul ad de Medicina, Uni e sidad de Cádiz, Spain.
*These au ho s sha e he i s au ho ship.
&These au ho s sha e he las au ho ship.
Gonzalo Re illa-González: g. e illa[email p o ec ed].uk.
ORCID: 0000-0002-5512-8381
+P esen add ess: Leeds Ins i u e o Ca dio ascula and Me abolic Medicine (LICAMM), School o
Medicine, Uni e si y o Leeds. Leeds LS2 9JT. Uni ed Kingdom.
Jian Shi: [email protected].
ORCID: 0000-0003-2179-8579
El on J. R. Vasconcelos: e. [email protected].
ORCID: 0000-0001-5130-6622
Juan U eña: ju [email protected]
ORCID: 0000-0003-4086-5941
Ma ía del Ca men González-Mon elongo: mca [email protected] /
ma iadelca men.go[email p o ec ed].
ORCID: 0000-0003-2332-6153
†P esen add ess: Unidad de In es igación, Ins i u o de In es igación e Inno ación Biomédica de Cádiz
(INiBICA), Hospi al Uni e si a io Pue a del Ma , A da. Ana de Viya 21, 11009 Cádiz, Spain; and Á ea
de Fisiología, Facul ad de Medicina, Uni e sidad de Cádiz, C/D . Ma añon 3, 3 d Floo , 11002, Cádiz,
Spain.
An onio Cas ellano: acas [email protected].
ORCID: 0000-0003-3955-5137
#Co espondence o: ac[email p o ec ed], Tel.: +(34)-955923059 (A.C.); mca [email protected], Tel.:
+(34)-956003111 (M.C.G.M.)
Highligh s
SAH induces delayed changes in gene exp ession in he ce eb al a e ies.
In lamma ion and he immune sys em a e up- egula ed on day 7 a e SAH.
The pa hways ela ed o asospasm (VSP) a e al e ed a e bleeding.
Vascula al e a ions and VSP could be p oduced by mul iple mechanisms a e SAH.
Abs ac
Aneu ismal suba achnoid hemo hage (aSAH) is a neu o ascula disease cha ac e ized
by blood eleased in o he suba achnoid space due o up u e o he ce eb al a e ies. A e
REVISED Manusc ip Click he e o iew linked Re e ences
he onse o bleeding, seconda y associa ed asospasm (VSP) emains a d ama ic side
e ec ha causes se e e como bidi ies. We analyzed al e a ions in he exp ession p o iles
o a e ies om a a model o SAH using mic oa ay and bioin o ma ics app oaches. A
single injec ion au ologous blood a model, p e iously cha ac e ized in ou labo a o y,
was used. We pe o med a o al RNA ex ac ion and a mic oa ay analysis o ce eb al
a e ies om animals 7 days a e su ge y o s udy he delayed ansc ip ional changes
induced by SAH. To assess he unc ional ela ionship be ween di e en ly exp essed
genes, we un a combina ion o gene en ichmen ools: GSEA, ClueGO, and
Clus e P o ile . Ou esul s showed ha in SAH animals, he gene se s ela ed o
in lamma ion and immune sys em ac i a ion we e up- egula ed; genes ela ed o he
pa hways in ol ed in he egula ion o muscle con ac ion had hei exp ession dis u bed;
and he gene ca ego ies associa ed wi h DNA damage and epai we e o e ep esen ed.
In conclusion, ou esul s sugges ha , a e he SAH insul , mul iple mechanisms, a he
han a single cause, a e ac i a ed a he same ime in he ce eb al essels o igge
ascula al e a ions.
Keywo ds
Suba achnoid hemo hage (SAH), ce eb al a e ies, mic oa ay analysis, unc ional
en ichmen , immune esponse.
In oduc ion
Aneu ismal suba achnoid hemo hage (aSAH) is a neu opa hology ha occu s when he
b ain a e ies up u e and subsequen ly blood is eleased in o he suba achnoid space.
Al hough i s wo ldwide incidence has dec eased om 10.2 pe 100,000 pe son-yea in
1980 o 6.1 in 2010, i is s ill a majo neu o ascula disease wi h a high case a ali y a e
(E minan e al., 2019). Pa ien s who su i e he hemo hagic insul ha e a high disease-
speci ic bu den; up o 19 % will become disabled o wo k and could su e cogni i e,
unc ional and beha io al complica ions (Al-Khindi e al., 2010; Eagles e al., 2019;
Nieuwkamp e al., 2009). Damages a e aSAH a e classi ied as ea ly ce eb al inju y
(ECI), hose ha occu up o 72 h a e he insul , o delayed ce eb al inju y (DCI), hose
appea ing om day 3 up o 14 a e hemo hage (Macdonald and Schweize , 2017).
