pha maceu ics
A icle
No el Use o PLGA Mic osphe es o C ea e an Animal Model
o Glaucoma wi h P og essi e Neu o e inal Degene a ion
Da id Ga cia-He anz 1,2,†, Ma ia Jesus Rod igo 3,4,5,† , Manuel Subias 4,5 , Te esa Ma inez-Rincon 4,5,
Sil ia Mendez-Ma inez 4,5 , I ene B a o-Osuna 1,2,3,6 , Aina Bone 7,8,9, Jesus Rube e 7,8,9,
Julian Ga cia-Feijoo 1,3,10,11, Luis Pablo 3,4,5, Elena Ga cia-Ma in 3,4,5 and Rocío He e o-Van ell 1,2,3,6,*


Ci a ion: Ga cia-He anz, D.;
Rod igo, M.J.; Subias, M.;
Ma inez-Rincon, T.;
Mendez-Ma inez, S.; B a o-Osuna, I.;
Bone , A.; Rube e, J.; Ga cia-Feijoo, J.;
Pablo, L.; e al. No el Use o PLGA
Mic osphe es o C ea e an Animal
Model o Glaucoma wi h P og essi e
Neu o e inal Degene a ion.
Pha maceu ics 2021,13, 237.
h ps://doi.o g/10.3390/
pha maceu ics13020237
Academic Edi o : And ew U quha
Recei ed: 16 Decembe 2020
Accep ed: 28 Janua y 2021
Published: 8 Feb ua y 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1
Inno a ion, The apy and Pha maceu ical De elopmen in Oph halmology (InnO al) Resea ch G oup, UCM,
28040 Mad id, Spain; [email p o ec ed] (D.G.-H.); [email p o ec ed] (I.B.-O.); [email p o ec ed] (J.G.-F.)
2Depa amen o de Fa macia Galénica y Tecnología Alimen a ia, Facul ad de Fa macia,
Uni e sidad Complu ense de Mad id (UCM), IdISSC, 28040 Mad id, Spain
3Thema ic Resea ch Ne wo k in Oph halmology (O a ed), Ca los III Na ional Ins i u e o Heal h,
28040 Mad id, Spain; [email p o ec ed] (M.J.R.); lpablo@uniza .es (L.P.);
[email p o ec ed] (E.G.-M.)
4Depa men o Oph halmology, Miguel Se e Uni e si y Hospi al, 50009 Za agoza, Spain;
[email p o ec ed] (M.S.); [email p o ec ed] (T.M.-R.);
[email p o ec ed] (S.M.-M.)
5Miguel Se e Oph halmology Resea ch G oup (GIMSO), A agon Heal h Resea ch Ins i u e (IIS A agon),
50009 Za agoza, Spain
6Ins i u o Uni e si a io de Fa macia Indus ial (IUFI), Facul ad de Fa macia, Uni e sidad Complu ense de
Mad id (UCM), 28040 Mad id, Spain
7Cen e o Animal Bio echnology and Gene The apy (CBATEG), Uni e si a Au ònoma de Ba celona,
08193 Bella e a, Spain; [email p o ec ed] (A.B.); [email p o ec ed] (J.R.)
8CIBER o Diabe es and Associa ed Me abolic Diseases (CIBERDEM), 28029 Mad id, Spain
9Depa men o Animal Heal h and Ana omy, School o Ve e ina y Medicine, Uni e si a Au ònoma de Ba celona,
08193 Bella e a, Spain
10 Se icio de O almología, Hospi al Clínico San Ca los, 28040 Mad id, Spain
11 Depa amen o de Inmunología, O almología y ORL, Facul ad de Medicina, Uni e sidad Complu ense de
Mad id (UCM), IdISSC, 28040 Mad id, Spain
*Co espondence: [email p o ec ed]; Tel.: +34-91-394-1739; Fax: +34-91-394-1736
† These au ho s con ibu ed equally o his wo k.
