BRIEF RESEARCH REPORT
published: 22 Augus 2019
doi: 10.3389/ neu .2019.00911
F on ie s in Neu ology | www. on ie sin.o g 1Augus 2019 | Volume 10 | A icle 911
Edi ed by:
Oli ie De an e,
Cen e Hospi alie Uni e si ai e de
G enoble, F ance
Re iewed by:
A is eidis H. Ka sanos,
Uni e si y o Ioannina, G eece
Fabien Chau eau,
INSERM U1028 Cen e de Reche che
en Neu osciences de Lyon, F ance
*Co espondence:
F ancisco Campos
[email p o ec ed]
Ramón Iglesias-Rey
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
S oke,
a sec ion o he jou nal
F on ie s in Neu ology
Recei ed: 22 May 2019
Accep ed: 06 Augus 2019
Published: 22 Augus 2019
Ci a ion:
A gibay B, Campos F, Pe ez-Ma o M,
Viei es-P ado A, Co ea-Paz C,
López-A ias E, Da Sil a-Candal A,
Mo eno V, Mon e o C, Sob ino T,
Cas illo J and Iglesias-Rey R (2019)
Ligh -Emi ing Diode
Pho obiomodula ion A e Ce eb al
Ischemia. F on . Neu ol. 10:911.
doi: 10.3389/ neu .2019.00911
Ligh -Emi ing Diode
Pho obiomodula ion A e Ce eb al
Ischemia
Bá ba a A gibay1, F ancisco Campos1*, Ma ía Pe ez-Ma o1, Alba Viei es-P ado1,
Cla a Co ea-Paz1, Es eban López-A ias1, And és Da Sil a-Candal1, Vicen e Mo eno2,3,
Ca los Mon e o2,3, Tomás Sob ino1, José Cas illo1and Ramón Iglesias-Rey1*
1Clinical Neu osciences Resea ch Labo a o y, Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS), Clinical Uni e si y
Hospi al, Uni e sidade de San iago de Compos ela, San iago de Compos ela, Spain, 2Op ics A ea, Depa men o Applied
Physics, Facul y o Physics, Uni e si dade de San iago de Compos ela, San iago de Compos ela, Spain, 3Facul y o Op ics
and Op ome y, Uni e si dade de San iago de Compos ela, San iago de Compos ela, Spain
Pho obiomodula ion (PBM) he apy is a p omising he apeu ic app oach o se e al
pa hologies, including s oke. The biological e ec s o PBM o he ea men o ce eb al
ischemia ha e p e iously been explo ed as a neu op o ec i e s a egy using di e en
ligh sou ces, wa eleng hs, and inciden ligh powe s. Howe e , he capabili y o PBM
as a no el al e na i e he apy o s imula e he eco e y o he inju ed neu onal issue
a e ischemic s oke has been poo ly explo ed. The aim o his s udy was o in es iga e
he low-le el ligh i adia ion he apy by using Ligh Emi ing Diodes (LEDs) as po en ial
he apeu ic s a egy o s oke. The LED pho obiomodula ion (con inuous wa e, 830 nm,
0.2–0.6 J/cm2) was i s ly e alua ed a di e en ene gy densi ies in C17.2 immo alized
mouse neu al p ogeni o cell lines, in o de o obse e i his ea men had any e ec on
cells, in e ms o p oli e a ion and iabili y. Then, he PBM-LED e ec (con inuous wa e,
830 nm, 0.28 J/cm2a b ain co ex) on long- e m eco e y (12 weeks) was analyzed in
ischemic animal model by means lesion educ ion, beha io al de ici s, and unc ional
magne ic esonance imaging ( MRI). Analysis o cellula p oli e a ion a e PBM was
signi ican ly inc eased (1 mW) in all di e en exposu e imes used; howe e , his e ec
could no be eplica ed in i o expe imen al condi ions, as PBM did no show an in a c
educ ion o unc ional eco e y. Despi e he p omising he apeu ic e ec desc ibed o
PBM, u he p eclinical s udies a e necessa y o op imize he he apeu ic window o his
no el he apy, in e ms o he mechanism associa ed o neu o eco e y and o educe he
isk o ailu e in u u es clinical ials.
Keywo ds: unc ional eco e y, ischemic s oke, pho obiomodula ion he apy, magne ic esonance imaging,
animal model, in ace eb al hemo hage
INTRODUCTION
Pho obiomodula ion he apy has been in es iga ed in he pas ew yea s as an al e na i e
ea men o s oke and auma ic b ain inju y (TBI) in o de o p omo e a neu op o ec i e
e ec and issue egene a ion (1–5). The main bene i s a ibu ed o b ain PBM he apy a e
ela ed o di e en biological p ocesses such as inc easing ce eb al me abolic unc ion, s imula ing
neu ogenesis and synap ogenesis, and neu op o ec ion ia an i-in lamma o y, and an ioxidan
biological signaling (5,6).