DCI is p oduced by a e y occlusion and ce eb al damage in poo ly i iga ed egions o
he b ain. One o i s main causes is ce eb al asospasm (VSP), which occu s as a
consequence o he e ec o he eleased blood on he walls o he essels (B ami e al.,
2019; C owley e al., 2011; Egea-Gue e o e al., 2015; Mossa-Basha e al., 2019).
Ce eb al a e ies a e s uc u ed in h ee main laye s, whose al e a ions could induce VSP
unde pa hological condi ions. VSP could be p oduced by inc easing myocy e
con ac ili y ia Ca2+-dependen and Ca2+-independen mechanisms. Howe e , in some
ascula pa hologies i can also be p oduced by ac i a ing molecula pa hways ela ed o
in lamma ion and ascula emodeling (Liu and Khalil, 2018).
Exis ing s udies on ce eb al a e y ansc ip ion p o iles a e SAH a e limi ed. In a
models o ECI, an inc ease in he exp ession o genes ela ed o in lamma ion,
ex acellula ma ix emodeling, and apop osis was obse ed (Vikman e al., 2007, 2006).
Fu he mo e, in a canine model o DCI, genes ela ed o in lamma ion and Ca2+ egula ion
we e also up- egula ed (Sasaha a e al., 2008). The pu pose o he p esen wo k was o
in es iga e, using ansc ip omic assays and bioin o ma ic app oaches, SAH-induced
delayed al e a ions in he gene exp ession p o ile o he a ce eb al a e ies ha could
explain he pa hological p ocesses ha cause VSP.
Ma e ial and me hods
Animals
Male wis a a s (400 g app oxima ely and six mon hs old) we e di ided in o h ee g oups:
con ol, sham7d, and SAH7d. We ha e analyzed his ime poin acco ding o p e ious
esul s om ou labo a o y showing an inc ease in smoo h muscle myocy e exci abili y
on day 7 a e su ge y (Re illa-González e al., 2024a). The expe imen s we e ca ied ou
acco ding o he Spanish legisla ion on he p o ec ion o expe imen al animals and we e
app o ed by he local animal ca e commi ee acco ding o he Eu opean Di ec i e
2010/63/EU and he Spanish Royal Dec ee 53/2013. Be o e and a e su ge y, he animals
we e housed in sepa a e cages in a d y place, away om sou ces o in ec ion and su gical
a eas. A s able empe a u e be ween 23 and 27 ºC and a ligh /da k cycle we e main ained
o 12 h. They ecei ed ood and wa e ad libi um be o e and a e su ge y.
Su gical p ocedu es and sac i ice
Su ge y was pe o med ollowing p ocedu es p e iously published by ou g oup (Egea-
Gue e o e al., 2015; Muñoz-Sánchez e al., 2012). Be o e p ocedu es, he animals we e
anes he ized wi h a p epa a ion composed o 96 µg/g Ke amine (Ke ola ©, P ize , 50
mg/mL), 4.5 µg Xylazine (Rompun©, Baye , 20 mg/mL) and 0.2 µg/g A opine (A opine
Baye © 1 mg/mL). The op imal dose o achie e a good deg ee o analgesia and seda ion
(2.5 µL/g) was injec ed in ape i oneally. Subsequen ly, he head, neck, and ail we e
sha ed and he animals we e immobilized in a decubi us-p one posi ion in a s e eo axic
ame (S oel ing©). Du ing su ge y, animal b ea hing, empe a u e, and an app op ia e
deg ee o seda ion and analgesia we e con inuously moni o ed. Sham and SAH g oups
we e gene a ed using a 25G needle (BD Biosciences) and 1 mL sy inge (BD Plas ipak©),
moun ed on he a m o he s e eo axic ame. 100 µL o ce eb ospinal luid was emo ed
om he cis e na magna h ough he pos e io a lan o-occipi al memb ane. Then, in he
SAH g oup, 100 µL o blood was ob ained om he caudal ein and immedia ely injec ed
in acis e nally. In he sham g oup, he same p ocedu es we e pe o med, bu ins ead o
blood, he same olume o saline (0.9 % NaCl) was injec ed. A e su ge y, bo h animals
we e e u ned o hei cages o moni o he p ope awakening. The con ol g oup did no
ecei e any su ge y. On day 7 he animals we e sac i iced by exsanguina ion a e
injec ion o a le hal dose o sodium hiopen al. Fo RNA analysis, we ollowed he
ex ac ion me hod p e iously epo ed (Re illa-González e al., 2024a). La ge a e ies o
he ce eb al su ace di ec ly exposed o blood we e used. Whole b ains we e ex ac ed
and he a e ies we e cleaned unde a magni ying glass in ice-cold Hank’s solu ion ee
o RNAses, immedia ely ozen in liquid ni ogen and s o ed a -80 ºC. Hank’s solu ion
composi ion was (in mM): 125 NaCl, 5.36 KCl, 0.44 KH2PO4, 0.34 Na2HPO4, 5
NaHCO3, 10 glucose, 1.45 suc ose, 10 HEPES, a pH 7.4.
