F on ie s in Neu oana omy 01 on ie sin.o g
E alua ion o he neu op o ec i e
e icacy o he g amine de i a i e
ITH12657 agains NMDA-induced
exci o oxici y in he a e ina
JohnnyDi Pie domenico
1†, Alejand oGallego-O ega
1†‡,
Ma íaNo e-Muñoz
1, Bea izVidal-Villegas
1‡, IsaacB a o
2,3,
Ma íaBoluda-Ruiz
1, Jose ManuelBe nal-Ga o
1,
I ánFe nandez-Bueno
4, JoseCa losPas o -Jimeno
4, Ma ía
PazVillegas-Pé ez
1, Ma celinoA ilés-T igue os
1
*, C is obalde
los Ríos
2,3* and ManuelVidal-Sanz
1
1 Depa amen o de O almología, Uni e sidad de Mu cia e IMIB-A ixaca, Mu cia, Spain, 2 Ins i u o de
In es igación Sani a ia, Hospi al Uni e si a io de la P incesa, Mad id, Spain, 3 Depa amen o de
Ciencias Básicas de la Salud, Uni e sidad Rey Juan Ca los, Alco cón, Spain, 4 Ins i u o Uni e si a io de
O almobiología Aplicada (IOBA), Re ina G oup, Uni e sidad de Valladolid, Valladolid, Spain
Pu pose: The aim o his s udy was o in es iga e, he neu op o ec i e e ec s o
a new G amine de i a i e named: ITH12657, in a model o e inal exci o oxici y
induced by in a i eal injec ion o NMDA.
Me hods: Adul Sp ague Dawley a s ecei ed an in a i eal injec ion o 100 mM
NMDA in hei le eye and we e ea ed daily wi h subcu aneous injec ions o
ITH12657 o ehicle. The bes dose– esponse, he apeu ic window s udy, and
op imal ea men du a ion o ITH12657 we e s udied. Based on he bes su i al o
B n3a + RGCs ob ained om he abo e-men ioned s udies, he p o ec i e e ec s o
ITH12657 we e s udied in i o ( e inal hickness and ull- ield Elec o e inog aphy),
and ex i o by quan i ying he su i ing popula ion o B n3a + RGCs, αRGCs and
hei sub ypes α-ONsRGCs, α-ON RGCs, and α-OFFRGCs.
Resul s: Adminis a ion o 10 mg/kg ITH12657, s a ing 12 h be o e NMDA
injec ion and dispensed o 3  days, esul ed in he bes signi ican p o ec ion
o B n3a  +  RGCs agains NMDA-induced exci o oxici y. In i o, ITH12657-
ea ed a s showed signi ican p ese a ion o e inal hickness and unc ional
p o ec ion agains NMDA-induced e inal exci o oxici y. Ex i o esul s showed
ha ITH12657 a o ded a signi ican p o ec ion agains NMDA-induced
exci o oxici y o he popula ions o B n3a  +  RGC, αRGC, and αONs-RGC, bu
no o he popula ion o αOFF-RGC, while he popula ion o α-ON RGC was
ully esis an o NMDA-induced exci o oxici y.
Conclusion: Subcu aneous adminis a ion o ITH12657 a 10 mg/kg, ini ia ed
12  h be o e NMDA-induced e inal inju y and con inued o 3  days, esul ed in he
bes p o ec ion o B n3a +  RGCs, αRGC, and αONs-RGC agains exci o oxici y-
induced RGC dea h. The popula ion o αOFF-RGCs was ex emely sensi i e
while α-ON RGCs we e ully esis an o NMDA-induced exci o oxici y.
KEYWORDS
NMDA exci o oxici y, glaucoma, e ina ganglion cell, neu op o ec ion, B n3a + RGCs,
SD-OCT, αRGCs, e ina calcium blocke
OPEN ACCESS
EDITED BY
Jose Paulo And ade,
Uni e si y o Po o, Po ugal
REVIEWED BY
Edua do Puelles,
Miguel He nández Uni e si y o Elche, Spain
Glyn Chidlow,
Royal Adelaide Hospi al, Aus alia
Luis Pe ez de Se illa,
Uni e si y o Cali o nia, Los Angeles,
Uni edS a es
*CORRESPONDENCE
Ma celino A ilés-T igue os
[email p o ec ed]
C is obal de los Ríos
[email p o ec ed]
†These au ho s sha e i s au ho ship
‡PRESENT ADDRESSES
Alejand o Gallego-O ega,
Depa men o Oph halmology, Da id Ge en
School o Medicine, S ein Eye Ins i u e,
Uni e si y o Cali o nia, Los Angeles, Los
Angeles, Uni ed S a es
Bea iz Vidal-Villegas,
Depa men o Oph halmology, S Thomas’
Hospi al, Guy’s and S Thomas T us , London,
Uni ed Kingdom
RECEIVED 08 No embe 2023
ACCEPTED 16 Janua y 2024
PUBLISHED 13 Feb ua y 2024
CITATION
Di Pie domenico J, Gallego-O ega A,
No e-Muñoz M, Vidal-Villegas B, B a o I,
Boluda-Ruiz M, Be nal-Ga o JM,
Fe nandez-Bueno I, Pas o -Jimeno JC,
Villegas-Pé ez MP, A ilés-T igue os M, de los
Ríos C and Vidal-Sanz M (2024) E alua ion o
he neu op o ec i e e icacy o he g amine
de i a i e ITH12657 agains NMDA-induced
exci o oxici y in he a e ina.
F on . Neu oana . 18:1335176.
doi: 10.3389/ nana.2024.1335176
COPYRIGHT
© 2024 Di Pie domenico, Gallego-O ega,
No e-Muñoz, Vidal-Villegas, B a o, Boluda-
Ruiz, Be nal-Ga o, Fe nandez-Bueno,
Pas o -Jimeno, Villegas-Pé ez, A ilés-
T igue os, de los Ríos and Vidal-Sanz. This is
an open-access a icle dis ibu ed unde he
e ms o he C ea i e Commons A ibu ion
License (CC BY). The use, dis ibu ion o
ep oduc ion in o he o ums is pe mi ed,
p o ided he o iginal au ho (s) and he
copy igh owne (s) a e c edi ed and ha he
o iginal publica ion in his jou nal is ci ed, in
acco dance wi h accep ed academic
p ac ice. No use, dis ibu ion o ep oduc ion
is pe mi ed which does no comply wi h
hese e ms.
TYPE O iginal Resea ch
PUBLISHED 13 Feb ua y 2024
DOI 10.3389/ nana.2024.1335176
Di Pie domenico e al. 10.3389/ nana.2024.1335176
F on ie s in Neu oana omy 02 on ie sin.o g
Highligh s
• Adminis a ion o 10 mg/kg ITH12657, s a ing 12 hou s be o e
NMDA injec ion and dispensed o 3 days, esul ed in he bes
signi ican p o ec ion o B n3a+RGCs agains NMDA-
induced exci o oxici y.
• ITH12657- ea men showed a signi ican p ese a ion o e inal
hickness when compa ed o ehicle- ea men .
• T ea men wi h ITH12657 esul ed, in smalle educ ions o
pSTR and b-wa e ampli udes compa ed o hose ea ed wi h
ehicle, indica ing ha ITH12657 ea men o e ed unc ional
p o ec ion agains NMDA-induced e inal exci o oxici y.
• ITH12657 a o ded a signi ican p o ec ion agains NMDA-
induced exci o oxici y o he popula ions o B n3a+RGC, αRGC,
and αONs-RGC, bu no o he popula ion o αOFF-RGC.
1 In oduc ion
The e ina has been la gely used o explo e he esponse o adul
cen al ne ous sys em (CSN) neu ons o inju y, p o ec ion and
egene a ion (McKe ache e  al., 1990a,b; Whi eley e  al., 1998;
A iles-T igue os e al., 2000; Vidal-Sanz e al., 2002, 2015b, 2017;
Lindq is e al., 2004).
The e a e se e al ma ke s ha allow he iden i ica ion o la ge
popula ions o oden RGCs (pan-ma ke s) such as B n3a o RBPMS
(Nadal-Nicolas e al., 2023) o speci ic ela ed RGCs g oups, also named
subclasses, such as he in insically pho osensi i e RGCs (Vidal-Villegas
e al., 2021a,b) o he al a RGCs (αRGCs; Gallego-O ega e al., 2022),
among o he s (T an e al., 2019). Immunohis ochemical s udies using a
combina ion o ma ke s allow he s udy, in pa allel bu independen ly,
o how di e en RGCs espond o di e en e inal inju ies (Vidal-Sanz
e al., 2015a; Agudo-Ba iuso e al., 2016; Di Pie domenico e al., 2022b)
and p o ec ion (Valien e-So iano e  al., 2015; Ro e e e  al., 2016;
Sanchez-Migallon e al., 2018). Fo ins ance, he exp ession o B n3a by
oden RGCs has allowed o iden i ica ion o he main popula ion o
RGCs, which accoun s o app oxima ely 96% o he RGC popula ion
(Nadal-Nicolas e al., 2014).
Recen s udies ha e sugges ed up o 46 di e en ypes o oden
e inal ganglion cells (RGCs), based on a ious c i e ia such as
hei shape, hei esponse o ligh , hei b ain connec ions and he
molecula exp ession o pa icula genes (Nassi and Callaway,
2009; Sanes and Masland, 2015; Baden e al., 2016; Rheaume e al.,
2018; Ch is ensen e al., 2019; Sweeney e al., 2019; Goe z e al.,
2022; Huang e  al., 2022). Indeed, he use o eadily a ailable
immunocy ochemical ma ke s has allowed o iden i y he αRGCs,
a subclass o RGCs well-cha ac e izaed by hei la ge size, as
conduc ing eloci y and mono-s a i ied dend i ic a bo s wi hin
di e en s a a o he inne e inal laye (Gallego-O ega e al.,
2021, 2022). Mo eo e , his class acqui es pa icula ele ance
because i may cons i u e he oden o hologue o he midge
RGCs, he mos abundan popula ion o RGCs subse ing isual
acui y and colo ision in p ima es (Hahn e  al., 2023). This
popula ion in he albino a accoun s o app oxima ely 2.2% o all
RGCs (Gallego-O ega e al., 2021) wi h ou ecen ly desc ibed
sub ypes acco ding o hei s a i ica ion in he inne laye and
unc ional esponse o ligh ; ON sus ained (αONsRGCs), ON
ansien (αON RGCs), OFF sus ained (αOFFsRGCs) and OFF
ansien (αOFF RGCs; Gallego-O ega e  al., 2022). Fo he
p esen s udies, weiden i y and quan i y αRGCs, αONsRGCs,
αON RGCs and αOFFRGCs.
Glu ama e is he main exci a o y neu o ansmi e in he (CNS),
including he e ina whe e i media es ansmission h ough he main
di ec pa hway, om pho o ecep o s o bipola s and RGCs ha e
glu ama e ecep o s, including he NMDA ecep o s, and hei
o e s imula ion can cause neu onal exci o oxici y, ha is cell dea h by
excessi e ac i a ion. The NMDA ecep o s a e pe meable o calcium
and sodium and ha e impo an oles in neu onal plas ici y, lea ning
and memo y. Howe e , excessi e NMDA ecep o s imula ion may
esul in changes in he Na+/K+ balance and en y o la ge amoun s o
Ca
2+
in o he cell (Mane e al., 1989) which may esul in di ec
damage by ac i a ing enzymes ha damage DNA and cell memb anes
(S a o skaya and K is al, 2005) o by inducing apop osis h ough
ac i a ion o cAMP (Ha dingham e  al., 2002). Glu ama e
exci o oxici y is belie ed o play an impo an ole in he loss o RGCs
in a ious e inal inju ies (Lucas and Newhouse, 1957; Choi, 1988),
such as glaucoma (D eye e al., 1996; Vo we k e al., 2004; Izzo i
e al., 2006; Tezel, 2013), ansien ischemia (Lam e al., 1997) and
op ic ne e inju y (Schue au e al., 2000; Ke me e al., 2001). In
addi ion, can also play a key ole in many CNS diseases ha in ol e
neu onal dea h (Almasieh e al., 2012). Animal models o NMDA-
induced e inal exci o oxici y a e o en used o in es iga e molecula
mechanisms o RGC apop osis and he way o p o ec hem
(Kobayashi e al., 2017; Fah en hold e al., 2018; Picha a am e al.,
2018; Lambuk e  al., 2019). The e ec s o NMDA-media ed
exci o oxici y on RGCs ha e been p e iously in es iga ed in adul a s
(Gomez-Vicen e e al., 2015; Vidal-Villegas e al., 2019) and mice
(DePa is e al., 2012; Wang e al., 2018). Calcium channel blockade
can imp o e RGC su i al a e exci o oxici y-induced inju y by
educing calcium in lux in o neu ons and p e en ing cell damage.
Calcium channel blocke s can ac on di e en ypes o channels, such
as L, T, N o P/Q channels. Examples o calcium channel blocke s a e
nimodipine, ni edipine and e apamil (Chen e al., 2022; E angeliou
e al., 2023).