A gibay e al. Pho obiomodula ion A e Ce eb al Ischemia
In he ield o s oke, PBM applied in he acu e phase has
been sugges ed as a p omising he apeu ic app oach o inducing
unc ional eco e y. Thus, some s udies ha e demons a ed
ha 808–660 nm Low Le el Lase The apy (LLLT) applied
a e ischemia on expe imen al animals imp o ed neu ological
a ing sco es wi hou inc easing body empe a u e, by di ec
illumina ion o he skin on sha ed animals (7–10).
On he o he hand, i has been shown ha ansc anial lase
he apy wi hin 24 h om s oke onse is a sa e p ocedu e, in
e ms o mo ali y o occu ence o ad e se e ec s, when i is
used on s oke pa ien s (11,12). LLLT used alone o combined
wi h he h omboly ic ea men o s oke ( ecombinan issue
plasminogen ac i a o ) did no inc ease he isk o hemo hagic
ans o ma ion (13). Despi e expe imen al e idences and human
sa e y, a clinical ial designed o analyze he bene icial e ec nea -
in a ed lase he apy in s oke has showed nega i e esul s, in
pa because many o he pa ame e s used ( he apeu ic ime-
window, lase in ensi y) we e no su icien ly op imized o use
in animal p eclinical s udies (14).
Ligh emi ing diodes (LEDs) de ices ha e eme ged as an
inno a i e sou ce o b ain PBM o a wide ange o neu ological
condi ions, and hei use has been app o ed by he US Food
and D ugs Adminis a ion. Di e en s udies abou LED-PBM
ha e been mainly es ed on cell in i o assays (15–18), o
in expe imen al animal s udies like e inal al e a ions (19,20),
and i s use in neu onal inju es ha e been e en es ed in
pa hologies as TBI and s oke as a neu op o ec an app oach,
wi h con adic o y esul s (5,9,21).
In his ega d, he biological e ec s o PBM o he ea men
o s oke ha e been widely explo ed using di e en ligh sou ces,
wa eleng hs, and inciden powe s in p eclinical s udies. Howe e ,
s udies we e mainly ocused on he neu op o ec i e e ec s, while
neu o eco e y p ocesses we e e alua ed only by unc ional es s
o mo ali y a e, and he di ec e ec o PBM on eco e y o
he damage neu onal issue was no accu a ely es ed so a .
In he ield o magne ic esonance imaging (MRI), he in e es
in unc ional magne ic esonance imaging ( MRI) has been
inc easing signi ican ly as a nonin asi e ool o e alua e neu al
ac i i y and he e icacy o neu o eco e y he apies (22).
The e o e, o e alua e i LED-PBM could induce any posi i e
e ec s o on neu onal eco e y a e s oke, in his s udy we ha e
s udied i s he e ec o PBM a di e en ene gy densi ies in
cell cul u e, in e ms o p oli e a ion and iabili y. Secondly, we
ha e s udied he long- e m eco e y e ec o LED-PBM in an
animal model o ansien ischemic s oke by means o educ ion
o ischemic lesion size, and inally we ha e es ed o he i s ime
unc ional eco e y de e mined by MRI in combina ion wi h
unc ional beha io al es s.
MATERIALS AND METHODS
Cellula in i o Analysis o LED
Pho obiomodula ion
I is well-known ha neu onal g ow h and p oli e a ion is one
o he mos ele an physiological mechanisms in ol ed in he
bene icial e ec s o ligh (1,3). In his s udy, we ha e pe o med
a p e ious in i o e alua ion abou he e ec o LED-PBM a
di e en ene gy densi ies on cellula p oli e a ion and iabili y.
Fo ha pu pose, C17.2 immo alized mouse neu al
p ogeni o cell line was used. C17.2 cells, p o ided by P o .
Ma hias Hoehn (Max-Planck Ins i u e, Cologne, Ge many),
we e cul u ed in DMEM (78%), e al bo ine se um (10%), ho se
se um (5%), penicillin-s ep omycin (1%) (Gibco In i ogen,
Paisley, UK), and ampho e icin-B (1%) (Sigma-Ald ich, S .
Louis, MO, USA).