To al RNA ex ac ion and mic oa ay analysis
The homogeniza ion o he a e ies was pe o med using a Poly on PT4000 (Kinema ica)
o 5 seconds in RLT bu e om he RNeasy® Mic o Ki (Qiagen). Subsequen ly, he
samples we e incuba ed a oom empe a u e o 5 min and hen cen i uga ed a 12,000
X g, o 3 min, un il he supe na an was cla i ied. The RNA isola ion p ocess was
comple ed using he QIAcube pla o m (Qiagen). Once he RNA was isola ed, i was
elu ed in 15 µL o RNAses- ee wa e , quan i ied using a Nanod op 2000 (The mo) and
inally ozen a -80 ºC. To al RNA was ampli ied and labeled using he GeneChip® WT
PLUS Reagen Ki (The mo). Ampli ica ion was ca ied ou using 100 ng o o al RNA.
The ampli ied cDNA was quan i ied, agmen ed and labeled o hyb idiza ion wi h
GeneChip® Cla iom S Ra A ay (The mo). The a ays we e washed and labeled in a
GeneChip® Fluidics S a ion 450 (The mo) and scanned wi h GeneChip® Scanne 3000
(The mo). Finally, da a we e analyzed wi h he TAC 4 p og am (The mo), se ing he
P- alue < 0.05 and Fold-change > 1.3 as h esholds o Di e en ially Exp essed Genes
selec ion (DEGs). Di e ences be ween g oups we e e alua ed using eBayes es . The da a
discussed in his publica ion ha e been deposi ed in NCBI's Gene Exp ession Omnibus
and a e accessible h ough GEO Se ies accession numbe GSE266601.
Valida ion o mic oa ay da a using RT-qPCR
Da a om he mic oa ay analysis we e alida ed by RT-qPCR. A de ailed desc ip ion o
he alida ion p ocess is included in “Supplemen a y ma e ials”.
En ichmen analysis
DEGs lis s we e submi ed o a gene se en ichmen assay (GSEA) wi h GSEA 4.1
(Moo ha e al., 2003; Sub amanian e al., 2005) and he ollowing lib a ies: KEGG C2
7.4, Reac ome C2 7.4, GO Biological p ocesses C5 7.4 y GO Molecula Func ions
C5 7.4. Addi ionally, o e ep esen a ion analyzes we e pe o med using he
Clus e P o ile Bioconduc o package (Wu e al., 2021) and ClueGo (Bindea e al., 2009)
as al e na i e ools o mo e comp ehensi e unc ional analysis elucida ion. As in he
GSEA analysis, GO, KEGG, and Reac ome we e he main unc ional anno a ion conso ia
elied on, se ing an adjus ed P- alue h eshold o 0.05 o bo h ools.
Resul s
Sample dis ibu ion in a SAH animal model
To assess changes in gene exp ession caused by SAH, we ha e pe o med a mic oa ay
analysis o cha ac e ize gene exp ession p o iles in he h ee sample g oups unde s udy:
con ol, sham7d, and SAH7d. P incipal componen analysis (PCA) showed clea
di e ences be ween he di e en g oups. The con ol and SAH7d g oups we e placed in
dis al posi ions, indica ing impo an di e ences in hei exp ession p o iles, while he
sham7d g oup was loca ed hal way, indica ing an in e media e exp ession p o ile (Fig.
1A). The ollowing con ol es s we e un o e i y he quali y o he samples: posi i e s
nega i e AUC, o compa e he in on con ols o he exon con ols; hyb idiza ion con ol,
o e alua e sample hyb idiza ion e iciency on gene exp ession a ays; and labeling
con ols, o moni o he a ge labeling p ocess. All samples used in he s udy passed hese
sc eenings, allowing hei use o u he analysis (Fig. 1B-D).