ITH12657 (1-benzyl-5-me hyl-3-(pipe idin-1-ylme hyl-1H-
indole, 2) is a G amine de i a i e ha exhibi s neu op o ec i e
p ope ies agains Alzheime ’s disease (AD; Laja in-Cues a e al.,
2016; Gonzalez e al., 2018) and ep esen a hope ul s a egy o
he ea men o neu odegene a ion. Among he mechanisms o
ac ion exe ed by ITH12657 a e he blockade o ol age-dependen
calcium channels (VGCC), which p e en s excess calcium en y
in o neu ons and associa ed cell damage, and he p e en ion o
inhibi ion o p o ein phospha ase 2A (PP2A), an enzyme ha
egula es Tau p o ein phospho yla ion o neu o ib illa y angles
(Gonzalez e al., 2018).
In his wo k, we u he in es iga e he esponse o wo di e en
RGC popula ions, he gene al B n3a- and he α-RGCs wi h hei
sub ypes, o e inal exci o oxici y induced by in a i eal injec ion o
100 mM NMDA and p o ec ion wi h sys emic adminis a ion o
ITH12567. The pu pose o his s udy in adul albino a s was wo old.
In an ini ial g oup o expe imen s, wecha ac e ized he p o ec ion
a o ded by ITH12657 on he su i al o B n3a
+
RGCs ollowing a
single in a i eal injec ion o 5 μL con aining 100 mM NDMA. These
s udies indica ed ha he bes dose, ime window and leng h o
ea men egime o ITH12657 we e 10 mg/kg, s a ing 12 h be o e
in a i eal NMDA injec ion and main ained o 3 addi ional days
Di Pie domenico e al. 10.3389/ nana.2024.1335176
F on ie s in Neu oana omy 03 on ie sin.o g
ollowing in a i eal NMDA. A second g oup o expe imen s u he
in es iga ed he neu op o ec i e e ec s o ITH12657 on he e ina,
and his was assessed longi udinally in i o wi h mo phological and
unc ional echniques, and ex i o by coun ing and mapping in e inal
whole-moun s o al numbe s o su i ing B n3a
+
RGCs, αRGCs and
hei sub ypes (αONsRGCs, αON RGCs o αOFFRGCs).
2 Me hods
2.1 Animal handling
Adul emale albino Sp ague Dawley (SD; n = 144) a s aged
3 mon hs and weighing app oxima ely 180–220 g we e used since
he e is su icien e idence in he li e a u e o indica e ha da a
ob ained om emale oden s a e no mo e a iable han hose om
male oden s (Becke e al., 2016; Bee y, 2018), o a be e compa ison
wi h p e ious published wo k, and hei be e docili y and smalle
size. The a s we e b ed and main ained in he Expe imen al Animal
Facili y o he Uni e si y o Mu cia unde con olled ligh (12-h ligh –
da k cycles wi h ligh in ensi y wi hin he cages anging om 5 o
30 lx) and empe a u e condi ions (23–24°C), and wi h access o ood
and wa e ad libi um.
The animals we e ea ed acco ding o he cu en Eu opean and
na ional egula ions and, speci ically, acco ding o Di ec i e 86/609/
EEC, 2010/63/EU on he p o ec ion o animals used o scien i ic
pu poses, he R.D.1201/2005 on he p o ec ion o animals used o
expe imen al and o he scien i ic pu poses, he Law 32/2007 o he
ca e o animals, in hei exploi a ion, anspo , expe imen a ion, and
slaugh e , he Associa ion o Resea ch in Vision and Oph halmology
(ARVO) guidelines o he use o animals in oph halmic and isual
sys em expe imen a ion. Animal expe imen s we e app o ed by he
Uni e si y o Mu cia E hical animal s udies commi ee (P o ocols
A13171103, A13170110 and A13170111).
2.2 Expe imen al design
Ra s we e di ided in o wo main g oups o animals. A i s g oup
(n = 96) was used o de e mine he bes dose, he apeu ic window and
egime o ITH12657 ea men , ha is, o: (i) cha ac e ize he dose–
esponse cu e (n = 36) (6 subg oups) wi h di e en concen a ions o
ITH12657 (n = 6 pe subg oup), sac i iced a 7 days a e NMDA-
induced exci o oxici y: ehicle, 1, 3, 10, 30, o 60 mg/kg; (ii) de e mine
he he apeu ic window o ITH12657 (n = 30) wi h 5 subg oups
depending on he ime o ea men ini ia ion (n = 6 pe subg oup),
sac i iced 7 days a e NMDA-induced exci o oxici y: 12 h be o e, 1 h
be o e, 12 h a e , 24 h a e o ehicle; and (iii) o in es iga e he
op imal ea men du a ion (n = 30), ITH12657 was adminis e ed o
1, 2, 3, o 7 days a e NMDA injec ion (Figu e1A). The second g oup
(n = 48) was used o in es iga e he ITH12657 a o ded p o ec ion
longi udinally in i o using unc ional ( ull- ield ERG was eco ded a
3, 7, 10, 14, and 21 days) and mo phological analysis [Op ical
cohe ence omog aphy (OCT) was pe o med a 7, 14, and 21 days]
(n = 16) and ex i o by quan i ying a di e en pe iods o 7, 14 o
21 days he su i al o se e al RGC ypes; he gene al popula ion o
B n3a
+
RGCs, he popula ion o αRGCs (OPN
+
RGCs) and
hei sub ypes αONsRGCs (OPN
+
Tb 2
+
RGCs), αON RGCs
(OPN
+
B n3a
−
Tb 2
−
RGCs) and αOFFRGCs (OPN
+
B n3a
+
RGCs). Two
subg oups (one ea ed wi h ehicle, and one ea ed wi h ITH12657
10 mg/kg) (n = 8 pe subg oup) we e analyzed a 7, 14 o 21 days a e
NMDA-induced exci o oxici y. The g oup analyzed a 21 days (n = 16)
was used o longi udinal analysis o OCT and ull- ield
elec o e inog aphy (ERG; Figu e1B).
2.3 NMDA-induced exci o oxici y
Animals we e placed unde gene al anes hesia and ecei ed an
in a i eal injec ion (IVI) o ei he 5 μL o NMDA (100 mM) o saline
in o hei le eye, while he igh eye se ed as an un ea ed in ac
con ol. A 30G needle was used o c ea e a punc u e app oxima ely
1 mm pos e io o he limbus, h ough which a 32G Hamil on sy inge
needle was inse ed and ad anced owa d he cen e o he globe,
aking ca e o a oid lens damage. Any subjec s ha expe ienced
e inal inju y o ca a ac o ma ion du ing he in a i eal injec ion
we e excluded om he s udy.
2.4 Adminis a ion o ITH12657
Animals ecei ed a daily subcu aneous (sc) injec ion o 1 mL o
ei he ITH12657 o i s ehicle (saline wi h 1% DMSO) o 3 days. The
ITH12657 molecule was syn hesized acco ding o p e iously
es ablished p ocedu es and p o ided by Gonzalez e al. (2018).
2.5 In i o analysis
2.5.1 Op ical cohe ence omog aphy (OCT)
The e inal hickness was analyzed longi udinally in i o using a
Spec alis SD-OCT sys em (Heidelbe g, Ge many) speci ically
adap ed o i s use in a eyes (Ro e e e al., 2015; Nadal-Nicolas e al.,
2018). This g oup o animals (n = 16) was ea ed wi h ITH12657
10 mg/kg (n = 8) o ehicle (n = 8) and analyzed a 7, 14 and 21 days
a e NMDA exci o oxici y. A he end o he OCT analysis, his g oup
which was also assessed unc ionally, was used o ex i o analysis o
RGC su i al. Gene al anes hesia was adminis e ed and 1%
opicamide d ops (Colicu si opicamide 1%; Alcon-Cusí, S.A.,
Ba celona, Spain) we e ins illed in bo h eyes o induce myd iasis.
Imaging was pe o med using wo op ions: 31 B-scans (240-mic on
a ea) o quan i y o al and ou e e inal hickness and he pe ipapilla y
ing B-scan o Ganglion Cell Complex (GCC) hickness.
To measu e inne and o al e inal hickness we ollowed
p e iously desc ibed me hods ha a e s anda d in he labo a o y
(O in-Ma inez e al., 2014; Ro e e e al., 2015; Vidal-Villegas
e al., 2019). In b ie , ou measu emen s o o al ( om he ne e
ibe laye o he e inal pigmen epi helium) and inne ( om he
inne limi ing memb ane o he ou e bounda y o he inne
nuclea laye ) hickness we e aken on h ee lines o he scans
(supe io , in e io , and h ough he op ic ne e). On he uppe and
lowe lines, hese measu emen s we e aken a ou equidis an
poin s, while on he cen al line, which includes he op ic disc, wo
measu emen s we e aken on ei he side o he op ic disc. This
esul ed in a o al o 12 measu emen s (4 measu emen s on 3 lines)
o o al and inne hickness pe subjec .
Di Pie domenico e al. 10.3389/ nana.2024.1335176
F on ie s in Neu oana omy 04 on ie sin.o g
2.5.2 Elec ophysiology
Full- ield Elec o e inog aphy (ERG) was pe o med acco ding o
p e iously published me hods (Ala con-Ma inez e al., 2009, 2010;
Gallego-O ega e al., 2020, 2021). B ie ly, a e 12 h o da k adap a ion,
a s we e anaes he ized and bo h eyes we e dila ed wi h opical
myd ia ic (T opicamida 1%; Alcon-Cusí, S.A. Ba celona, Spain).
Sco opic and pho opic esponses we e eco ded simul aneously in
bo h eyes using Bu ian–Allen co neal bipola elec odes. A d op o
me hylcellulose (Me hocel
®
2%; No a is Labo a o ies CIBA Vision,
Annonay, F ance) was applied be ween he co nea and he elec odes
o imp o e signal conduc i i y. The e e ence elec ode was placed in
he mou h and a needle a he base o he ail se ed as a g ound
elec ode. Re inal ganglion cell (RGC)-media ed esponses we e
eco ded using ligh lashes anging om − 4.4 log cd·s/m
2
sco opically, od-media ed esponses we e eco ded a − 2.5 log cd·s/
m2, while mixed (a- and b-wa es) esponses we e eco ded a − 0.5 log
cd·s/m2. Cone-media ed esponses we e elici ed using 0.5 log cd·s/m2
lashes on a 30 cd/m
2
od-sa u a ing backg ound. Elec ical signals
we e digi ized a 20 kHz using a Powe Lab da a acquisi ion boa d (AD
Ins umen s, Chalg o e, UK) and s anda d ERG wa es we e analyzed
acco ding o he guidelines o he In e na ional Socie y o Clinical
Elec ophysiology o Vision (ISCEV). A o al o 16 a s we e used o
he elec ophysiological s udies, hese we e di ided in o wo g oups
ea ed wi h ITH12657 (n = 8) o ehicle (n = 8) and analyzed
longi udinally a 3, 7, 10, 14 o 21 days. This g oup had also been
analyzed in i o o OCT. A he end o hese s udies, his g oup was
used o ex i o his ological RGC su i al analysis a 21 days.
2.6 Ex i o analysis
2.6.1 Iden i ica ion o di e en RGC popula ions
In o de o s udy he idiosync a ic esponses o inju y-induced
e inal degene a ions and neu op o ec ion o RGCs, we analyse ew
ypes o RGCs o which immunoys ochemical ools a e a ailable. The
popula ion o B n3a exp essing RGCs, we e immunos ained wi h
B n3a an ibodies (Mouse an i-B n3a, MAB1585 Millipo e, Me ck
Bu ling on, MA, USA). To iden i y he alpha RGCs (αRGCs) and hei
sub ypes ON sus ained (αONsRGCs), ON ansien (αON RGCs), and
OFF (αOFFRGCs), all e inas we e exposed o a ious combina ions
o an ibodies and subsequen ly di e en wa eleng h luo opho e-
conjuga ed seconda y an ibodies ollowing ecen ly desc ibed
me hods (Gallego-O ega e al., 2021, 2022). In b ie : (i) an ibodies
agains os eopon in (OPN) (Goa an i-Os eopon in, AF808 Bio ech,
Bio ech Spain, Ba celona, Spain) we e used o de ec he αRGC
popula ion, (ii) colocaliza ion o OPN and Tb 2 (T-box ansc ip ion
ac o T-b ain 2) (Rabbi an i-Tb 2, AB23345 Abcam, Camb idge,
UK) was used o de ec αONsRGCs, (iii) colocaliza ion o OPN and
B n3a was used o de ec αOFFRGCs, and (i ) posi i e signal o OPN
bu nega i e o B n3a and Tb 2 was used o de ec αON RGCs
(Figu e2).
FIGURE1
Expe imen al design o he analysis o he p o ec i e e ec s o he compound ITH12657in an NMDA-induced e inal exci o oxici y model. (A) The i s
g oup (n  =  96) was used o de e mine he bes dose, he apeu ic window, and ea men egime o ITH12657. (B) The second g oup (n  =  48) was
designed o in es iga e ITH12657-a o ded p o ec ion, longi udinally in i o and by quan i ying he su i al o di e en RGC ypes a 7, 14 o 21  days.