To analyze he e ec o LED-PBM on cells, an 830 nm LED
a ay lamp (Quan um Spec ali e 830 nm LED, Quan um De ices
Inc., WI, USA) was used as ligh sou ce. C17.2 cells we e seeded
(100,000 cells/well) in 6-well pla es and placed a 15 cm om
he LED sou ce o PBM. Based on p e ious s udies (15–17),
ligh exposu e was applied 2 consecu i e days a 1, 5, and
10 mW du ing 6, 12, o 18 min, ob aining ene gy in ensi ies
om 0.2 o 6 J/cm2(Supplemen a y Table 1). Non-exposed cells
(con ol) we e main ained unde he same condi ions as he
18 min exposed cells bu wi hou ligh s imula ion.
To de e mine he in luence o he PBM on he cellula
p oli e a ion, o al cell coun was pe o med wi h ypan blue
s aining (STEMCELL Technologies, G enoble, F ance) and a
Neubaue coun ing chambe (Blaub and, Sigma-Ald ich, S .
Louis, MO, USA) 24 h a e he las PBM. Samples we e dilu ed
1:5 wi h phospha e bu e ed saline (PBS) and 1:2 wi h T ypan
blue. Cell coun was pe o med by using an in e ed mic oscope
(Olympus IX51, Shinjuku, Tokyo, Japan).
Fo assessing he iabili y o he cells a e PBM, supe na an s
we e collec ed om well cul u e, including a nega i e con ol
o lysed cells. Cell iabili y was de e mined by means o he
lac a e dehyd ogenase assay (LDH) (Lac a e Dehyd ogenase
Assay Ki . Sigma-Ald ich, S . Louis, MO, USA), ollowing he
manu ac u e ’s p o ocol. In b ie , supe na an s we e cen i uged
a 1,000 pm o 5 min and u he incuba ed wi h LDH eagen s
o 20 min. Nex , he pla e was ead in Syne gy2 (Bio ek
Ins umen s, Ve mon , USA) a 490 nm and he iabili y a e was
calcula ed wi h espec o con ol and lysed alues.
Animals S udies
All expe imen al p o ocols in ol ing he use o esea ch
animals ha e been app o ed by he Uni e si y o San iago de
Compos ela (Spain) e hics commi ee ( e : 15010/2019/004) and
we e pe o med acco ding o he guidelines o he Animal
Wel a e Commi ee o he hos ins i u ion and in acco dance
wi h applicable legisla ion o he Eu opean Union (86/609/EEC,
2003/65/EC, 2010/63/EU, RD 1201/2005 and RD 53/2013) and
ollowing he ARRIVE Guidelines o animal expe imen s.
Male Sp ague–Dawley a s (Ha lan Labo a o ies, Ba celona,
Spain) weighing 300 ±30 g (12 weeks old) we e used in his
s udy. Animals we e kep in a con olled en i onmen a 22 ±
1◦C and 60 ±5% humidi y, wi h 12/12 h ligh /da kness cycles.
Animals we e ed ad libi um wi h s anda d die pelle s and ap
wa e . All animals we e housed indi idually.
All su gical p ocedu es and MRI s udies we e conduc ed
unde se o lu ane (Abbo Labo a o ies, IL, USA) anes hesia
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A gibay e al. Pho obiomodula ion A e Ce eb al Ischemia
(3–4%) using a ca ie 65:35 gas mix u e o N2O:O2. Fo
MRI expe imen s, animals we e seda ed wi h subcu aneous
adminis a ion o mede omidine (Domi o R
) (0.05 mg/kg bolus
and 0.1 mg/kg/h in usion) du ing MRI da a acquisi ion.
Respi a ion a e was con inuously eco ded non-in asi ely, and
body empe a u e was kep cons an by a eedback-con olled
hea ing pad. A e he expe imen , seda ion was an agonized
wi h an in ape i oneal injec ion o a ipamezole (0.1 mg/kg). No
animal died du ing he ollow-up pe iod.
Su gical P ocedu es o T ansien Focal
Ischemia
T ansien middle ce eb al a e y occlusion ( MCAO) (60 min o
occlusion) was induced by in aluminal occlusion o he middle
ce eb al a e y wi h silicon handmade su u es (head diame e
300–350 µm), ollowing he p ocedu e desc ibed elsewhe e (23,
24). A lase -Dopple low p obe ( ip diame e 1 mm) a ached o
a low me e (Pe iFlux 5000; Pe imedAB, S ockholm, Sweden)
was placed on he hinned skull (a ea =0.8 mm2, hickness
=0.2 mm) o e he MCA e i o y (4 mm la e al o B egma)
o con inuously eco d he ela i e ce eb al blood low (CBF)
du ing he occlusion. Only animals wi h a educ ion o CBF
highe han 60%, and wi h e ec i e epe usion a e e ie ing
he ilamen , we e included in he s udy.