Fig. 1 PCA analysis and quali y con ol. (A) PCA analysis showing he dis ibu ion o he samples o each
g oup. The con ol and SAH7d g oups we e placed in dis al posi ions, while he sham7d g oup occupied
an in e media e posi ion. (B-D) Pos s Neg AUC, hyb idiza ion and labeling con ol es s we e used o
analyze he quali y o he di e en samples. E e y sample passed quali y con ol es s.
T ansc ip ional changes in ce eb al a e ies a e induc ion o SAH
Once we con i med he dis ibu ion o he samples and he quali y o he mic oa ays, we
pe o med a gene di e en ial exp ession analysis using TAC4 so wa e o s udy how he
gene exp ession p o ile could be al e ed a e SAH. Fo his pu pose, we conside ed as
di e en ially exp essed genes hose wi h a old-change <-1.3 and >1.3 and a P- alue
<0.05. Volcano plo s show he dis ibu ion o he di e en ially exp essed genes (Fig. 2A).
A o al numbe o 23,188 genes we e analyzed. Fig. 2B shows he numbe o genes
di e en ially exp essed in a wo-by- wo compa ison o he expe imen al g oups, and
de ails o he gene dis ibu ion a e summa ized in Table 1. In he compa ison o SAH7d
s. con ol, a o al numbe o 1,706 genes we e di e en ially exp essed, 801 we e up-
egula ed and 905 we e down- egula ed unde he SAH condi ion. In sham7d s. con ol,
1,246 genes we e di e en ially exp essed, 522 we e up- egula ed, and 724 we e down-
egula ed in sham. Finally, in SAH7d s. sham7d, 817 genes we e di e en ially
exp essed, whe e 438 genes we e up- egula ed and 379 we e down- egula ed in SAH
g oup. The Venn diag am shows he logical ela ionships be ween he di e en ially
exp essed genes o each compa ison (Fig. 2C). Genes included in he AB a ea (226
genes) ep esen he g oup o in e es . These genes we e di e en ially exp essed in bo h
compa isons, SAH7d s. con ol and SAH7d s. sham7d, and we e he e o e genes whose
exp ession changes a e associa ed wi h bleeding (Supplemen a y Table 1). Among hese
genes, we ha e iden i ied some ha could be in ol ed in a ious p ocesses such as he
unc ioning o Rho amily GTPases and he ac i a ion and egula ion o he immune
sys em (de ails a e summa ized in Table 2). F om he mic oa ay da a, we alida ed by
RT-qPCR hose genes included in he “Rho GTPases amily and i s egula ion” and
“ac i a ion and egula ion o immune sys em” gene se s ha showed he g ea es change
in exp ession (>1.5 old-change) in he compa ison o he SAH7d s. sham7d g oups. The
RT-qPCR esul s con i med, in mos cases, he mic oa ay da a (Supplemen a y Figu e
1). C3, IL10RA and LCP2, included in he “ac i a ion and egula ion o immune sys em”
gen se , ha we e up- egula ed in he mic oa ay analysis, we e also signi ican ly up-
egula ed in he RT-qPCR alida ion. ARHGAP4, included in he “Rho GTPases amily
and i s egula ion” gen se and ha was up- egula ed in he mic oa ay, showed a simila
end in he alida ion, al hough i did no each s a is ical signi icance (P=0.103).
Howe e , he RAC2 gene, belonging o he same gene se , s ood ou , as i was he one
ha showed he g ea es change in exp ession in he mic oa ay, bu did no show any
signi ican change in exp ession in he qPCR alida ion.
Fig. 2 Mic oa ay analysis o genes ha a e up- egula ed and down- egula ed in he di e en compa isons.
(A) Volcano plo s show di e ences in exp ession be ween expe imen al g oups. Fold-change <-1.3 and
>1.3 and P- alue< 0.05. (B) Di e en ially exp essed genes be ween compa isons. G ay ba s ep esen he
o al numbe o di e en ial exp essed genes (ba s: ed, up- egula ed genes; g een, down- egula ed genes).
The numbe s abo e he ba s indica e di e en ially exp essed genes. (C) Venn diag am o he di e en
compa isons showing he numbe o speci ic di e en ially exp essed genes in each compa ison. n= 3
con ol animals, n= 5 sham7d animals, and n= 5 SAH7d animals.