Di Pie domenico e al. 10.3389/ nana.2024.1335176
F on ie s in Neu oana omy 05 on ie sin.o g
2.6.2 Image analysis
Immunos ained e inal whole-moun s we e analyzed and imaged
using an epi luo escence mic oscope (Leica DM6-B; Leica
Mic osys ems, We zla , Ge many) acco ding o p e iously desc ibed
me hods (Gallego-O ega e al., 2022; Di Pie domenico e al., 2022a).
B ie ly, o econs uc e inal la moun s, mul iple ames we e
acqui ed o each il e in a con iguous as e scan pa e n (x10) wi h
no o e lap o spacing be ween images. Indi idual images we e ocused
be o e acquisi ion and ob ained using he same ocus o each speci ic
il e . To acqui e highe magni ica ion images o he e ina, x40 and
x60 objec i es we e used.
2.6.3 Quan i ica ion and co-exp ession analysis
To de e mine in la -moun ed e inas he o al numbe o RGCs
immunolabeled wi h B n3a, weused s anda d compu e ou ines
de eloped in ou labo a o y (O in-Ma inez e  al., 2015; Nadal-
Nicolas e al., 2018). To coun αRGCs and di e en αRGC sub ypes,
he labeled and/o colocalized cells we e manually ma ked on each
e inal pho omon age and he o al numbe o ma ks pe e ina was
quan i ied using Image P oPlus so wa e (IPP5.1 o Windows; Media
Cybe ne ics, Sil e Sp ing, MD, USA) as p e iously desc ibed
(Gallego-O ega e al., 2022).
2.6.4 Topog aphical dis ibu ions
The opog aphic dis ibu ion o B n3a+RGCs, αRGCs and αRGC
sub ypes was s udied using isodensi y o neighbo maps acco ding o
p e iously desc ibed me hods (Salinas-Na a o e al., 2009; Galindo-
Rome o e al., 2013; Valien e-So iano e al., 2014; Gallego-O ega
e  al., 2022). All maps we e plo ed using SigmaPlo so wa e
(SigmaPlo 11.0 o Windows; Sys a So wa e, Inc., Richmond, CA,
USA) and a colo code was used o ep esen he da a.
2.6.5 S a is ic
RGC numbe s a e exp essed as mean ± s anda d de ia ion (SD) o
he mean. S a is ical compa isons be ween di e en g oups we e done
using one-way analysis o a iance (ANOVA-Tukey’s Pos hoc es s),
and compa isons be ween wo g oups we e done using S uden ’s - es
wi h he so wa e G aph Pad P ism® o Windows (Ve sion 5.01;
G aphPad So wa e Inc., La Jolla, CA, EEUU). A alue o p ≤ 0.05 was
conside ed s a is ically signi ican .
3 Resul s
3.1 ITH12657 dose, ime window and
leng h o ea men
The i s g oup o expe imen s was designed o cha ac e ize
he dose/ esponse cu e, he apeu ic window, and
neu op o ec i e e ec s o ITH12657 as a neu op o ec an o
RGCs agains NMDA-induced exci o oxici y. These s udies
indica ed ha 10 mg/kg ITH12657, s a ing 12 h be o e
FIGURE2
Immunohis ochemical de ec ion o αRGCs popula ions. Rep esen a i e luo escence mic og aphs o os eopon in (A), B n3a (B), Tb 2 (C), and he
me ge (D) colocaliza ion o RGC om con ol animals. Scale ba , 50  μm.

Di Pie domenico e al. 10.3389/ nana.2024.1335176
F on ie s in Neu oana omy 06 on ie sin.o g
in a i eal NMDA injec ion and main ained o 3 addi ional
days a o ded he bes p o ec ion on he su i al o B n3a+RGCs
ollowing a single in a i eal injec ion o 100 mM NMDA.
3.1.1 ITH12657 dose– esponse
To analyze he p o ec i e e ec s o ITH12657, wede e mined
he lowes concen a ion whe e he bes esul s we e ob ained.
Following in a i eal injec ion in he le eye o (5 μL o 100 mM
NMDA) o ehicle, di e en doses o he compound ITH12657
we e adminis e ed (1, 3, 10, 30 o 60 mg/kg) (n = 6 pe
g oup), and e inas we e examined a 7 days o
B n3a+RGC su i al.
All expe imen al g oups showed di e ences when compa ed
wi h he con ol (in ac igh eyes) g oup (One-way ANOVA,
p < 0.001) (Figu e3) indica ing ha in a i eal injec ion (IVI) o
NMDA esul ed in subs an ial B n3a
+
RGC loss (28% su i al)
(Figu e3). The ITH12657- ea ed g oups showed signi ican ly
g ea e o al numbe s o su i ing B n3a+RGCs when compa ed
o he ehicle- ea ed ones, hus showing e ec i e ITH12657
a o ded p o ec ion agains 100 mM NMDA-induced
exci o oxici y (Figu e 3). Mo eo e , he 10 mg/kg ITH12657-
ea ed g oup showed highe su i al han he 3 o 30 mg/kg
ITH12567- ea ed g oups. Thus, o he es o he s udy weused
he ITH12567 10 mg/kg dose ha esul ed in he highes (72%)
su i al o B n3a+RGCs (Figu e3).
3.1.2 The apeu ic window o ITH12657
We nex in es iga ed he he apeu ic window o compound
ITH12657. Fi e g oups o animals (n = 6 pe g oup) we e injec ed
wi h NMDA in he le eye while he igh eye was used as con ol,
and ehicle (daily injec ion) o ITH12657 was adminis e ed (i.p.)
wi h di e en ime egimes: (i) 12 h be o e, jus be o e and he
ollowing 6 days; (ii) 1 h be o e and he ollowing 6 days; (iii) 12 h
a e and he ollowing 6 days and; and (i ) 24 h a e and he
ollowing 5 days.
As o he p e ious dose– esponse s udy, we ound ha all
expe imen al (le eye) g oups p esen ed signi ican di e ences in o al
numbe s o su i ing B n3a+RGCs when compa ed o he con ol ( igh
eye) g oup (One-way ANOVA, p < 0.0001), and all ITH12657- ea ed
g oups showed signi ican ly g ea e o al numbe s o B n3a+RGCs when
compa ed o he ehicle- ea ed g oups (One-way ANOVA, p < 0.0001;
Figu e4). The g oup ea ed 12 h be o e, jus be o e and he ollowing
6 days a e NMDA in a i eal injec ion, showed he highes B n3a
+
RGC
su i al, when compa ed o he o he g oups. The g oup ea ed wi h
ITH12657 1 h a e NDMA-induced exci o oxici y showed no signi ican
di e ences wi h he g oup ea ed 12 h la e (34,448 ± 7,647 B n3a+RGC)
o 24 h la e (33,363 ± 66.53 B n3a+RGC) (Figu e4). Thus, o e all, he bes
p o ec i e e ec s we e ob ained when he ITH12657 ea men s a ed
12 h p io o NMDA in a i eal injec ion.
3.1.3 Leng h o ITH12657 ea men
Once he ideal concen a ion (10 mg/kg) and he apeu ic window
(12 h be o e in a i eal injec ion o NMDA) we e ound,
wede e mined he op imal leng h o ITH12657 ea men . Fo his
pu pose, ou g oups o animals (n = 6 pe g oup) we e injec ed wi h
NMDA in he le eye while he igh eye was used as con ol and
ITH12657 was injec ed sc. 12 h be o e in a i eal injec ion o NMDA,
bu o di e en leng hs o ime: (i) g oup1 ecei ed a single injec ion
12 h p io o NMDA injec ion, (ii) g oup2 ecei ed ea men 12 h
p io and jus p io o NMDA injec ion (2 injec ions), (iii) g oup3
ecei ed ea men 12 h p io , jus p io o NMDA injec ion, and e e y
day un il day 3 pos -NMDA injec ion (5 injec ions), and (i ) g oup4
ecei ed ea men un il 7 days pos -NMDA injec ion (9 injec ions).
All ITH12657 ea ed g oups exhibi ed signi ican di e ences in
mean o al numbe s o B n3a
+
RGCs when compa ed o he con ol ( igh
eyes) (One-way ANOVA, p < 0.0001) o he ehicle- ea ed g oups o eyes
(One-way ANOVA, p < 0.0001) (Figu e5). Quan i ica ion o B n3a
+
RGCs
in ITH12657- ea ed g oups e ealed ha animals ea ed un il 3 days
pos in a i eal injec ion o NMDA showed he highes su i al when
compa ed o he o he g oups (One-way ANOVA, p < 0.04) (Figu e5).
FIGURE3
Dose– esponse cu e. His og am showing o al B n3a+RGCs su i ing a e in a i eal injec ion (IVI) o NMDA-induced exci o oxici y and
subcu aneous ea men wi h di e en doses o he compound ITH12657. The su i al a e a 7  days was highe in he g oup in which ITH1657 was
adminis e ed a a dose o 10/mg/kg. *Signi ican di e ences p  <  0.05 (One-way ANOVA).
Di Pie domenico e al. 10.3389/ nana.2024.1335176
F on ie s in Neu oana omy 07 on ie sin.o g
3.2 In i o and ex i o assessmen o
ITH12657-a o ded e inal p o ec ion
agains NMDA-induced exci o oxici y
In a second g oup o expe imen s, weemploy in i o unc ional
and mo phological echniques, as well as ex i o immunohis ochemical
me hods o assess he e ec s o in a i eal NMDA-induced
exci o oxici y and p o ec ion a o ded wi h sys emic ITH12657in he
adul a e ina. Weassessed quan i a i ely in he same e inal whole-
moun s he su i al o B n3a
+
RGCs, αRGCs, α-ONs-RGCs, α-ON -
RGCs and α-OFF-RGCs, a 7, 14 o 21 days. These s udies indica ed
ha ITH12657 a o ded mo phological and unc ional longi udinal
p o ec ion o he e ina. Mo eo e , he quan i a i e analysis o RGCs
indica e ha ITH12657 a o ded signi ican p o ec ion o
B n3a+RGCs, αRGCs and αONsRGCs, bu no o αOFFRGCs, while
he αON RGCs we e ully esis an o NMDA-induced e inal inju y.
3.2.1 In i o s udy o e inal hickness.
ITH12657-a o ded p o ec ion agains
NMDA-induced exci o oxici y
The esul s ob ained in i o wi h SD-OCT showed ha o al and
inne e inal hickness we e educed signi ican ly in all expe imen al
g oups when compa ed o he con ol g oup, wi h e inal hinning
mainly due o inne e ina educ ion. To al e inal hickness in he
FIGURE4
The apeu ic window. His og am o he s udy o he he apeu ic window o he adminis a ion o ehicle o he compound ITH12657 a e in a i eal
injec ion (IVI) o 5  μL o 100  mM NMDA in he le eye. The su i al a e was highes in he g oup in which ea men s a ed 12  h be o e exci o oxici y.
One-way ANOVA analysis signi ican di e ences **p <  0.0001 and *p  <  0.05.
FIGURE5
Leng h o ea men . His og am o he s udy o he leng h o ea men o he adminis a ion o ehicle o he compound ITH12657 a e in a i eal
injec ion o 5  μL o 100  mM NMDA o ehicle in he le eye. The su i al a e was highe in he g oup in which ea men s a ed 12  h p io o NMDA
in a i eal injec ion and was main ained un il day 3. *Signi ican di e ences p  <  0.0001 (One-way ANOVA).
Di Pie domenico e al. 10.3389/ nana.2024.1335176
F on ie s in Neu oana omy 08 on ie sin.o g
NMDA-injec ed e inas was signi ican ly lowe when compa ed o he
con ol g oup (in ac igh e inas) a all ime in e als s udied, wi h
an a e age hinning o 10.3, 8.65 and 13.65% a 7, 14 and 21 days,
espec i ely (Figu e 6). Fu he mo e, e inal hickness was
signi ican ly highe in he e inas o he ITH12657- ea ed animals
han in he e inas o he ehicle- ea ed animals; he ehicle- ea ed
e inas showed an inne e inal hickness educ ion o 13.6, 9.46% o
24.54% a 7, 14 o 21 days, espec i ely (Figu e6), while he e inas o
ITH12657- ea ed animals showed educ ions o 6.8, 8.2% o 14.4%
a 7, 14, o 21 days, espec i ely (S uden ’s - es , p < 0.001) (Figu e6).
O e all, hese esul s indica e ha ITH12657 ea men signi ican ly
educed NMDA-induced hinning o he e ina, ac ing mainly on he
inne laye s.
3.2.2 Re inal unc ional p o ec ion wi h ITH12657
a e NMDA-induced exci o oxici y
To analyze he e ec s o NMDA-induced e inal exci o oxici y and
o in es iga e whe he ITH12657 a o ds unc ional p o ec ion o he
e ina, we eco ded longi udinally ull- ield elec o e inog ams
analyzing he pSTR (p oduced by RGCs), he a-wa e (p oduced by
pho o ecep o s) and he b-wa e (p oduced by cone bipola cells, CBs)
o he mixed esponse (Gallego-O ega e al., 2020). Fo his analysis,
weemployed he same g oup o 16 animals used o he SD-OCT
longi udinal analysis, and eco dings we e ob ained a 3, 7, 10, 14, and
21 days a e NMDA injec ion.