Ligh Pene a ion In o he Ra B ain
Be o e es ing he e ec o LED-PBM on ischemic animal
models, he pene a ion a e o ligh h ough di e en a b ain
issues ( oden skin, skull, and b ain) was pe o med. Fo his
pu pose, he LED a ay lamp p e iously used in cellula s udies
was employed (830 nm, 10 mW/cm2). To de e mine he ligh
pene a ion, a Newpo 835 Lase Pico-Wa Digi al Powe Me e
wi h 818 UV de ec o (Newpo Co po a ion, F anklin, MA,
USA) was used.
Ligh pene a ion h ough issues was measu ed wi h isola ed
a samples o skin, skull, and b ain issues. To e alua e he
in luence o he powe in he pene a ion o ligh h ough
di e en issue samples, he LED lamp was loca ed 15 cm
away om he sample. To pe o m his analysis, a sample
holde was made om black Plexiglas R
, and consis s o a
base wi h an 8 mm hole in i s cen e whe e we placed he
samples and 3 black Plexiglas R
walls. Two pho ode ec o s le he
simul aneous measu emen o ligh powe ansmi ed h ough
he di e en issues we placed on he holde , and a po ion o he
inciden ligh .
Ligh pene a ion was calcula ed and pe o med 3 imes (n=
2 animals). Da a we e p ocessed using a p og am designed o
ha pu pose in C++ (Bo land, Texas, USA). Fi s , we measu ed
he powe wi hou any sample be ween he ligh sou ce and he
de ec o (ligh in mW/cm2ac oss o ai ) and hen we calcula ed
he pe cen age o ansmi ed ligh when samples we e placed
on he sys em (ligh in mW/cm2ac oss o issue). Rela i e
pene a ion pe cen age alues we e calcula ed using he ollowing
o mula ob ained om a p e ious s udy (25):
100 % XLigh ac oss issue [mW
cm2]
Ligh [mW
cm2](1)
In i o LED Pho obiomodula ion
Neu o eco e y Analysis
Because TBI ep esen s he ype o neu ological damage closes o
ischemic inju y, we used simila con inues LED PBM p o ocol in
ou ischemic animal model (5). In ou case, in o de o educe
as much as possible he s ess ul e ec s ha LED-PBM could
induce on he expe imen al animals and in e e e in he MRI
expe imen s (26), we decided o es wo di e en con inuous
LED-PBM p o ocols; G oup 1: 30 min o ligh ea men , 1
day/week du ing 12 weeks (n=6), and G oup 2: 30 min o
ligh ea men , 3 days/week du ing 12 weeks (n=6). An
addi ional ischemic g oup wi hou ea men was used as con ol
G oup (n=6). Animals we e andomly assigned o he h ee
expe imen al g oups. LED-PBM was ini ia ed 24 h a e he onse
o ischemia in awake and eely mo ing condi ions. Head animals
we e sha ed be o e ligh s imula ion and he ligh exposu e was
pe o med indi idually.
Based on he cellula in i o expe imen s and ligh
pene a ion analysis on ce eb al issues, LED-PBM was induced
wi h a powe densi y o 10 mW/cm2 o 1 cm2o a ea (a 15 cm o
he sou ce) wi h a ligh in ensi y o 18 J/cm2.
Magne ic Resonance Imaging
Magne ic Resonance Imaging and MRI s udies we e conduc ed
on a 9.4 T ho izon al bo e magne (B uke BioSpin, E lingen,
Ge many) wi h 20 cm wide ac i ely shielded g adien coils (440
mT/m). MR images we e acqui ed using a c oss-coil se up,
consis ing o a linea bi dcage esona o o 7 cm diame e ,
o ansmission, and a 2 ×2 a ayed su ace coil o
signal de ec ion.
In a c olume was de e mined by means o T2-weigh ed
images acqui ed a 24 h (de ined as 0) and 1, 3, 5, 7, and 12 weeks
a e he onse o ischemia using a mul i-slice mul i-spin-echo
(MSME) sequence wi h he ollowing acquisi ion pa ame e s:
epe i ion ime (TR) =3 s, 16 echoes wi h echo ime (TE) =9 ms,
75 KHz spec al bandwid h, lip angle (FA) =180◦, 14 slices o
1 mm hickness, ield o iew (FOV) o 19.2 ×19.2 mm2(wi h
sa u a ion bands o supp ess signal ou side his FOV), a ma ix
size o 192 ×192 (iso opic in-plane esolu ion o 0.1 mm/pixel),
and implemen ed wi hou a supp ession op ion.
All images we e p ocessed using he ImageJ so wa e
(Rasband, W.S., ImageJ, NIH) on an independen compu e
wo ks a ion. In a c olumes we e quan i ied om T2-weigh ed
images a e aged all echoes by a esea che blinded o he
ea men g oups.