Abs ac :
P og essi e degene a ion o neu o e inal issue wi h main ained ele a ed in aocula
p essu e (IOP) o simula e ch onic glaucoma was p oduced by in acame al injec ions o poly (lac ic-
co-glycolic) acid (PLGA) mic osphe es (Ms) in a eyes. The igh eye o 39 a s ecei ed di e en
sizes o PLGA-Ms (2
µ
L suspension; 10% w/ ): 14 wi h 38–20
µ
m Ms (Ms38/20 model) and 25 wi h
20–10
µ
m pa icles (Ms20/10 model). This no el glaucoma animal model was compa ed o he
episcle al ein scle osis (EPI) model (25 eyes). Injec ions we e pe o med a baseline, wo, ou and
six weeks. Clinical signs, IOP, e ina and op ic ne e hicknesses (using
in i o
op ical cohe ence
omog aphy; OCT), and his ological s udies we e pe o med. An IOP inc emen was obse ed in
all h ee g oups, howe e , he alues ob ained om he PLGA-Ms injec ion esul ed lowe wi h a
be e p ese a ion o he ocula su ace. In ac , he injec ion o Ms20/10 c ea ed a gen le , mo e
p og essi e, and mo e sus ained inc ease in IOP. This IOP al e a ion was co ela ed wi h a signi ican
dec ease in mos OCT pa ame e s and in his ological ganglion-cell coun o he h ee condi ions
h oughou he eigh -week ollow-up. In all cases, p og essi e degene a ion o he e ina, e inal
ganglion cells and op ic ne e, simula ing ch onic glaucoma, was de ec ed by OCT and co obo a ed
by his ological s udy. Resul s showed an al e na i e glaucoma model o he well-known episcle al
ein model, which was simple o pe o m, mo e ep oducible and easie o moni o in i o.
Keywo ds:
PLGA mic osphe es; in aocula p essu e; glaucoma animal model; in acame al injec-
ion; op ic ne e; neu odegene a ion
Pha maceu ics 2021,13, 237. h ps://doi.o g/10.3390/pha maceu ics13020237 h ps://www.mdpi.com/jou nal/pha maceu ics
Pha maceu ics 2021,13, 237 2 o 20
1. In oduc ion
Glaucoma is a degene a i e op ic neu opa hy in which i e e sible ision loss is
p oduced by he g adual dea h o e inal ganglion cells (RGC). I has been es ima ed
ha his neu odegene a i e disease will a ec up o 111.8 million people by 2040 [
1
].
Inc eased in aocula p essu e (IOP) is he uni e sal isk ac o and IOP luc ua ions ha e
been associa ed wi h immune-media ed p og ession o glaucoma [
2
–
4
]. Howe e , he
mechanism by which ele a ed IOP leads o RGC dea h emains unclea .
In o de o b oaden and imp o e knowledge o glaucoma ous pa hologies, se e al an-
imal models ha e been de eloped in ecen decades applying a signi ican inc ease in IOP
seconda y o a dec ease in ou low o aqueous humo . To his, ei he lase , cau e iza ion,
liga u e and/o episcle al ein scle osis o mechanical blockage o he abecula mesh-
wo k wi h obs uc i e subs ances (hyalu onic acid o pa amagne ic, la ex o polys y ene
beads injec ed in o he an e io chambe ) ha e been used [
5
]. All hese models p oduce
a sudden, sho inc ease in IOP esul ing in limi ed RGC and axonal damage. S udies
using non-biodeg adable beads o c ea e glaucoma animal models include a wide ange o
pa icle sizes, concen a ions o adminis e ed suspensions and injec ion equencies [
6
–
13
].
Al hough injec ions o hese pa icles in o he an e io chambe ha e been epo ed as
causing RGC and op ic ne e degene a ion, undesi ed e ec s such as synechia and co neal
opaci y we e also epo ed [14].
Al hough op ic ne e degene a ion and RGC apop osis ha e been analyzed in glau-
coma models using his ological s udies and compu a ional echnologies [
15
–
17
], disc epan-
cies in e inal dea h dis ibu ion s ill emain. Fu he mo e, ecen s udies ha e e en shown
immune-media ed [18] con ala e al eye degene a ion [17].
The biodeg adable alipha ic polyes e co-polyme poly (lac ic-co-glycolic) acid (PLGA)
is app o ed by he U.S. Food and D ug Adminis a ion (FDA) and he Eu opean Medicines
Agency o se e al clinical applica ions, mainly o d ug deli e y sys ems [
19
]. In an
aqueous en i onmen , PLGA deg ades by hyd olysis, p oducing lac ic and glycolic acid
me abolized by K ebs’s cycle o CO2and H2O being easily elimina ed om he body [20].