Re inal unc ion in he le expe imen al eyes was impai ed
ollowing in a i eal injec ion o NMDA (Figu e 7). The e we e
signi ican di e ences be ween he mean ampli udes o he pSTR in
he con ala e al (in ac igh ) e sus he expe imen al (le ) eyes ha
ecei ed an in a i eal injec ion o NMDA and hose ea ed wi h
ehicle. Th ee days a e NMDA adminis a ion, he e was an 80%
educ ion (0.042 ± 0.006 con ol s. 0.008 ± 0.0034 ehicle 3d)
(One-way ANOVA, p < 0.001) in he mean pSTR ampli ude o animals
ecei ing ehicle as ea men when compa ed o con ol. The dec ease
in he mean ampli ude o he pSTR was main ained h oughou he
pe iod o s udy (0.008 ± 0.0034 ehicle 3d s. 0.01 ± 0.04 ehicle 21d)
(One-way ANOVA, p > 0.05) (Figu e7).
Howe e , he educ ion in mean pSTR ampli ude was signi ican ly
smalle in animals ha we e ea ed wi h ITH12657 han in hose
ea ed wi h ehicle, a all ime-in e als s udied (Figu e7), sugges ing
ha ITH12657 a o ds unc ional pe manen p o ec i e p ope ies on
RGC unc ionali y a e NMDA-induced exci o oxici y. A e y simila
pa e n was obse ed in he b-wa e o he mixed esponse. The le
e inas p esen ed signi ican di e ences when compa ed wi h he
ampli ude o he con ol ( igh ) e inas. Howe e , such educ ion in
he ehicle- ea ed g oup was g ea e (0.55 ± 0.11 con ol s. 0.18 ± 0.07
ehicle 3d) han in he ITH12657- ea ed one (0.36 ± 0.008 ITH12657
3d). The b-wa e ampli udes o he animals ha had ecei ed he
ehicle showed an ini ial educ ion o 68%, whe eas hose ea ed wi h
ITH12657 we e educed by only 35%, wi h no u he p og ession
du ing he es o he s udy (One-way ANOVA, p < 0.01; Figu e7). The
a-wa e o he mixed esponse, which assesses he unc ional s a us o
pho o ecep o s, did no show signi ican changes in any o he s udy
g oups o ime-in e als analyzed a e NMDA-induced exci o oxici y
(One-way ANOVA, p > 0.05; Figu e7). Thus, i is emp ing o sugges
ha in a i eal injec ion o NMDA does no esul in pho o ecep o
damage ha can be egis e ed unc ionally wi h ERG sho ly, up o
21 days a e inju y.
3.2.3 Ex i o analysis o he neu op o ec i e
e ec s o ITH12657 on B n3a+RGCs a e
NMDA-induced exci o oxici y
NMDA-induced exci o oxici y esul s in he degene a ion o he
B n3a
+
RGC popula ion, as al eady desc ibed (Vidal-Villegas e al., 2019;
Milla-Na a o e al., 2021). One pu pose o he p esen s udies was o
in es iga e he neu op o ec i e e ec s o sys emically adminis e ed
ITH12657 agains NMDA-induced exci o oxici y, in he B n3a
+
RGC
popula ion. The e inas om he g oups o animals ea ed wi h ehicle
unde wen a apid and massi e loss o B n3a+RGCs, al eady e iden by
7 days a e NMDA in a i eal injec ion, wi h a educ ion o 73% o i s
o iginal alue when compa ed o con ol e inas (82,061 ± 3,584 con ol
s. 21,794 ± 57,222 ehicle 3d) (One-way ANOVA, p < 0.05). Such
educ ions we e main ained a 14 (19,556 ± 5,119) and 21 days
(21,794 ± 5,080) wi h no signi ican di e ence be ween hem (One-way
ANOVA, p > 0.05) sugges ing ha ollowing he ini ial loss o
B n3a
+
RGCs obse ed by 7 days, he e was no u he loss (Figu e8;
Table1). T ea men wi h ITH12657 signi ican ly p e en ed NMDA-
induced degene a ion o B n3a
+
RGCs. When o al numbe s o
B n3a
+
RGCs we e compa ed be ween he ITH12657- ea ed g oups
e sus ehicle- ea ed g oups, signi ican ly g ea e di e ences we e
ob ained a all ime in e als analyzed wi h a su i al o 78, 69% o 58%
o he B n3a+RGC popula ion a 7, 14 o 21 days, espec i ely (One-way
ANOVA, p < 0.05; Figu e 8; Table 1). When he ITH12657 ea ed
g oups we e compa ed a 7 (63,639 ± 7,274) and 14 (56,744 ± 5,995)
days, he e we e signi ican di e ences (One-way ANOVA, p < 0.05), bu
no signi ican di e ences we e ound be ween he 14 and he 21 days
g oups (56,744 ± 5,995 s. 47,450 ± 7,255) (One-way ANOVA, p > 0.05),
sugges ing ha he ITH12657 a o ded p o ec ion a 14 days was
main ained (Figu e8J; Table1).
In he con ol (in ac igh ) e inas, he dis ibu ion o
BRN3a+RGCs showed he ypical highe densi ies in he uppe e ina
along a s ip loca ed app oxima ely 1 mm abo e he op ic disc wi h
maximum alues in he supe io - empo al quad an , coinciding wi h
he exis ence o he e inal isual s eak (Nadal-Nicolas e al., 2009;
Salinas-Na a o e al., 2009; O in-Ma inez e al., 2010), a dis ibu ion
ha did no change wi h he ime-in e als s udied (Figu es9A–C).
Howe e , in he expe imen al (le ) e inas, he dis ibu ion o RGCs,
bo h in he ehicle- and in ITH12657- ea ed g oups, was educed a
all imes s udied, in ag eemen wi h he abo e-shown quan i a i e
esul s, and was di use h oughou he e ina (Figu es 9D–I).
Ne e heless, i was clea a all ime in e als analyzed ha he
ITH12657- ea ed e inas showed highe B n3a+RGCs densi ies a all
ime in e als analyzed when compa ed o ehicle- ea ed e inas
(Figu es9G–I).
3.2.4 Ex i o analysis o he p o ec ion a o ded
by ITH12657 agains NMDA exci o oxici y in he
αRGC popula ion and i s sub ypes
3.2.4.1 P o ec ion a o ded by ITH12657 on αRGCs agains
NMDA exci o oxici y
The αRGCs we e iden i ied wi h OPN an ibodies. In con ol
e inas αRGCs showed he ypical he e ogeneous dis ibu ion wi hin
he e ina wi h highe densi ies in he empo al hemi e ina
(Figu e10A) and mean o al numbe s o 2,135 ± 155 OPN
+
RGCs
(αRGCs) (n = 48; Figu e10E), and hese a e in ag eemen wi h a ecen
epo om ou labo a o y (Gallego-O ega e al., 2022).
Di Pie domenico e al. 10.3389/ nana.2024.1335176
F on ie s in Neu oana omy 09 on ie sin.o g
Se en days a e in a i eal injec ion o NMDA, he expe imen al
(le ) e inas ea ed wi h ehicle showed a 26% su i al o he αRGC
popula ion when compa ed wi h con ol ( igh in ac ) e inas
(559 ± 90 OPN+RGCs 7d ehicle s 2,135 ± 155 OPN+RGCs con ol),
whe eas he e inas ea ed wi h ITH12657 p esen ed a 60% su i al
(1,294 ± 241 OPN
+
RGCs 7d ITH12657 s. 2,135 ± 155 OPN
+
RGCs
con ol) (Figu e10; Table1).
When he ehicle- ea ed g oups we e compa ed a di e en
su i al in e als, he e we e no signi ican di e ences (One-way
ANOVA, p > 0.05). Tha is, ollowing in a i eal injec ion o NMDA
he e was a apid and massi e educ ion o OPN
+
RGCs, al eady a
7 days, which did no p og ess u he wi h ime (559 ± 90 OPN+RGCs
7d ehicle s. 729 ± 191 OPN+RGCs 21d ehicle). The g oups ea ed
wi h ITH12657 showed signi ican di e ences be ween 7 (60%
su i al) and 14 (39% su i al) days (1,294 ± 241 OPN
+
RGCs 7d
ITH12657 s. 832 ± 133 OPN
+
RGCs 14d ITH12657, One-way
ANOVA, p < 0.005), wi h no signi ican u he educ ion be ween 14
and 21 days, hus, sugges ing ha p o ec ion a o ded by ITH12657 a
14 days was pe manen (Figu e10).
3.2.4.2 P o ec ion a o ded by ITH12657 agains NMDA
exci o oxici y on αONsRGCs
Cells doubly labeled wi h OPN and Tb 2 we e do ed manually
o e e inal wholemoun s. Such colocaliza ion iden i ies α ON
sus ained RGCs (α-ONsRGCs) a sub ype o αRGC ha co esponds
o he M4 ype o he in insically pho osensi i e RGCs (K iege e al.,
FIGURE6
Re inal hickness. In i o images ob ained by op ical cohe ence omog aphy (SD-OCT) o o al and inne e inal hickness. Rep esen a i e images o
he undus (A,E,I). Rep esen a i e images o he con ala e al (B,F,J) and expe imen al e inal laye s, analyzed a 7, 14 o 21  days a e NMDA injec ion
ea ed wi h ehicle (C,G,K) o ITH12657 (D,H,L). In panel (B), he ed lines show he measu emen s made in he o al e ina and he blue lines show he
inne e ina measu emen s. (M) His og ams showing o al and inne e inal hickness in con ol ( igh ) (n  =  16 o each ime poin ), and in expe imen al
(le ) e inas ea ed wi h ehicle (in blue) o ITH12657 (in ed) a 7 (n  =  8), 14 (n  =  8), and 21 (n  =  8) days a e in a i eal injec ion o 100 nM NMDA in he
le eye. S uden ’s - es s a is ical signi icance *p  <  0.001, #p <  0.001, o †p  <  0.001.
Di Pie domenico e al. 10.3389/ nana.2024.1335176
F on ie s in Neu oana omy 16 on ie sin.o g
Ou in i o esul s also demons a e ha ollowing glu ama e gic
o e s imula ion he e a e p og essi e his ological and unc ional
changes, as e lec ed by e inal hinning and al e a ions o he main
elec ophysiological wa es. Al e a ions in elec o e inog aphic wa es
we e obse ed as ea ly as 7 days and pe sis ed un il 21 days pos -
injec ion, sugges ing ha NMDA-induced exci o oxici y p oduces
immedia e unc ional al e a ions in he e ina, as shown ana omically
in he p esen and p e ious s udies (Vidal-Villegas e al., 2019). In
con as , animals ea ed sys emically wi h ITH12657 did no exhibi
such ab up unc ional damage, and he e was only a 40% educ ion
in pSTR and a 35% educ ion in b-wa e ampli ude. Thus, ou
his ological da a on RGC su i al and ou elec ophysiological
da a sugges ha ea men wi h ITH12657 no only inc eases cell
iabili y bu also imp o es unc ionali y ollowing NMDA-
induced exci o oxici y.
The p og essi e his ological and unc ional changes ollowing
in a i eal injec ion o NMDA a e consis en wi h p e ious
s udies om o he esea ch g oups (Ke me e  al., 2001;
Ha dingham e al., 2002; Gomez-Vicen e e al., 2015; Huang
e al., 2018; Wang e al., 2018) including ou labo a o y (Vidal-
Villegas e al., 2019) ha in es iga ed RGC loss. A 7 days pos -
injec ion, he e was a 74% educ ion in he B n3a
+
RGC
popula ion, wi h no subsequen educ ions obse ed in animals
ha ecei ed only ehicle ea men . This loss is p obably due o
he ac i a ion o NMDA ecep o s, which igge a massi e in lux
o Ca
2+
ha ac s as a second messenge o ini ia e apop o ic
neu onal dea h pa hways (Leb un-Julien e al., 2009). Howe e ,
he exac signaling pa hways in ol ed in NMDA-induced RGC
dea h a e no ully unde s ood (Fah en hold e  al., 2018).
ITH12657 signi ican ly p e en ed he de imen al mo phological
and unc ional e ec s induced by NMDA in a i eal injec ion.
I is emp ing o sugges ha ITH12657 may exe i s p o ec ion
by blocking ol age-dependen calcium channels, p e en ing
excessi e calcium in lux and subsequen cell dea h ia pa hways
ac i a ed by NMDA-induced exci o oxici y. Addi ionally,
ITH12657 p e en s inhibi ion o PP2A, an enzyme in ol ed in
Tau p o ein syn hesis, which is essen ial o axonal main enance
and in acellula anspo .
4.3 Ex i o p o ec ion a o ded by ITH
12657
In he second g oup o expe imen s, we u he in es iga ed ex
i o he neu op o ec i e e ec s o sys emic adminis a ion o ehicle
o he no el molecule ITH12657 on he e ina agains NMDA-induced
exci o oxici y, and his was assessed wi h o al coun s o su i ing
B n3a
+
RGCs, αRGCs and hei sub ypes (αONsRGCs; αON RGCs
and αOFFRGCs).