BOLD MRI Me hodology
Func ional MRI was achie ed using BOLD con as , s a ing a
leas 1 h a e he induc ion o he seda ion. MRI expe imen s
we e pe o med 1 week be o e MCAO and 0 (24 h), 1, 3,
5, 7, and 12 weeks a e ischemia, acqui ing se s o 115
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A gibay e al. Pho obiomodula ion A e Ce eb al Ischemia
consecu i e spin-echo Echo-Plana -Images (SE-EPI) wi h TE/TR
=30/3,000 ms, 5 slices o 2 mm hickness, FOV o 19.2 ×19.2
mm2co e ed by a 64 ×64 ma ix, i.e., an in-plane esolu ion
o 0.3 mm/pixel. Home-buil s eel needle (30 G) elec odes we e
placed subcu aneously in o bo h o epaws. Al e na ing unila e al
o epaw s imula ion was pe o med using ec angula pulses
(2 mA, 8 Hz, 0.3 ms, STG2004 s imula o om Mul i Channel
Sys ems, Ge many) ollowing a pa adigm in which a block,
composed by a 45 s es ing pe iod and 15 s ac i a ion pe iod,
was epea ed i e imes, ending wi h an addi ional 45 s es ing
pe iod o a o al expe imen al ime o 5 min 45 s (5 ×[45
OFF +15 ON] +45 OFF). BOLD MRI echnique is based on
he di e en ial magne ic p ope ies o oxygena ed (diamagne ic)
and deoxygena ed (pa amagne ic) hemoglobin. Upon neu al
ac i a ion, changes in local CBF, ce eb al blood olume (CBV),
and ce eb al me abolic a e o oxygen consump ion (CMRO2)
leads o a locally inc eased a io o oxygena ed o e deoxygena ed
hemoglobin, esul ing in an enhancemen in T2∗-weigh ed signal
in ensi y (27).
BOLD MRI was conduc ed, al e na ing h ee imes
be ween each hemisphe e, and animals we e allowed
o es o 10 min be ween s imula ion sessions. A e
p e-p ocessing, he BOLD signal was modeled as he
con olu ion o he applied s imula ion design (block o
e en - ela ed design) wi h he hemodynamic esponse
unc ion (HRF) in o de o ha e a be e es ima ion o
he ue design- ela ed BOLD signal. Finally, s a is ics was
pe o med on he model es ima es de ining b ain egions
ac i a ed (27).
S a is ical pa ame ic ac i a ion maps we e cons uc ed wi h
he so wa e STIMULATE (28). The ime cou se o each
pixel du ing o epaw s imula ion was examined using a pai ed
S uden ’s - es (p<0.05) acco ding o p e ious s udies (29,
30), compa ing he mean baseline signal alue wi h he mean
o he s imula ed signal alue. Only clus e s ha included a
leas ou adjacen ac i a ed pixels we e conside ed as posi i e
ac i a ion a eas.
Senso imo o Tes : Cylinde Tes
A cylinde es (31) was used o e alua e he unc ional de ici
and was pe o med 1 week p io o MCAO ( o assess he basal
locomo o symme y o he animals) and 24 h (de ined as 0), 1,
3, 5, 7, and 12 weeks a e ischemia du ing he da kness cycle
(Supplemen a y Ma e ial).
B ain His ological Analysis
Animals we e sac i iced 12 weeks a e su gical ea men , and
b ain issue was p ocessed o his ological analysis, ollowing
he p ocedu e desc ibed elsewhe e (32). Neu onal nuclea
p o ein (Fox3) and glial ib illa y acidic p o ein (GFAP) (Sigma-
Ald ich) labeling we e combined wi h DAPI s ain (The mo
Fishe Scien i ic, Wal ham, MA, USA). In addi ion, neu ogenesis
was examined in he s ia al egion by immunolabeling a
p oli e a ing cell ma ke (Ki-67, Abcam) and doubleco in (DCX;
an imma u e p ogeni o cell ma ke ) (Dako, Ba celona, Spain).
Dobleco in and Ki-67 posi i e, GFAP-posi i e and Fox3-posi i e
cells and nuclei we e coun ed manually in ImageJ so wa e
(Supplemen a y Ma e ial).
S a is ical Analysis
All da a a e p esen ed as he mean and s anda d de ia ion o
he mean (mean ±SD). The Kolmogo o -Smi no es was
pe o med in o de o check he no mali y o dis ibu ions.
Two-way analysis o a iance (ANOVA) ollowed by pos -hoc
Bon e oni e alua ion was used o mul iple g oups o de e mine
signi ican di e ences. S a is ical signi icance was se a p<
0.05. The s a is ical analysis was conduc ed using IBM SPSS
S a is ics o Macin osh, Ve sion 18.0. (SPSS Inc., IL, USA).