In oph halmic he apy, he e is al eady an in aocula PLGA implan app o ed o
clinical use (Ozu dex) [
21
]. This biodeg adable polyme can also be used o p oduce
mic osphe es (Ms). Fo se e al decades now, in aocula adminis a ion o Ms loaded wi h
di e en ac i e subs ances has been unde e alua ion as a he apeu ic ool o diseases
a ec ing he pos e io segmen o he eye [
20
,
22
]. The deg ada ion a e o PLGA Ms anges
om weeks o mon hs and is in luenced by unable ac o s such as polyme molecula
weigh , lac ic:glycolic a io, Ms size and shape, po osi y, and pH en i onmen [23,24].
This pape p esen s a new ch onic animal glaucoma model c ea ed wi h epea ed
injec ions o biodeg adable nonloaded PLGA Ms in o he an e io chambe o a eyes. This
p oduces a p og essi e and ch onic inc ease in IOP (up o eigh weeks) and, consequen ly,
a neu odegene a i e p ocess ha simula es he condi ions appea ing in glaucoma pa ien s.
To ou knowledge, nonloaded biodeg adable mic osphe es ha e ne e been used o his
pu pose un il now. The de eloped animal model shows a degene a ion o pho o ecep o s
and e inal ganglion cells ha simula es human glaucoma.
2. Ma e ials and Me hods
2.1. Ma e ials
Poly (D,L-lac ide-co-glycolide) (PLGA) (50:50; inhe en iscosi y: 0.16–0.24 dL/g) was
ob ained om E onik España, G anolle s, Spain. Poly inyl alcohol (PVA; 67,000 g/mol)
(pu chased om Me ck KgaA, Da ms ad , Ge many) and me hylene chlo ide (ob ained om
Pan Reac Appli Chem, Ba celona, Spain) we e also employed in mic osphe e manu ac u e.
2.2. Manu ac u e o PLGA Mic osphe es
The PLGA Ms we e ob ained using he oil-in-wa e (O/W) emulsion sol en ex ac ion-
e apo a ion echnique. Thus, PLGA (400 mg) was i s dissol ed in me hylene chlo-
ide (2 mL). Once dissol ed, he o ganic polyme solu ion was emulsi ied wi h 5 mL o
Pha maceu ics 2021,13, 237 3 o 20
PVA Milli-Q wa e solu ion (1% w/ ) using a homogenize (Poly on RECO, Kinema ica,
GmbHT PT3000, Luce ne, Swi ze land) a 7000 pm o 1 min. The emulsion o med was
hen pou ed in o 100 mL o PVA Milli-Q wa e solu ion (0.1% w/ ) and magne ically s i ed
o 3 h o allow o ganic sol en e apo a ion and, subsequen ly, Ms ma u a ion.
A e wa ds, he Ms we e washed wi h Milli-Q wa e o emo e he PVA and we e
sepa a ed in o wo g anulome ic ac ions (38–20
µ
m and 20–10
µ
m) using h ee sie es
(mesh size 38, 20 and 10
µ
m). Finally, he Ms we e eeze-d ied ( eezing:
−
60
◦
C/15 min;
d ying: −60 ◦C/12 h/0.1 mBa ) and s o ed a −30 ◦C in d y condi ions un il use.
2.3. Mic osphe e Cha ac e iza ion
2.3.1. P oduc ion Yield Pe cen age (PY%)
The p oduc ion yield pe cen age was calcula ed using he ollowing Equa ion (1):
PY% = (amoun o mic osphe es)/( o al amoun o polyme s) ×100 (1)
2.3.2. Mo phological E alua ion
Mo phological e alua ion was pe o med on he eeze-d ied Ms using scanning
elec on mic oscopy (SEM; Jeol, JSM-6335F, Tokyo, Japan). Gold spu e -coa ing was
applied o he samples p io o obse a ion.
2.3.3. Mean Pa icle Size and Pa icle Size Dis ibu ion
Dual ligh sca e ing (Mic o ac S3500 Se ies Pa icle Size Analyze , Mon gome y ille,
PA, USA) was employed o de e mine mean pa icle size and pa icle size dis ibu ion.
Th ee di e en measu emen s o each sample we e ob ained. The esul s we e exp essed
as olume mean diame e s (±s anda d de ia ion).
2.4. Animals
All wo k wi h animals was app o ed by he E hics Commi ee o Animal Resea ch
(PI34/17, 27 June 2017) and was ca ied ou in s ic acco dance wi h he ARVO S a e-
men o he Use o Animals in Oph halmic and Vision Resea ch on he p emises o he
Biomedical Resea ch Cen e o A agon (CIBA). The animals we e housed unde con olled
condi ions (12-h da k/ligh cycle, empe a u e 22
◦
C, ela i e humidi y 55%). S anda d
cages wi h en i onmen al en ichmen , wa e and ood ad libi um we e used.