4.3.1 P o ec ion o B n3a+RGCs a o ded by ITH
12657
In a i eal injec ion o NMDA esul ed by 7 days in he loss o
app oxima ely 74% o he B 3a
+
RGC popula ion wi hou u he loss,
as p e iously epo ed (Vidal-Villegas e  al., 2019). Howe e ,
ITH12657 ea men esul ed in signi ican p o ec ion wi h a su i al
o 58% o he B n3a
+
RGC popula ion a 14 days, which was pe manen .
I is emp ing o sugges ha he p o ec ion a o ded by ITH12567
could bemedia ed by i s capaci y o block ol age-dependen calcium
channels, hus p e en ing excessi e calcium in lux and subsequen cell
dea h ia pa hways ac i a ed by NMDA injec ion. Mo eo e ,
ITH12657 p e en s inhibi ion o PP2A, an enzyme in ol ed in Tau
p o ein syn hesis, which is essen ial o axonal main enance and
in acellula anspo (Gonzalez e al., 2018).
4.3.2 Di e en ial esponses o αRGCs and hei
sub ypes o NMDA-induced inju y and p o ec ion
wi h ITH 12657
In ehicle- ea ed g oups o a s, ollowing he NMDA IVI, he
αRGC (OPN
+
RGC) popula ion unde wen a 73% loss a 7 days wi h
no u he loss du ing he es o he s udy, whe eas ea men wi h
ITH12657 educed his loss o 40% a 7 days and o 60% a 14 days,
wi h such p o ec ion main ained o up o 21 days. The α-ONsRGC
(OPN
+
Tb 2
+
RGC) popula ion also exhibi ed di e en esponses o
NMDA-induced exci o oxici y and p o ec ion wi h ITH12657. The
e inas ecei ing NMDA IVI and ehicle ea men , showed an
app oxima ely 28% su i al o he o iginal popula ion o α-ONsRGC
by 7 days, wi h no signi ican u he losses wi h ime. By con as , he
FIGURE12
OPN+B n3a+RGC and OPN+Tb 2−B n3a−RGC popula ions. (A) Rep esen a i e his og am o he OPN+B n3a+RGC popula ion (mean  ±  SD) compa ing
con ol eyes wi h expe imen al eyes NMDA-injec ed ea ed wi h ehicle (blue) o ITH12657 ( ed). (B) Rep esen a i e his og am o he
OPN+Tb 2−B n3a−RGC popula ion (mean ±  SD) compa ing con ol wi h expe imen al NMDA-injec ed eyes ea ed eyes wi h ehicle (blue) o ITH12657
( ed). ns, no s a is ically di e ence One-way ANOVA, p  >  0.05.

Di Pie domenico e al. 10.3389/ nana.2024.1335176
F on ie s in Neu oana omy 17 on ie sin.o g
e inas ea ed wi h ITH12657 showed p o ec ion a 7 days ha
esul ed in he su i al o app oxima ely 54% o he OPN+Tb 2+RGC
popula ion, wi h no signi ican a ia ions a inc easing su i al
in e als, sugges ing ha p o ec ion a o ded by ITH12657 o
OPN
+
Tb 2
+
RGCs was pe manen (Figu e 11). The α-OFF RGCs
(OPN
+
B n3a
+
RGCs) showed o beex emely sensi i e o NMDA-
induced exci o oxici y wi h only app oxima ely 2% o he o iginal
popula ion emaining by 7 days and no esponse o ITH12657
ea men (Figu e12A). The α-ON RGCs (OPN
+
Tb 2
−
B n3a
−
RGCs),
on he con a y, we e ully esis an o NMDA-induced exci o oxici y
(Figu e12B), as has been p e iously documen ed o he melanopsin
exp essing RGCs M1-M3 (Vidal-Villegas e al., 2019).
Thus, i is in e es ing ha αRGCs, a subclass ha in a s comp ises
app oxima ely 2.2% o all RGCs aced om he in a-o bi al op ic
ne e (Nadal-Nicolas e al., 2015; Gallego-O ega e al., 2021) and
includes 4 o he 46 known ypes o RGCs (Rheaume e al., 2018; T an
e  al., 2019; Goe z e  al., 2022), exhibi a di e en ial esponse o
NMDA-induced exci o oxici y. A 7 days in animals ha ecei ed
ehicle ea men , he o al popula ion was educed by 73, 72 o 98%,
o he αRGC, he αONsRGC o he αOFFRGC popula ions,
espec i ely, while he RGCα-ON popula ion was ully esis an o
NMDA-induced exci o oxici y. I is in e es ing ha αONsRGCs,
which co esponds o M4, one ype o ipRGCs (Schmid e al., 2014;
K iege e al., 2017; Sonoda e al., 2020), does no show he ypical
esilience shown o o he ipRGCs (M1-M3) o ligh induced
pho o oxici y (Ga cia-Ayuso e al., 2017), acu e ocula hype ension
(Ro e e e al., 2016), op ic ne e inju y (Nadal-Nicolas e al., 2015;
Sanchez-Migallon e al., 2018; Gallego-O ega e al., 2021; Vidal-
Villegas e al., 2021a) o NMDA-induced exci o oxici y (Vidal-Villegas
e al., 2019). Mo eo e , αONsRGCs ha e been shown o bepa icula ly
esis an o ansien ocula hype ension (Zhao e al., 2023) pe haps
due o hei abili y o modi y hei synap ic connec ions (El-Dana
and Hube man, 2015; Ou e al., 2016; Della San ina e al., 2021), bu
no o NMDA-induced exci o oxici y. Mo eo e , αRGC sub ypes ha e
been shown o exhibi di e en esilience o op ic ne e inju y, wi h
he wo sus ained ypes being mo e esis an han he wo ansien
ypes (T an e al., 2019), while hei esilience o NMDA-induced
exci o oxici y di e s based on hei ON s OFF esponses, wi h a
esilience o he ON- s a g ea agili y o he OFF- αRGC sub ypes.
Thus, o e all, ou p esen esul s con i m he suscep ibili y o di e en
RGCs o inju y (Vidal-Villegas e al., 2019; Gallego-O ega e al., 2021;
Vidal-Villegas e al., 2021a) and add new da a ega ding he esponse
o B n3a
+
RGCs, αRGCs and hei sub ypes o NMDA-induced
exci o oxici y and ITH12567 a o ded p o ec ion.
An e ol ing concep o e he las yea s ela es o he idiosync a ic
esponses o RGCs o inju y and p o ec ion (Vidal-Sanz e al., 2015a;
Agudo-Ba iuso e al., 2016). Indeed, i is cu en ly hough ha o he
many di e en ypes o RGCs p esen in he oden e ina, a la ge
numbe exhibi a di e en ial esponse o inju y and p o ec ion (Duan
e al., 2015; Honda e al., 2019; T an e al., 2019; Vidal-Villegas e al.,
2019; Gallego-O ega e al., 2021; Vidal-Villegas e al., 2021b; Zhao
e al., 2023). Following op ic ne e c ush in mice, RNA sequencing
e ealed ha no all RGC ypes espond simila ly o inju y (T an e al.,
2019). Ou s udy suppo s hese indings, as we epo ha di e en
αRGC ypes exhibi a ying esponses o inju y, wi h αONsRGC being
mo e esis an , αOFFRGCs being ex emely suscep ible and
αON RGC being o ally esis an . These esul s a e consis en wi h
o he s udies ha ha e shown ha he OFF pa hway is mo e sensi i e
o NMDA exci o oxici y, wi h di ec ion-selec i e RGC-ONs being
mo e esis an han di ec ion-selec i e RGC-OFFs (Milla-Na a o
e al., 2021). Mo eo e , he esponse o ITH12657 a o ded p o ec ion
also a ied g ea ly be ween αRGs and hei sub ypes, so ha inal
su i al a 21 days in animals ha ecei ed ITH12657 ea men was
34, 45 o 2%, o he αRGC, he αONsRGC o he αOFFRGC
popula ions, espec i ely, while he RGCα-ON popula ion was ully
esis an o NMDA-induced exci o oxici y.
A p e ious s udy epo ed ha αRGCs, immunode ec ed using an
OPN an ibody, a e comple ely esis an and esilien o NMDA
induced exci o oxici y (Ch is ensen e al., 2019; Honda e al., 2019).
Al hough αRGCs exhibi low exp ession o calcium-pe meable
glu ama e ecep o s, high concen a ions o NMDA can s ill induce
dea h in hese neu ons. This may be he explana ion o he di e ences
be ween ou esul s and hose epo ed by Honda e al. (2019) and
Honda e al. (2019) who used a 100- old lowe concen a ion o
NMDA han we did. Addi ionally, hei s udy was conduc ed on
pigmen ed mouse e inas, and he suscep ibili y o cells in he albino
a e ina may di e . Fu he mo e, hei cell coun ing was pe o med
by sampling e inal egions whe eas wecoun ed he o al numbe s o
cells in e inal whole-moun s.
T ea men wi h he ITH12657 molecule had bene icial e ec s on
neu op o ec ion o bo h he αRGC and B n3a
+
RGC popula ions.
Blockade o ol age-dependen calcium channels by ITH12657 may
ha e p e en ed excessi e calcium in lux and subsequen cell dea h,
esul ing in inc eased su i al o αRGCs and hei sub ype αONsRGCs
in ITH12657- ea ed animals compa ed o hose ea ed wi h ehicle
alone. Howe e , his e ec was no obse ed in he αOFFRGC
popula ion. The high suscep ibili y o he αOFFRGC popula ion o
NMDA and he high concen a ion (100 mM) used in his s udy may
ha e masked any neu op o ec i e e ec o ITH12657. Fu he s udies
a e needed o analyze i a lowe concen a ions o NMDA, ITH12657
would beneu op o ec i e on he αOFFRGC popula ion.
In conclusion, ollowing in a i eal injec ion o NMDA o induce
exci o oxici y in he albino a e ina, sys emic adminis a ion o
ITH12657 (10 mg/kg) p e en ed signi ican e inal hinning, imp o ed
he unc ionali y o RGCs and e inal in e neu ons, and p o ec ed
B n3a
+
RGCs, αRGCs and αONsRGCs popula ions, bu no
αOFFRGCs, while αON RGCs appea ed esis an o inju y, u he
adding o p e ious indings abou he idiosync asy o di e en RGCs
o inju y and p o ec ion (Vidal-Sanz e al., 2015a; Agudo-Ba iuso
e  al., 2016; Vidal-Villegas e  al., 2019, 2021a; Gallego-O ega
e al., 2021).
4.4 Limi a ions o he s udy
Neu op o ec ion s udies in he e ina need de ailed knowledge
abou he esponse o di e en ypes o RGCs o inju y and p o ec ion,
as well as new s a egies designed o impinge on a ious inju y-
ac i a ed dea h pa hways.
I is emp ing o sugges ha he p o ec i e e ec s o ITH12657
a e due o i s capaci y o block excess in acellula calcium in lux.
Howe e , in his s udy he mechanism o ac ion o ITH-12657 was no
in es iga ed, so addi ional expe imen s need o bedesigned o a o d
his pu pose.
Ou s udies ely only in he immunohis ochemical iden i ica ion o
RGCs by he exp ession o co-exp ession o speci ic ma ke s, such as
Di Pie domenico e al. 10.3389/ nana.2024.1335176
F on ie s in Neu oana omy 18 on ie sin.o g
B n3a, Os eopon in o Tb 2 o iden i y B n3a + RGCs, αRGCs and hei
sub ypes αONsRGCs, αON RGCs and αOFFRGCs, espec i ely. Bu i is
well known ha NMDA-induced exci o oxici y can causes degene a ion
o o he neu onal popula ions in he e ina such us amac ine cells, and
ha inju ed neu ons may modi y he exp ession o ypical ma ke s
(Vidal-Villegas e al., 2019). Fo his eason, a no e o cau ion should
beconside ed when in e p e ing he ex i o and in i o esul .
5 Conclusion
We epo o he i s ime he dele e ious e ec s o in a i eal
NMDA-induced exci o oxici y as well as he p o ec i e e ec s o
subcu aneous adminis a ion o ITH12657, o blun mo phological
and unc ional degene a ion o he e ina as well as he loss o
B n3a
+
RGCs, αRGCs and hei sub ype αONsRGCs. These esul s
p o ide u he e idence o he idiosync a ic esponses o inju y and
p o ec ion o he main sub ypes o a main class o e inal neu ons,
he αRGCs.
Da a a ailabili y s a emen
The aw da a suppo ing he conclusions o his a icle will
bemade a ailable by he au ho s, wi hou undue ese a ion.
E hics s a emen
The animal s udy was app o ed by Uni e si y o Mu cia E hical
animal s udies commi ee. The s udy was conduc ed in acco dance
wi h he local legisla ion and ins i u ional equi emen s.