S a is ical analysis was pe o med by a blinded esea che o he
ea men . Sample size was based on expec ed a iances: α=0.05
(con idence le el: 95%) and β=0.20 (powe : 80%).
To achie e a powe o 80% o de ec di e ences in he con as
o he hypo hesis null H0 (LED-PBM ea men could educe
he ischemic lesion by 30%) aking in o accoun ha he le el o
FIGURE 1 | In i o s udy o e alua e: (A) cellula p oli e a ion; cell coun was ound signi ican inc eased a low emission powe s and exposu es imes. (B) Viabili y;
pho os imulal ion did no p oduce oxic side e ec s in i o. A wo-way analysis o a iance (ANOVA) ollowed by pos -hoc Bon e oni e alua ion was used. (n=4;
*p<0.5; **p<0.01; ***p<0.001). Lamp emission powe a h ee di e en exposu e ime (6, 12, and 18 min).
F on ie s in Neu ology | www. on ie sin.o g 4Augus 2019 | Volume 10 | A icle 911
A gibay e al. Pho obiomodula ion A e Ce eb al Ischemia
signi icance is 5%, he numbe o animal in each g oup is n=6.
The calcula ion has been made wi h he p og am Ene 3.0.
RESULTS
In i o Analysis o Pho obiomodula ion
Analysis o cellula p oli e a ion a e PBM was signi ican ly
inc eased wi h a densi y powe o 1 mW in all di e en exposu e
imes es ed, howe e highe in ensi ies did no show highe
e ec s (Figu e 1A). To de e mine i he PBM was associa ed wi h
cellula dea h, LHD assay was pe o med (Figu e 1B). Al hough
i did no achie e a signi ican di e ence, a highe cell dea h was
obse ed only in hose cells ea ed o 18 min wi h 5 mW o
ligh exposu e. Based on hese in i o esul s, we es ablished 0.2–
0.6 J/cm2(Supplemen a y Table 1) as he op imal ligh ene gy
in ensi y needed o induce any bene icial e ec on he neu onal
issue in he in i o animal s udy.
In i o Pho obiomodula ion E ec on
Func ional Reco e y and Ischemic Lesion
A o al numbe o 20 animals we e used in he s udy: 2 animals
in ol ed in he ligh pene a ion analysis in o he b ain, and 18
animals in he neu o eco e y analysis. Two animals died du ing
he su ge y, and 3 animals did no mee he inclusion c i e ia in
ela ion o he educ ion o CBF.
Ligh pene a ion was de e mined h ough he di e en
issues, esul ing 16 ±3% h ough he sha ed skin, 10
±3% h ough he skull, 0.9 ±0.4% h ough igh b ain
hemisphe e (ischemic), and 0.9 ±0.4% h ough le b ain
hemisphe e (heal hy).
Analysis o ligh pene a ion s udy h ough di e en biological
issues om a ( oden skin, skull, and b ain) showed ha he
decay o ligh in ensi y a e along he deep b ain issue esul ed
abou 2.5 ×10−2J/cm2. Based on his analysis, ligh PBM
in ensi y was adjus ed o 18 J/cm2 ha allowed inducing on b ain
co ex wi h a ligh in ensi y o 0.28 J/cm2, close o ha alues
es ablished in he p e ious in i o s udy (0.2–0.6 J/cm2).
Func ional MRI was pe o med in all animals p io o
he MCAo and all o hem exhibi ed bila e al BOLD signal
(Figu e 2). Ne e heless, 12 weeks a e PBM ea men , animals
did no show he eco e y BOLD signal in he ischemic
hemisphe e wi h none o he p o ocols used. In line wi h MRI
esul s, analysis o he p o ile o la e al index ( ha de e mina es
he o elimb asymme y) analyzed du ing he pe iod o ollow up,
did no show a signi ican imp o emen in he unc ional de ici
in he ea ed g oups espec o he con ol g oup (Figu e 3A).
FIGURE 2 | Func ional b ain eco e y obse a ion. (A) Rep esen a i e BOLD images ob ained du ing p e-ce eb al ischemia, 5 and 12 weeks a e he insul in bo h
hemisphe es. (B) Al e na ing unila e al o epaw s imula ion was pe o med using ec angula pulses. BOLD MRI was conduc ed al e na ing h ee imes be ween each
hemisphe e, and animals we e allowed o es o 10 min be ween s imula ion sessions. The ime cou se o each pixel du ing o epaw s imula ion was examined using
a pai ed S uden ’s - es (p<0.05). Only clus e s ha included a leas ou adjacen ac i a ed pixels we e conside ed as posi i e ac i a ion a eas. (n=6
animals/g oup). All animals exhibi ed BOLD signal in bo h hemisphe es (measu ed sepa a ely) p io o he ce eb al ischemia. The animals we e ollowed o 12 weeks
and all o hem exhibi ed BOLD signal in he con ala e al side bu none o hem showed BOLD ac i a ion in he ipsila e al side.