SAH7d s. con ol
To al
Passed Fil e
Up-Regula ed
Down-Regula ed
Coding
21848
1598
752
846
Mul iple Complex
858
61
35
26
Non-Coding
310
25
3
22
Unassigned
142
17
7
10
Pseudogene
28
5
4
1
P ecu so mic oRNA
2
0
0
0
sham7d s. con ol
To al
Passed Fil e
Up-Regula ed
Down-Regula ed
Coding
21848
1186
495
691
Mul iple Complex
858
33
20
13
Non-Coding
310
17
2
15
The ac i a ion o he immune sys em and in lamma ion a e bleeding a e side e ec s
obse ed in bo h pa ien s and animal models o SAH (G is e al., 2019; Ridwan e al.,
2021; Sa a aj e al., 2018). Fu he mo e, ac i a ion o immune p ocesses in ce eb al
essels is conside ed one o he causes o SAH (Li e al., 2017; Wang e al., 2017). Ou
mic oa ay esul s, alida ed using RT-qPCR, showed ha in lamma o y pa hways we e
al e ed in SAH7d animals s. con ol and sham7d g oups, and we e consis en wi h
p e ious mic oa ay s udies pe o med in ea ly- and long- e m SAH animal models
(Sasaha a e al., 2008; Vikman e al., 2007, 2006). In addi ion, ou esul s align wi h
bioin o ma ic s udies pe o med in human b ain and aneu ismal wall issues showing he
ac i a ion o immune sys em in he suba achnoid hemo hage en i onmen (Ku ki e al.,
2011; Rosell e al., 2011; Ye e al., 2022). An inc ease in long- e m immune esponse
a e aSAH is also obse ed in pa ien s (Bacigaluppi e al., 2020), and ecen esul s om
ou labo a o y showed an inc ease in he numbe o monocy es and hei adhesion o he
endo helium, 5 days a e bleeding in pa ien s who de eloped VSP (Re illa-González e
al., 2024b). The en ichmen o hese ca ego ies, oge he wi h hese p e ious esul s, is a
ele an inding in his pa hology. The ac i a ion o he immune esponse and he
in lamma o y p ocesses induced by SAH in he ce eb al a e ies could be ela ed o VSP
and a poo p ognosis o pa ien s a e SAH. These p ocesses could be a ge ed in new
he apeu ic app oaches ha allow be e pa ien managemen .
The Rho amily is a amily o small GTPases ha media es se e al in acellula signaling
p ocesses, including VSMC con ac ion o elaxa ion h ough he Ca2+ sensi iza ion
mechanism (Liu and Khalil, 2018). Ou mic oa ay esul s showed an inc ease in he
exp ession o genes associa ed wi h nega i e egula ion o hese p o eins (ARAP3 and
ARHGAP4) and a dec ease in genes associa ed wi h posi i e egula ion (ARHGEF4).
Fu he mo e, o he genes ha codi y o he membe s o his amily we e up- egula ed
(RAC2 and RHOG). RT-qPCR alida ion da a showed a posi i e end in ARHGAP4 ha
ma ched he mic oa ay esul s. Howe e , RAC2 exp ession le els in SAH animals did
no show his co ela ion. The easons ha could explain his disc epancy may include
di e ences in he sensibili y and speci ici y o he echniques, backg ound o alse
posi i es in mic oa ay, he low numbe o samples and di e ences in he sample quali y.
P e ious esul s om ou labo a o y showed ha he RhoA/ROCK pa hway is
unc ionally in ol ed in he egula ion o he smoo h muscle con ac ion in esponse o
long depola iza ions and in asocons ic ion o he ce eb al a e ies o SAH animals 5
days a e bleeding (Egea-Gue e o e al., 2015). Fu he mo e, he esul s o expe imen s
ca ied ou in ou labo a o y on pe iphe al blood mononuclea cells om pa ien s showed
ha RhoA unc ion is signi ican ly inc eased 4 days a e bleeding in pa ien s who
de eloped VSP (González-Mon elongo e al., 2018). These esul s sugges ha he e is a
ime window o RhoA/ROCK ac i a ion, peaking 4-5 days a e SAH and hen declining.