Au ho con ibu ions
JD: Concep ualiza ion, Da a cu a ion, Fo mal analysis,
In es iga ion, Me hodology, W i ing – o iginal d a , W i ing – e iew
& edi ing. AG-O: Da a cu a ion, Fo mal analysis, In es iga ion,
Me hodology, W i ing – o iginal d a . MN-M: Da a cu a ion, Fo mal
analysis, In es iga ion, W i ing – o iginal d a . BV-V: Da a cu a ion,
In es iga ion, W i ing – o iginal d a . IB: In es iga ion, Resou ces,
W i ing – o iginal d a . MB-R: Da a cu a ion, W i ing – o iginal
d a . JB-G: In es iga ion, W i ing – o iginal d a . IF-B: Funding
acquisi ion, W i ing – e iew & edi ing. JP-J: Funding acquisi ion,
W i ing – e iew & edi ing. MV-P: Funding acquisi ion, W i ing
– e iew & edi ing. MA-T: Fo mal analysis, Funding acquisi ion,
Supe ision, Valida ion, W i ing – e iew & edi ing. CdlR:
In es iga ion, Me hodology, W i ing – e iew & edi ing. MV-S:
Concep ualiza ion, Da a cu a ion, Fo mal analysis, Funding
acquisi ion, In es iga ion, Me hodology, Supe ision, Valida ion,
W i ing – o iginal d a , W i ing – e iew & edi ing.
Funding
The au ho (s) decla e inancial suppo was ecei ed o he
esea ch, au ho ship, and/o publica ion o his a icle. This esea ch
was unded by PID2019-106498GB-I00 unded by Minis e io de
Ciencia, Inno ación y Uni e sidades (MCIN)/AEI/ 10.13039/501100
011033 o MV-S and MA-T; (Re iB ain) RED2018-102499-T o MV-S;
and by he Ins i u o de Salud Ca los III (PI19/01724 o CdlR) and
co- unded wi h he Eu opean Regional De elopmen Fund (ERDF)
wi hin he “Plan Es a al de In es igación Cien í ica y Técnica y de
Inno ación 2017–2020” (FIS/PI 18–00754) o MV-P.
Acknowledgmen s
Sho accoun s o his wo k ha e been p esen ed a in e na ional
cong esses and published in abs ac o ma (Di Pie domenico
e al., 2022c).
Con lic o in e es
The au ho s decla e ha he esea ch was conduc ed in he
absence o any comme cial o inancial ela ionships ha could
becons ued as a po en ial con lic o in e es .
The au ho (s) decla ed ha hey we e an edi o ial boa d membe
o F on ie s, a he ime o submission. This had no impac on he pee
e iew p ocess and he inal decision.
Publishe ’s no e
All claims exp essed in his a icle a e solely hose o he
au ho s and do no necessa ily ep esen hose o hei a ilia ed
o ganiza ions, o hose o he publishe , he edi o s and he
e iewe s. Any p oduc ha may be e alua ed in his a icle, o
claim ha may be made by i s manu ac u e , is no gua an eed o
endo sed by he publishe .
Re e ences
Agudo-Ba iuso, M., Nadal-Nicolas, F. M., Madei a, M. H., Ro e e, G.,
Vidal-Villegas, B., and Vidal-Sanz, M. (2016). Melanopsin exp ession is an indica o o
he well-being o melanopsin-exp essing e inal ganglion cells bu no o hei iabili y.
Neu al Regen. Res. 11, 1243–1244. doi: 10.4103/1673-5374.189182
Akopian, A., A lasz, T., Pan, F., Wong, S., Zhang, Y., Volgyi, B., e al. (2014). Gap
junc ion-media ed dea h o e inal neu ons is connexin and insul speci ic: a po en ial
a ge o neu op o ec ion. J. Neu osci. 34, 10582–10591. doi: 10.1523/
JNEUROSCI.1912-14.2014
Ala con-Ma inez, L., A iles-T igue os, M., Galindo-Rome o, C., Valien e-So iano, J.,
Agudo-Ba iuso, M., Villa Pde, L., e al. (2010). ERG changes in albino and pigmen ed
mice a e op ic ne e ansec ion. Vis. Res. 50, 2176–2187. doi: 10.1016/j.
is es.2010.08.014
Ala con-Ma inez, L., de la Villa, P., A iles-T igue os, M., Blanco, R.,
Villegas-Pe ez, M. P., and Vidal-Sanz, M. (2009). Sho and long e m axo omy-induced
ERG changes in albino and pigmen ed a s. Mol. Vis. 15, 2373–2383.
Almasieh, M., Wilson, A. M., Mo que e, B., Cue a Va gas, J. L., and Di Polo, A.
(2012). The molecula basis o e inal ganglion cell dea h in glaucoma. P og. Re in. Eye
Res. 31, 152–181. doi: 10.1016/j.p e eye es.2011.11.002
A iles-T igue os, M., Sau e, Y., Lund, R. D., and Vidal-Sanz, M. (2000). Selec i e
inne a ion o e ino ecipien b ains em nuclei by e inal ganglion cell axons
Di Pie domenico e al. 10.3389/ nana.2024.1335176
F on ie s in Neu oana omy 19 on ie sin.o g
egene a ing h ough pe iphe al ne e g a s in adul a s. J. Neu osci. 20, 361–374. doi:
10.1523/JNEUROSCI.20-01-00361.2000
Baden, T., Be ens, P., F anke, K., Roman Roson, M., Be hge, M., and Eule , T. (2016).
The unc ional di e si y o e inal ganglion cells in he mouse. Na u e 529, 345–350. doi:
10.1038/na u e16468
Becke , J. B., P ende gas , B. J., and Liang, J. W. (2016). Female a s a e no mo e
a iable han male a s: a me a-analysis o neu oscience s udies. Biol. Sex Di e . 7:34.
doi: 10.1186/s13293-016-0087-5
Bee y, A. K. (2018). Inclusion o emales does no inc ease a iabili y in oden
esea ch s udies. Cu . Opin. Beha . Sci. 23, 143–149. doi: 10.1016/j.cobeha.2018.06.016
Be son, D. M., Cas ucci, A. M., and P o encio, I. (2010). Mo phology and mosaics
o melanopsin-exp essing e inal ganglion cell ypes in mice. J. Comp. Neu ol. 518,
2405–2422. doi: 10.1002/cne.22381
Boccuni, I., and Fai less, R. (2022). Re inal glu ama e neu o ansmission: om
physiology o pa hophysiological mechanisms o e inal ganglion cell degene a ion. Li e
12:638. doi: 10.3390/li e12050638
Chen, H., Chen, S., Zhang, H., Wang, S., Li, Y., and Meng, X. (2022). N-me hyl-D-
aspa a e ecep o -media ed spinal co d ischemia- epe usion inju y and i s p o ec i e
mechanism. Folia Neu opa hol. 60, 308–315. doi: 10.5114/ n.2022.118340
Choi, D. W. (1988). Glu ama e neu o oxici y and diseases o he ne ous sys em.
Neu on 1, 623–634. doi: 10.1016/0896-6273(88)90162-6
Ch is ensen, I., Lu, B., Yang, N., Huang, K., Wang, P., and Tian, N. (2019). The
suscep ibili y o e inal ganglion cells o glu ama e gic exci o oxici y is ype-speci ic.
F on . Neu osci. 13:219. doi: 10.3389/ nins.2019.00219
Della San ina, L., Yu, A. K., Ha is, S. C., Solino, M., Ga cia Ruiz, T., Mos , J., e al.
(2021). Disassembly and ewi ing o a ma u e con e ging exci a o y ci cui ollowing
inju y. Cell Rep. 36:109463. doi: 10.1016/j.cel ep.2021.109463
DePa is, S., Cap a a, C., and G imm, C. (2012). In insically pho osensi i e e inal
ganglion cells a e esis an o N-me hyl-D-aspa ic acid exci o oxici y. Mol. Vis. 18,
2814–2827.
Di Pie domenico, J., Gallego-O ega, A., Ma inez-Vacas, A., Ga cia-Be nal, D.,
Vidal-Sanz, M., Villegas-Pe ez, M. P., e al. (2022a). In a i eal and sub e inal syngeneic
bone ma ow mononuclea s em cell ansplan a ion imp o es pho o ecep o su i al
bu does no amelio a e e inal unc ion in wo a models o e inal degene a ion. Ac a
Oph halmol. 100, e1313–e1331. doi: 10.1111/aos.15165
Di Pie domenico, J., Hende son, D. C. M., Giamma ia, S., Smi h, V. L., Jame , A. J.,
Smi h, C. A., e al. (2022b). Age and in aocula p essu e in mu ine expe imen al
glaucoma. P og. Re in. Eye Res. 88:101021. doi: 10.1016/j.p e eye es.2021.101021
Di Pie domenico, J., Muñoz, M. N., O ega, A. G., Ruiz, M. B., Ga o, J. M. B.,
Pe ez, M. P. V., e al. (2022c). Neu op o ec i e e ec s o ITH-IB6 agains exci o oxici y-
induced e inal inju y. Ac a Oph halmol 100. doi: 10.1111/j.1755-3768.2022.0243
D eye , E. B., Zu akowski, D., Schume , R. A., Podos, S. M., and Lip on, S. A. (1996).
Ele a ed glu ama e le els in he i eous body o humans and monkeys wi h glaucoma.
A ch. Oph halmol. 114, 299–305. doi: 10.1001/a choph .1996.01100130295012
Duan, X., Qiao, M., Bei, F., Kim, I. J., He, Z., and Sanes, J. R. (2015). Sub ype-speci ic
egene a ion o e inal ganglion cells ollowing axo omy: e ec s o os eopon in and
mTOR signaling. Neu on 85, 1244–1256. doi: 10.1016/j.neu on.2015.02.017
El-Dana , R. N., and Hube man, A. D. (2015). Cha ac e is ic pa e ns o dend i ic
emodeling in ea ly-s age glaucoma: e idence om gene ically iden i ied e inal
ganglion cell ypes. J. Neu osci. 35, 2329–2343. doi: 10.1523/JNEUROSCI.1419-14.2015
Es e ez, M. E., Foge son, P. M., Ila di, M. C., Bo ghuis, B. G., Chan, E., Weng, S., e al.
(2012). Fo m and unc ion o he M4 cell, an in insically pho osensi i e e inal ganglion
cell ype con ibu ing o geniculoco ical ision. J. Neu osci. 32, 13608–13620. doi:
10.1523/JNEUROSCI.1422-12.2012
E angeliou, E., Plemmenos, G., Chalazias, A., and Pipe i, C. (2023). Impac o TRP
channels in O al pa hology and he apeu ic a ge ing op ions: a na a i e e iew. Cu .
Top. Med. Chem. 23, 1559–1573. doi: 10.2174/1568026623666230331110443
Fah en hold, B. K., Fe nandes, K. A., and Libby, R. T. (2018). Assessmen o in insic
and ex insic signaling pa hway in exci o oxic e inal ganglion cell dea h. Sci. Rep.
8:4641. doi: 10.1038/s41598-018-22848-y
Fle che , E. L., Hack, I., B ands a e , J. H., and Wassle, H. (2000). Synap ic localiza ion
o NMDA ecep o subuni s in he a e ina. J. Comp. Neu ol. 420, 98–112. doi: 10.1002/
(SICI)1096-9861(20000424)420:1<98::AID-CNE7>3.0.CO;2-U
Galindo-Rome o, C., Jimenez-Lopez, M., Ga cia-Ayuso, D., Salinas-Na a o, M.,
Nadal-Nicolas, F. M., Agudo-Ba iuso, M., e al. (2013). Numbe and spa ial dis ibu ion
o in insically pho osensi i e e inal ganglion cells in he adul albino a . Exp. Eye Res.
108, 84–93. doi: 10.1016/j.exe .2012.12.010
Gallego-O ega, A., No e-Munoz, M., Di Pie domenico, J., A iles-T igue os, M., de
la Villa, P., Valien e-So iano, F. J., e al. (2022). Alpha e inal ganglion cells in pigmen ed
mice e ina: numbe and dis ibu ion. F on . Neu oana . 16:1054849. doi: 10.3389/
nana.2022.1054849
Gallego-O ega, A., No e-Munoz, M., Mi alles de Impe ial-Olle o, J. A.,
Be nal-Ga o, J. M., Valien e-So iano, F. J., de la Villa Polo, P., e al. (2020). Func ional
and mo phological al e a ions in a glaucoma model o acu e ocula hype ension. P og.