F on ie s in Neu ology | www. on ie sin.o g 5Augus 2019 | Volume 10 | A icle 911
A gibay e al. Pho obiomodula ion A e Ce eb al Ischemia
FIGURE 3 | In i o s udy: (A) Cylinde es esul s ob ained om he h ee s udied g oups. (B) Ischemic olume esul s o he h ee g oups; No signi ican di e ences
we e obse ed be ween g oups up o 12 weeks. (C) T2-weigh ed MR images o a ep esen a i e b ain o each pho os imula ed g oup.
None heless, he ea ed g oups showed a educ ion o he use o
he impai ed o elimb, bu he di e ence did no each s a is ical
signi icance compa ed o he con ol g oup.
Figu e 3B de ailed he e olu ion o he lesion sizes o ischemic
animals. All he animals included in he s udy p esen ed a basal
ischemic lesion (<18%) de e mined by MRI. Acco ding o ou
expe imen al design, a educ ion o 30% in he ischemic lesion
was no ound. In line wi h his, no signi ican di e ences in
olume educ ion we e obse ed o he g oups compa ed o
con ol. Unde hese expe imen al condi ions, analysis o in a c
olume showed ha LED pho os imula ion doesn’ educe he
ischemic lesion in he ea ed animals compa ed wi h he con ol
g oup (Figu e 3C). Besides, suba achnoid hemo hages we e
ound in all g oups (con ol g oup included) due o he la ge
ischemic lesions induced wi h he MCAO model.
His ological analysis o a neu onal ma ke (Fox3), glial ma ke
(GFAP), and neu ogenesis pe o med 12 weeks a e s oke onse
did no e eal exp ession di e ences in ei he immunolabeling
(Figu e 4). All animals used we e included in he s udy and no
mo ali y associa ed o PBM was obse ed.
DISCUSSION
In he p esen s udy, PBM wi h 830 nm LED a low ene gy
in ensi y (0.28 J/cm2) did no educe he in a c olume o
imp o e he unc ional eco e y 12 weeks a e he onse o he
ischemia. The elec ical ac i i y and he e o e MRI signal in
esponse o he o epaw s imula ion was obse ed exclusi ely in
he con ala e al hemisphe e, e idencing no unc ional eco e y
a e ce eb al ischemia in he ipsila e al side. O he he apeu ic
s udies wi h e y di e en ea men s, as s em cells, bu
whe e MRI was used o e alua e he unc ional eco e y a e
ischemic s oke ha e e idenced ha he egaining o BOLD
signal was p oduced be ween he 7 h and he 10 h week a e
he adminis a ion (33). The e o e, and al hough he he apy
is comple ely di e en , we coinsu ed ha 12 weeks we e a
p uden ial pe iod o see some e ec o LED-PBM h ough MRI.
Nume ous s udies ha e ound posi i e e ec s con e ed by
PBM om dec easing in lamma ion, alle ia ing oxida i e s ess,
and imp o ing mi ochond ial unc ion (5–10). Some s udies
ha e e idenced educ ion o ce eb al ischemic lesions, be e
unc ional ou come o cell neu ogenesis in di e en animal
models (8–10). Howe e , in line wi h ou esul s, hese PBM
he apies we e mainly applied wi hin he i s 6 h om s oke
onse , while no bene i was obse ed a longe ime-poin s.
Simila ly, he clinical Phase III (NEST-3) s udy abou he
he apeu ic e ec o PBM ha did no ind signi ican bene i in
s oke pa ien s (14).
In ou s udy, 24 h a e s oke was selec ed as he ime-
poin o s a he LED-PBM ea men , in o de o s udy epai -
based he apies complemen acu e he apies (in a enous o
in aa e ial ib inolysis, h ombec omy). Besides, wi hin he i s
24 h a e s oke, pa ien equi es clinical ca e, MRI, o Compu ed
F on ie s in Neu ology | www. on ie sin.o g 6Augus 2019 | Volume 10 | A icle 911
A gibay e al. Pho obiomodula ion A e Ce eb al Ischemia
FIGURE 4 | His ological analysis o neu ons (Fox3), as ocy es (GFAP), and neu ogenesis (DCX and Ki 67) o animals om each expe imen al g oup a 12 weeks a e
ischemic lesion. Co ical (*) and subco ical b ain egions (**).