These obse a ions a e consis en wi h a p e ious wo k ha desc ibed a peak in RhoA
exp ession on day 5 and a sligh dec ease on day 7 a e SAH (Miyagi e al., 2000). O he
au ho s ha e poin ed ou an inc ease in RhoA ansloca ion o he plasma memb ane in
acu e cell cul u es and animal models o SAH, and an enhancemen e ec media ed by
endo helin-1 (Chang e al., 2014; Wang e al., 2014; Wickman e al., 2003). All his
e idence sugges s ha he exp ession and unc ion o he RhoA/Rock pa hway ha e a
dynamic beha io o e ime, ac ing in pa allel wi h o he pa hways in ol ed in SAH.
Ex a asa ion o blood om ce eb al essels in SAH p oduces dele e ious e ec s in SAH.
These e ec s can be assessed by measu ing he exp ession o some p o eins ela ed o
b ain inju y (Ma z e al., 1996). Ou esul s o he GSEA analysis pe o med in ce eb al
a e ies showed ha he gene se s ela ed o DNA damage and epai we e en iched in he
SAH7d g oup ela i e o he con ol and sham7d g oups, indica ing ha hemo hage
ac i a es he p ocesses ela ed o cell inju y and dea h in myocy es. This obse a ion is in
ag eemen wi h p e ious esul s ha showed an inc ease in DNA agmen a ion and cell
inju y in b ains om ea ly- and delayed inju y animal models o SAH (Ma z e al., 2001,
2000, 1996). In addi ion, a p e ious mic oa ay s udy on ea ly e ec s pe o med in a a
model indica ed ha genes ela ed o apop osis we e also up- egula ed in pa hological
animals (Vikman e al., 2006). These esul s sugges ha DNA damage and apop osis a e
p ocesses ha occu a e SAH. Howe e , mo e esea ch is needed in his ield o
de e mine i hese p ocesses a e he cause o e ec s o pa hophysiological al e a ions.
In summa y, ou expe imen s show ha gene exp ession is al e ed in he ce eb al a e ies
a e SAH. P ocesses ela ed o in lamma ion and immune sys em ac i a ion a e up-
egula ed and en iched 7 days a e bleeding. Gene ca ego ies ela ed o pa hways ha
can in luence he VSP p ocess a e also dis u bed, and p ocesses ha induce DNA damage
in cells a e induced. These esul s sugges ha , a e SAH insul , ascula al e a ions and
VSP could be igge ed by dis up ing mul iple mechanisms a he same ime in he
ce eb al essels, suppo ing he in e ac ion be ween hem ins ead o a single cause o his
neu o ascula disease. These esul s also suppo ha ou animal model o SAH, induced
by au ologous in acis e nal injec ion o blood, is alid and ep oduces many o he
changes obse ed in pa ien s wi h SAH and o he animal models.
Conclusions
Classically, a emp s ha e been made o iden i y a single cause o asospasm a e SAH,
bu his s a egy has no esol ed VSP in a high pe cen age o pa ien s. Howe e , ou
esul s suppo ha a conce o al e a ions ha include changes in he immune sys em,
egula ion o con ac ion, and cellula in eg i y occu a he same ime a e he
hemo hagic insul . This highligh s ha he e iology o asospasm could be he combined
e ec o se e al al e a ions, which implies ha i s he apeu ic app oach should include
mul iple pe spec i es. Ou esul s p o ide new clues abou he mechanisms unde lying
he changes obse ed a e SAH and will help o pa e he way o new undamen al and
applied esea ch on his pa hology.
Funding
The s udy was suppo ed by he Resea ch G an s SAF2017-89474-R Minis e io de
Economía, Indus ia y Compe i i idad, Spain; JU and AC) and US-1381231 (P oyec os
I+D+I FEDER Andalucia 2014-2020; AC). This s udy is pa ly suppo ed by he B i ish
Hea Founda ion P ojec G an (PG/21/10595; J.S.). GR-G is he ecipien o a
p edoc o al ellowship o he “V Plan P opio de la Uni e sidad de Se illa” and
pos doc o al g an o he “Fundación Al onso Ma ín Escude o”.
Decla a ion o Compe ing In e es
The au ho s decla e no compe ing in e es s.
Acknowledgemen
The au ho s acknowledge D . F ancisco Jesús Mo ón-Ci an os (Genomics Se ice,
Ins i u o de Biomedicina de Se illa (IBiS), Spain) o his echnical suppo wi h he
mic oa ay; Raquel Gómez Díaz (Biomedical Resea ch Suppo Se ice (SAIBIS) o he
echnical suppo in he RT-qPCR alida ion expe imen s; and D s. Albe o Pascual and
Ma ía Isabel Ál a ez-Ve ga a (IBiS, Spain) o hei ad ice on he GSEA echnique.
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