B ain Res. 256, 1–29. doi: 10.1016/bs.pb .2020.07.003
Gallego-O ega, A., Vidal-Villegas, B., No e-Munoz, M., Salinas-Na a o, M.,
A iles-T igue os, M., Villegas-Pe ez, M. P., e al. (2021). 7,8-Dihyd oxi la one main ains
e inal unc ionali y and p o ec s a ious ypes o RGCs in adul a s wi h op ic ne e
ansec ion. In . J. Mol. Sci. 22:11815. doi: 10.3390/ijms222111815
Ga cia-Ayuso, D., Galindo-Rome o, C., Di Pie domenico, J., Vidal-Sanz, M.,
Agudo-Ba iuso, M., and Villegas Pe ez, M. P. (2017). Ligh -induced e inal degene a ion
causes a ansien down egula ion o melanopsin in he a e ina. Exp. Eye Res. 161,
10–16. doi: 10.1016/j.exe .2017.05.010
Ga cia-Layana, A., Figue oa, M. S., A ias, L., A aiz, J., Ruiz-Mo eno, J. M.,
Ga cia-A umi, J., e al. (2015). Indi idualized he apy wi h anibizumab in we age-
ela ed macula degene a ion. J. Oph halmol. 2015:412903. doi: 10.1155/2015/412903
Goe z, J., Jessen, Z. F., Jacobi, A., Mani, A., Coole , S., G ee , D., e al. (2022). Uni ied
classi ica ion o mouse e inal ganglion cells using unc ion, mo phology, and gene
exp ession. Cell Rep. 40:111040. doi: 10.1016/j.cel ep.2022.111040
Gomez-Vicen e, V., Lax, P., Fe nandez-Sanchez, L., Rondon, N., Esqui a, G.,
Ge main, F., e al. (2015). Neu op o ec i e e ec o au ou sodeoxycholic acid on
N-me hyl-D-aspa a e-induced e inal ganglion cell degene a ion. PLoS One
10:e0137826. doi: 10.1371/jou nal.pone.0137826
Gonzalez, D., A ibas, R. L., Viejo, L., Laja in-Cues a, R., and de Los Rios, C. (2018).
Subs i uen e ec o N-benzyla ed g amine de i a i es ha p e en he PP2A inhibi ion
and dissipa e he neu onal Ca(2+) o e load, as a mul i a ge s a egy o he ea men
o Alzheime ’s disease. Bioo g. Med. Chem. 26, 2551–2560. doi: 10.1016/j.
bmc.2018.04.019
Hahn, J., Mona a eshani, A., Qiao, M., Kao, A. H., Kolsch, Y., Kuma , A., e al. (2023).
E olu ion o neu onal cell classes and ypes in he e eb a e e ina. Na u e 624,
415–424. doi: 10.1038/s41586-023-06638-9
Ha dingham, G. E., Fukunaga, Y., and Bading, H. (2002). Ex asynap ic NMDARs
oppose synap ic NMDARs by igge ing CREB shu -o and cell dea h pa hways. Na .
Neu osci. 5, 405–414. doi: 10.1038/nn835
Honda, S., Nameka a, K., Kimu a, A., Guo, X., Ha ada, C., Mu akami, A., e al. (2019).
Su i al o alpha and in insically pho osensi i e e inal ganglion cells in NMDA-
induced neu o oxici y and a mouse model o No mal ension Glaucoma. In es .
Oph halmol. Vis. Sci. 60, 3696–3707. doi: 10.1167/io s.19-27145
Huang, W., Hu, F., Wang, M., Gao, F., Xu, P., Xing, C., e al. (2018). Compa a i e
analysis o e inal ganglion cell damage in h ee glaucoma ous a models. Exp. Eye Res.
172, 112–122. doi: 10.1016/j.exe .2018.03.019
Huang, W., Xu, Q., Su, J., Tang, L., Hao, Z. Z., Xu, C., e al. (2022). Linking
ansc ip omes wi h mo phological and unc ional pheno ypes in mammalian e inal
ganglion cells. Cell Rep. 40:111322. doi: 10.1016/j.cel ep.2022.111322
I o, A., Tsuda, S., Kunika a, H., Toshi umi, A., Sa o, K., and Nakazawa, T. (2019).
Assessing e inal ganglion cell dea h and neu op o ec i e agen s using eal ime imaging.
B ain Res. 1714, 65–72. doi: 10.1016/j.b ain es.2019.02.008
Izzo i, A., Bagnis, A., and Sacca, S. C. (2006). The ole o oxida i e s ess in glaucoma.
Mu a . Res. 612, 105–114. doi: 10.1016/j.m e .2005.11.001
Ke me , P., Klocke , N., and Bah , M. (2001). Modula ion o me abo opic glu ama e
ecep o s ails o p e en he loss o adul a e inal ganglion cells ollowing axo omy o
N-me hyl-D-aspa a e lesion in i o. Neu osci. Le . 315, 117–120. doi: 10.1016/
s0304-3940(01)02318-7
Kobayashi, M., Hi ooka, K., Ono, A., Nakano, Y., Nishiyama, A., and Tsujikawa, A.
(2017). The ela ionship be ween he enin-angio ensin-aldos e one sys em and NMDA
ecep o -media ed signal and he p e en ion o e inal ganglion cell dea h. In es .
Oph halmol. Vis. Sci. 58, 1397–1403. doi: 10.1167/io s.16-21001
Kopecky, B. J., Liang, R., and Bao, J. (2014). T- ype calcium channel blocke s as
neu op o ec i e agen s. P luge s A ch. 466, 757–765. doi: 10.1007/s00424-014-1454-x
K iege , B., Qiao, M., Rousso, D. L., Sanes, J. R., and Meis e , M. (2017). Fou alpha
ganglion cell ypes in mouse e ina: unc ion, s uc u e, and molecula signa u es. PLoS
One 12:e0180091. doi: 10.1371/jou nal.pone.0180091
Laja in-Cues a, R., Nancla es, C., A anz-Taga o, J. A., Gonzalez-La uen e, L.,
A ibas, R. L., A aujo de B i o, M., e al. (2016). G amine de i a i es a ge ing ca(2+)
channels and Se /Th phospha ases: a new dual s a egy o he ea men o
neu odegene a i e diseases. J. Med. Chem. 59, 6265–6280. doi: 10.1021/acs.
jmedchem.6b00478
Lam, T. T., Able , A. S., Kwong, J. M., and Tso, M. O. (1999). N-me hyl-D-
aspa a e (NMDA)--induced apop osis in a e ina. In es . Oph halmol. Vis. Sci.
40, 2391–2397.
Lam, T. T., Siew, E., Chu, R., and Tso, M. O. (1997). Amelio a i e e ec o MK-801 on
e inal ischemia. J. Ocul. Pha macol. The . 13, 129–137. doi: 10.1089/jop.1997.13.129
Lambuk, L., Iezhi sa, I., Aga wal, R., Baka , N. S., Aga wal, P., and Ismail, N. M.
(2019). An iapop o ic e ec o au ine agains NMDA-induced e inal exci o oxici y in
a s. Neu o oxicology 70, 62–71. doi: 10.1016/j.neu o.2018.10.009
Leb un-Julien, F., Duplan, L., Pe ne , V., Osswald, I., Sapieha, P., Bou geois, P., e al.
(2009). Exci o oxic dea h o e inal neu ons in i o occu s ia a non-cell-au onomous
mechanism. J. Neu osci. 29, 5536–5545. doi: 10.1523/JNEUROSCI.0831-09.2009
Li, Y., Schlamp, C. L., and Nickells, R. W. (1999). Expe imen al induc ion o e inal
ganglion cell dea h in adul mice. In es . Oph halmol. Vis. Sci. 40, 1004–1008.
Di Pie domenico e al. 10.3389/ nana.2024.1335176
F on ie s in Neu oana omy 20 on ie sin.o g
Li, S., Yang, C., Zhang, L., Gao, X., Wang, X., Liu, W., e al. (2016). P omo ing axon
egene a ion in he adul CNS by modula ion o he melanopsin/GPCR signaling. P oc.
Na l. Acad. Sci. U. S. A. 113, 1937–1942. doi: 10.1073/pnas.1523645113
Lindq is , N., Peinado-Ramonn, P., Vidal-Sanz, M., and Hallbook, F. (2004). GDNF,
e , GFRalpha1 and 2in he adul a e ino- ec al sys em a e op ic ne e ansec ion.
Exp. Neu ol. 187, 487–499. doi: 10.1016/j.expneu ol.2004.02.002
Lucas, D. R., and Newhouse, J. P. (1957). The oxic e ec o sodium L-glu ama e on
he inne laye s o he e ina. A.M.A. A ch. Oph halmol. 58, 193–201. doi: 10.1001/
a choph .1957.00940010205006
Manabe, S., Gu, Z., and Lip on, S. A. (2005). Ac i a ion o ma ix me allop o einase-9
ia neu onal ni ic oxide syn hase con ibu es o NMDA-induced e inal ganglion cell
dea h. In es . Oph halmol. Vis. Sci. 46, 4747–4753. doi: 10.1167/io s.05-0128
Mane , H., Fa a on, M., Guido i, A., and Cos a, E. (1989). Delayed inc ease o Ca2+
in lux elici ed by glu ama e: ole in neu onal dea h. Mol. Pha macol. 36, 106–112.
McKe ache , L., Vidal-Sanz, M., and Aguayo, A. J. (1990a). Slow anspo a es o
cy oskele al p o eins change du ing egene a ion o axo omized e inal neu ons in adul
a s. J. Neu osci. 10, 641–648. doi: 10.1523/JNEUROSCI.10-02-00641.1990
McKe ache , L., Vidal-Sanz, M., Essagian, C., and Aguayo, A. J. (1990b). Selec i e
impai men o slow axonal anspo a e op ic ne e inju y in adul a s. J. Neu osci.
10, 2834–2841. doi: 10.1523/JNEUROSCI.10-08-02834.1990
Milla-Na a o, S., Diaz-Tahoces, A., O uno-Liza an, I., Fe nandez, E., Cuenca, N.,
Ge main, F., e al. (2021). Visual Dis unc ion due o he selec i e e ec o glu ama e
agonis s on e inal cells. In . J. Mol. Sci. 22:6245. doi: 10.3390/ijms22126245
Nadal-Nicolas, F. M., Galindo-Rome o, C., Lucas-Ruiz, F., Ma sh-Ams ong, N.,
Li, W., Vidal-Sanz, M., e al. (2023). Pan- e inal ganglion cell ma ke s in mice, a s, and
hesus macaques. Zool. Res. 44, 1–23. doi: 10.24272/j.issn.2095-8137.2022.308
Nadal-Nicolas, F. M., Jimenez-Lopez, M., Salinas-Na a o, M., Sob ado-Cal o, P.,
Albu que que-Beja , J. J., Vidal-Sanz, M., e al. (2012). Whole numbe , dis ibu ion and
co-exp ession o b n3 ansc ip ion ac o s in e inal ganglion cells o adul albino and
pigmen ed a s. PLoS One 7:e49830. doi: 10.1371/jou nal.pone.0049830
Nadal-Nicolas, F. M., Jimenez-Lopez, M., Sob ado-Cal o, P., Nie o-Lopez, L.,
Cano as-Ma inez, I., Salinas-Na a o, M., e al. (2009). B n3a as a ma ke o e inal
ganglion cells: quali a i e and quan i a i e ime cou se s udies in nai e and op ic ne e-
inju ed e inas. In es . Oph halmol. Vis. Sci. 50, 3860–3868. doi: 10.1167/io s.08-3267
Nadal-Nicolas, F. M., Salinas-Na a o, M., Jimenez-Lopez, M., Sob ado-Cal o, P.,
Villegas-Pe ez, M. P., Vidal-Sanz, M., e al. (2014). Displaced e inal ganglion cells in
albino and pigmen ed a s. F on . Neu oana . 8:99. doi: 10.3389/ nana.2014.00099
Nadal-Nicolas, F. M., Sob ado-Cal o, P., Jimenez-Lopez, M., Vidal-Sanz, M., and
Agudo-Ba iuso, M. (2015). Long- e m e ec o op ic ne e axo omy on he e inal
ganglion cell laye . In es . Oph halmol. Vis. Sci. 56, 6095–6112. doi: 10.1167/io s.15-17195
Nadal-Nicolas, F. M., Vidal-Sanz, M., and Agudo-Ba iuso, M. (2018). The aging a
e ina: om unc ion o ana omy. Neu obiol. Aging 61, 146–168. doi: 10.1016/j.
neu obiolaging.2017.09.021
Nassi, J. J., and Callaway, E. M. (2009). Pa allel p ocessing s a egies o he p ima e
isual sys em. Na . Re . Neu osci. 10, 360–372. doi: 10.1038/n n2619
O in-Ma inez, A., Jimenez-Lopez, M., Nadal-Nicolas, F. M.,
Salinas-Na a o, M., Ala con-Ma inez, L., Sau e, Y., e al. (2010). Au oma ed
quan i ica ion and opog aphical dis ibu ion o he whole popula ion o S- and
L-cones in adul albino and pigmen ed a s. In es . Oph halmol. Vis. Sci. 51,
3171–3183. doi: 10.1167/io s.09-4861
O in-Ma inez, A., Salinas-Na a o, M., Nadal-Nicolas, F. M., Jimenez-Lopez, M.,
Valien e-So iano, F. J., Ga cia-Ayuso, D., e al. (2015). Lase -induced ocula hype ension
in adul a s does no a ec non-RGC neu ons in he ganglion cell laye bu esul s in
p o ac ed se e e loss o cone-pho o ecep o s. Exp. Eye Res. 132, 17–33. doi: 10.1016/j.
exe .2015.01.006
O in-Ma inez, A., Valien e-So iano, F. J., Ga cia-Ayuso, D., Ala con-Ma inez, L.,
Jimenez-Lopez, M., Be nal-Ga o, J. M., e al. (2014). A no el in i o model o ocal ligh
emi ing diode-induced cone-pho o ecep o pho o oxici y: neu op o ec ion a o ded by
b imonidine, BDNF, PEDF o bFGF. PLoS One 9:e113798. doi: 10.1371/jou nal.
pone.0113798
Ou, Y., Jo, R. E., Ullian, E. M., Wong, R. O., and Della San ina, L. (2016). Selec i e
ulne abili y o speci ic e inal ganglion cell ypes and synapses a e ansien ocula
hype ension. J. Neu osci. 36, 9240–9252. doi: 10.1523/JNEUROSCI.0940-16.2016
Peng, Y. W., Blacks one, C. D., Hugani , R. L., and Yau, K. W. (1995). Dis ibu ion o
glu ama e ecep o sub ypes in he e eb a e e ina. Neu oscience 66, 483–497. doi:
10.1016/0306-4522(94)00569-q
Picha a am, P., Palani, C. D., Pa el, C., Xu, Z., Shosha, E., Fouda, A. Y., e al. (2018).