Tomog aphy (CT) s udies, which would limi he possibili y o
implemen ing he PBM p o ocols.
We ha e e alua ed he e olu ion o he ischemic b ain by
using MRI and MRI a e in i o PBM, howe e ou s udy
also shows some weaknesses. Fi s , he use o an ischemic
cell model [oxygen-glucose dep i a ion (OGD)] would help
unde s and basic biochemical and cellula mechanisms in ol ed
in he e ec s o ligh . Second, i would be possible ha p olonged
indi idual housing o animal p e en ed op imal eco e y and
nega i ely impac ed o de ec a he apeu ic e ec . Thi d, a
deep his ological analysis would con ibu e o elucida e he
ligh e ec s in he damaged b ain a a cellula le el, o e en
o desc ibe possible endogenous angiogenic o neu ogenic
p ocesses s imula ed by he LED ligh . In addi ion, in his
s udy we ha e used a MCAO model ha induces la ge
ischemic lesion wi h a high senso imo o co ex a ec a ion,
ha can limi he unc ional eco e y as i has been epo ed
in o he s udies (33), and po en ially explain he lack o PBM
e ec obse ed in ou s animals. Indeed, his could also e lec
ha his ea men could be mo e app op ia e o smalle
s oke lesions.
Hund eds o clinical ials es ing p o ec i e agen s ha e ailed
despi e e icacy in expe imen al models. In his line, om a long
ime ago, nega i e and neu al s udies ha e been equi ed o help
mo e no el ea men s o human and educe he isk o ailu e
in u u es clinical ials (34,35). The e o e, hese new indings
y o con ibu e o op imize he po en ial use PBM as no el
and non-in asi e he apy o ischemic s oke be o e mo ing o
clinical s udies.
CONCLUSION
Unde ou expe imen al condi ions, we ha e no obse ed a
bene icial e ec o LED pho obiomodula ion applied 24 h a e
he onse o ischemia in animal models, mainly in e ms o
neu onal eco e y de e mined by MRI. Fu he p eclinical
s udies in his ield a e necessa y in o de o op imize he
he apeu ic window o his no el he apy, he mechanism
associa ed and o educe he isk o ailu e in u u es clinical ials.
DATA AVAILABILITY
All da ase s gene a ed o his s udy a e included in he
manusc ip /Supplemen a y Files.
AUTHOR CONTRIBUTIONS
BA, RI-R, and MP-M pe o med he MRI scanning and MRI
and p epa a ion o manusc ip . AV-P, AD, CC-P, and EL-A
con ibu ed in he animal su ge ies and beha io analysis
and p epa a ion o manusc ip . VM and CM designed ligh
expe imen s and analysis and con ibu ed o he p epa a ion o
he manusc ip . BA, JC, TS, and FC con ibu ed o he execu ion
o expe imen s, da a analysis, p epa a ion o manusc ip , and
expe imen al design. All au ho s ha e ead he manusc ip , ag ee
ha he wo k is eady o submission o a jou nal, and accep
esponsibili y o he manusc ip ’s con en .
F on ie s in Neu ology | www. on ie sin.o g 7Augus 2019 | Volume 10 | A icle 911
A gibay e al. Pho obiomodula ion A e Ce eb al Ischemia
FUNDING
This p ojec was pa ially suppo ed by g an s om he Spanish
Minis y o Economy and Compe i i eness (SAF2014-56336-R
and SAF2017-84267-R), Xun a de Galicia (Conselle ía
Educación: GRC2014/027 and IN607A2018/3), Ins i u o de
Salud Ca los III (PIE13/00024 and PI17/01103), Spanish
Resea ch Ne wo k on Ce eb o ascula Diseases RETICS-
INVICTUS PLUS (RD16/0019), and by he Eu opean
Union FEDER p og am. Fu he mo e, TS (CPII17/00027)
and FC (CP14/00154) a e ecipien s o esea ch con ac s
om Miguel Se e P og am o Ins i u o de Salud Ca los
III. The sponso s did no pa icipa e in s udy design,
collec ion, analysis, o in e p e a ion o he da a, in
w i ing he epo , o in he decision o submi he pape
o publica ion.
ACKNOWLEDGMENTS
We would like o hank Quan um De ices Inc. (Ba ne eld, WI,
USA) o his coope a ion o he acquisi ion o he LED sys em
used in his wo k.
SUPPLEMENTARY MATERIAL
The Supplemen a y Ma e ial o his a icle can be ound
online a : h ps://www. on ie sin.o g/a icles/10.3389/ neu .
2019.00911/ ull#supplemen a y-ma e ial
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Con lic o In e es S a emen : The au ho s decla e ha he esea ch was
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