Ta ge ing polyamine oxidase o p e en exci o oxici y-induced e inal
neu odegene a ion. F on . Neu osci. 12:956. doi: 10.3389/ nins.2018.00956
Qua ochi, L. E., S abio, M. E., Kim, I., Ila di, M. C., Michelle Foge son, P.,
Ley e , M. L., e al. (2019). The M6 cell: a small- ield bis a i ied pho osensi i e e inal
ganglion cell. J. Comp. Neu ol. 527, 297–311. doi: 10.1002/cne.24556
Rheaume, B. A., Je een, A., Bolise y, M., Sajid, M. S., Yang, Y., Renna, K., e al. (2018).
Single cell ansc ip ome p o iling o e inal ganglion cells iden i ies cellula sub ypes.
Na . Commun. 9:2759. doi: 10.1038/s41467-018-05134-3
Ro e e, G., Nadal-Nicolas, F. M., Agudo-Ba iuso, M., Sob ado-Cal o, P.,
Nie o-Lopez, L., Nucci, C., e al. (2015). Compa ison o e inal ne e ibe laye hinning
and e inal ganglion cell loss a e op ic ne e ansec ion in adul albino a s. In es .
Oph halmol. Vis. Sci. 56, 4487–4498. doi: 10.1167/io s.15-17145
Ro e e, G., Nadal-Nicolas, F. M., Wang, J., Be nal-Ga o, J. M., Ga cia-Ca illo, N.,
Villegas-Pe ez, M. P., e al. (2016). Melanopsin-con aining o non-melanopsin-
con aining e inal ganglion cells esponse o acu e ocula hype ension wi h o wi hou
b ain-de i ed neu o ophic ac o neu op o ec ion. In es . Oph halmol. Vis. Sci. 57,
6652–6661. doi: 10.1167/io s.16-20146
Salinas-Na a o, M., Mayo -To oglosa, S., Jimenez-Lopez, M., A iles-T igue os, M.,
Holmes, T. M., Lund, R. D., e al. (2009). A compu e ized analysis o he en i e e inal
ganglion cell popula ion and i s spa ial dis ibu ion in adul a s. Vis. Res. 49, 115–126.
doi: 10.1016/j. is es.2008.09.029
Sanchez-Migallon, M. C., Valien e-So iano, F. J., Nadal-Nicolas, F. M., Di
Pie domenico, J., Vidal-Sanz, M., and Agudo-Ba iuso, M. (2018). Su i al o
melanopsin exp essing e inal ganglion cells long e m a e op ic ne e auma in mice.
Exp. Eye Res. 174, 93–97. doi: 10.1016/j.exe .2018.05.029
Sanes, J. R., and Masland, R. H. (2015). The ypes o e inal ganglion cells: cu en
s a us and implica ions o neu onal classi ica ion. Annu. Re . Neu osci. 38, 221–246.
doi: 10.1146/annu e -neu o-071714-034120
Schmid , T. M., Alam, N. M., Chen, S., Ko uji, P., Li, W., P usky, G. T., e al. (2014). A
ole o melanopsin in alpha e inal ganglion cells and con as de ec ion. Neu on 82,
781–788. doi: 10.1016/j.neu on.2014.03.022
Schmid , T. M., Chen, S. K., and Ha a , S. (2011a). In insically pho osensi i e e inal
ganglion cells: many sub ypes, di e se unc ions. T ends Neu osci. 34, 572–580. doi:
10.1016/j. ins.2011.07.001
Schmid , T. M., Do, M. T., Dacey, D., Lucas, R., Ha a , S., and Ma ynia, A. (2011b).
Melanopsin-posi i e in insically pho osensi i e e inal ganglion cells: om o m o
unc ion. J. Neu osci. 31, 16094–16101. doi: 10.1523/JNEUROSCI.4132-11.2011
Schue au , F., Naska , R., Vo we k, C. K., Zu akowski, D., and D eye , E. B. (2000).
Ganglion cell loss a e op ic ne e c ush media ed h ough AMPA-kaina e and NMDA
ecep o s. In es . Oph halmol. Vis. Sci. 41, 4313–4316.
Silip andi, R., Canella, R., Ca migno o, G., Schia o, N., Zanella o, A., Zanoni, R., e al.
(1992). N-me hyl-D-aspa a e-induced neu o oxici y in he adul a e ina. Vis.
Neu osci. 8, 567–573. doi: 10.1017/s0952523800005666
Sonoda, T., Okabe, Y., and Schmid , T. M. (2020). O e lapping mo phological and
unc ional p ope ies be ween M4 and M5 in insically pho osensi i e e inal ganglion
cells. J. Comp. Neu ol. 528, 1028–1040. doi: 10.1002/cne.24806
S a o skaya, I. G., and K is al, B. S. (2005). The powe house akes con ol o he cell: is
he mi ochond ial pe meabili y ansi ion a iable he apeu ic a ge agains neu onal
dys unc ion and dea h? F ee Radic. Biol. Med. 38, 687–697. doi: 10.1016/j.
ee adbiomed.2004.11.032
Sweeney, N. T., James, K. N., Nis o ica, A., Lo ig-Roach, R. M., and Feldheim, D. A.
(2019). Exp ession o ansc ip ion ac o s di ides e inal ganglion cells in o dis inc
classes. J. Comp. Neu ol. 527, 225–235. doi: 10.1002/cne.24172
Tezel, G. (2013). Immune egula ion owa d immunomodula ion o neu op o ec ion
in glaucoma. Cu . Opin. Pha macol. 13, 23–31. doi: 10.1016/j.coph.2012.09.013
T an, N. M., Shekha , K., Whi ney, I. E., Jacobi, A., Benha , I., Hong, G., e al. (2019). Single-
cell p o iles o e inal ganglion cells di e ing in esilience o inju y e eal neu op o ec i e
genes. Neu on 104, 1039–1055.e12. doi: 10.1016/j.neu on.2019.11.006
Tsu uga, H., Mu a a, H., A aie, M., and Aiha a, M. (2023). Neu op o ec i e e ec o
he calcium channel blocke nil adipine on e inal ganglion cell dea h in a mouse ocula
hype ension model. Heliyon 9:e13812. doi: 10.1016/j.heliyon.2023.e13812
Valien e-So iano, F. J., Ga cia-Ayuso, D., O in-Ma inez, A., Jimenez-Lopez, M.,
Galindo-Rome o, C., Villegas-Pe ez, M. P., e al. (2014). Dis ibu ion o melanopsin
posi i e neu ons in pigmen ed and albino mice: e idence o melanopsin in e neu ons
in he mouse e ina. F on . Neu oana . 8:131. doi: 10.3389/ nana.2014.00131
Valien e-So iano, F. J., Nadal-Nicolas, F. M., Salinas-Na a o, M., Jimenez-Lopez, M.,
Be nal-Ga o, J. M., Villegas-Pe ez, M. P., e al. (2015). BDNF escues RGCs bu no
in insically pho osensi i e RGCs in ocula hype ensi e albino a e inas. In es .
Oph halmol. Vis. Sci. 56, 1924–1936. doi: 10.1167/io s.15-16454
Ve ma, M., Lizama, B. N., and Chu, C. T. (2022). Exci o oxici y, calcium and
mi ochond ia: a iad in synap ic neu odegene a ion. T ansl. Neu odegene . 11:3. doi:
10.1186/s40035-021-00278-7
Vidal-Sanz, M., A iles-T igue os, M., Whi eley, S. J., Sau e, Y., and Lund, R. D. (2002).
Reinne a ion o he p e ec um in adul a s by egene a ed e inal ganglion cell axons:
ana omical and unc ional s udies. P og. B ain Res. 137, 443–452. doi: 10.1016/
s0079-6123(02)37035-3
Vidal-Sanz, M., Galindo-Rome o, C., Valien e-So iano, F. J., Nadal-Nicolas, F. M.,
O in-Ma inez, A., Ro e e, G., e al. (2017). Sha ed and di e en ial e inal esponses
agains op ic ne e inju y and ocula hype ension. F on . Neu osci. 11:235. doi: 10.3389/
nins.2017.00235
Vidal-Sanz, M., Nadal-Nicolas, F. M., Valien e-So iano, F. J., Agudo-Ba iuso, M., and
Villegas-Pe ez, M. P. (2015a). Iden i ying speci ic RGC ypes may shed ligh on hei
idiosync a ic esponses o neu op o ec ion. Neu al Regen. Res. 10, 1228–1230. doi:
10.4103/1673-5374.162751
Di Pie domenico e al. 10.3389/ nana.2024.1335176
F on ie s in Neu oana omy 21 on ie sin.o g
Vidal-Sanz, M., Salinas-Na a o, M., Nadal-Nicolas, F. M., Ala con-Ma inez, L.,
Valien e-So iano, F. J., de Impe ial, J. M., e al. (2012). Unde s anding glaucoma ous
damage: ana omical and unc ional da a om ocula hype ensi e oden e inas. P og.
Re in. Eye Res. 31, 1–27. doi: 10.1016/j.p e eye es.2011.08.001
Vidal-Sanz, M., Valien e-So iano, F. J., O in-Ma inez, A., Nadal-Nicolas, F. M.,
Jimenez-Lopez, M., Salinas-Na a o, M., e al. (2015b). Re inal neu odegene a ion in
expe imen al glaucoma. P og. B ain Res. 220, 1–35. doi: 10.1016/bs.pb .2015.04.008
Vidal-Villegas, B., Di Pie domenico, J., Gallego-O ega, A., Galindo-Rome o, C.,
Ma inez-de-la-Casa, J. M., Ga cia-Feijoo, J., e al. (2021a). Sys emic ea men wi h
7,8-Dihyd oxi la one ac i a es T kB and a o ds p o ec ion o wo di e en e inal
ganglion cell popula ions agains axo omy in adul a s. Exp. Eye Res. 210:108694. doi:
10.1016/j.exe .2021.108694
Vidal-Villegas, B., Di Pie domenico, J., Mi alles de Impe ial-Olle o, J. A.,
O in-Ma inez, A., Nadal-Nicolas, F. M., Be nal-Ga o, J. M., e al. (2019).
Melanopsin(+)RGCs a e ully esis an o NMDA-induced exci o oxici y. In . J. Mol. Sci.
20:3012. doi: 10.3390/ijms20123012
Vidal-Villegas, B., Gallego-O ega, A., Mi alles de Impe ial-Olle o, J. A., Ma inez de
la Casa, J. M., Ga cia Feijoo, J., and Vidal-Sanz, M. (2021b). Pho osensi i e ganglion
cells: a diminu i e, ye essen ial popula ion. A ch. Soc. Esp. O almol. 96, 299–315. doi:
10.1016/j.o ale.2020.06.020
Vo we k, C. K., K eu z, M. R., Bocke s, T. M., B osz, M., D eye , E. B., and Sabel, B. A.
(1999). Suscep ibili y o e inal ganglion cells o exci o oxici y depends on soma size and
e inal eccen ici y. Cu . Eye Res. 19, 59–65. doi: 10.1076/cey .19.1.59.5336
Vo we k, C. K., Zu akowski, D., McDe mo , L. M., Maw in, C., and D eye , E. B.
(2004). E ec s o axonal inju y on ganglion cell su i al and glu ama e homeos asis.
B ain Res. Bull. 62, 485–490. doi: 10.1016/S0361-9230(03)00075-3
Wang, S., Gu, D., Zhang, P., Chen, J., Li, Y., Xiao, H., e al. (2018). Melanopsin-
exp essing e inal ganglion cells a e ela i ely esis an o exci o oxici y induced by
N-me hyl-d-aspa a e. Neu osci. Le . 662, 368–373. doi: 10.1016/j.neule .2017.10.055
Whi eley, S. J., Sau e, Y., A iles-T igue os, M., Vidal-Sanz, M., and Lund, R. D. (1998).
Ex en and du a ion o eco e ed pupilla y ligh e lex ollowing e inal ganglion cell
axon egene a ion h ough pe iphe al ne e g a s di ec ed o he p e ec um in adul
a s. Exp. Neu ol. 154, 560–572. doi: 10.1006/exn .1998.6959
Zhao, M., Toma, K., Kinde, B., Li, L., Pa el, A. K., Wu, K. Y., e al. (2023). Os eopon in
d i es e inal ganglion cell esiliency in glaucoma ous op ic neu opa hy. Cell Rep.
42:113038. doi: 10.1016/j.cel ep.2023.113038