Six y- ou Long E ans a s (40% males, 60% emales) aged 4 weeks and weighing
50–100 g
a he beginning o he expe imen we e used o his longi udinal and in e en-
ionis s udy. Long E ans a s we e used as being easy handling and o compa ison wi h
p e ious s udies ha ing used hese animals in glaucoma esea ch and e inal degene a-
ion [3,18,25–27].
2.5. Ocula Hype ension (OHT) Induc ion
All animals ecei ed a o al o 4 ocula hype ension injec ions adminis e ed in he
igh eye (RE) a baseline, 2, 4 and 6 weeks unde in ape i oneally seda i e (60 mg/kg o
Ke amine + 0.25 mg/kg o Dexmede omidine), along wi h opical eye d ops con aining
e acaine 1 mg/mL + oxibup ocaine 4 mg/mL (Anes esico doble Coli cusi, Alcon CusíSA,
Ba celona, Spain), unde asep ic su gical condi ions (iodized solu ion and e y h omycin
5 mg/g oin men (O almolosa Cusíe i omicina, Alcon CusíSA, Ba celona, Spain)).
Tempe a u e was con olled using wa m pads. Twen y- i e animals ecei ed a scle osing
injec ion in he episcle al eins as p e iously desc ibed [
28
]. The o he animals ecei ed
PLGA Ms (2
µ
L suspension, 10% w/ ) injec ed in o he an e io chambe o he eye by
supe o empo al co neal punc u e: 14 animals ecei ed Ms38/20 and 25 animals ecei ed
Ms20/10. Injec ions we e pe o med using a mic ome e ed Hamil on sy inge wi h a
glass mic opipe e.
Pha maceu ics 2021,13, 237 4 o 20
The injec ion was always done by an expe oph halmologis using he same en ance
doo in he pe iphe al co nea, so he co nea was al e ed as li le as possible. The e we e no
cen al co neal leukomas ha al e ed he co neal hys e esis.
2.6. Oph halmological S udies
2.6.1. In Vi o
Clinical signs such as ocula su ace edness, co neal al e a ions, in aocula in lam-
ma ion, in ec ion o ca a ac o ma ion, as well as IOP measu emen s using a Tonolab
onome e (Tonolab, Tiola Oy Helsinki, Finland), we e eco ded e e y week. This p oce-
du e was accomplished using a seda i e mix u e o 3% se o lu ane gas and 1.5% oxygen
o less han 3 min in o de o a oid hypo ension e ec s, as ecommended by o he au-
ho s [
29
]. The IOP alue was ob ained by a e aging h ee consecu i e measu emen s,
aken om he a e age o 6 ebounds.
Neu o e inal s uc u es we e analyzed using op ical cohe ence omog aphy (OCT
Spec alis, Heidelbe g Enginee ing, Heidelbe g, Ge many). Obse a ions we e pe o med
a baseline and hen biweekly up o week 8. A plane con ac lens was adap ed o he
a ’s co nea o acqui e high quali y images. The p o ocols employed we e as ollows:
e ina pos e io pole (R), e ina ne e ibe laye (RNFL) and ganglion cell laye (GCL)
wi h au oma ic segmen a ion. These p o ocols analyzed a ci cula a ea cen e ed on he
op ic disc using 61 b-scans. Subsequen ollow-up examina ions we e pe o med a his
same loca ion using he eye- acking so wa e and ollow-up applica ion. The e ina and
GCL we e analyzed on he basis o he 9 ETDRS (Ea ly T ea men Diabe ic Re inopa hy
S udy) a eas [
30
], which included a cen al (C) 1-mm ci cle cen e ed on he op ic disc
( hough no o ea exis s in a s) and inne (in e io ; II; supe io : IS; nasal: IN; empo al:
IT) and ou e (in e io : OI; supe io : OS; nasal: ON; empo al: OT) ings measu ing 2
and 3 mm in diame e , espec i ely, as well as o al olume (TV). The RNFL p o ocol
p o ided measu emen s o he 6 pe ipapilla y sec o s (in e o empo al: IT; in e onasal:
IN; supe o empo al: ST; supe onasal: SN; nasal: N; and empo al: T). Re inal hickness
composed om he inne limi ing memb ane o he e inal pigmen epi helium; RNFL om
he inne limi ing memb ane o he GCL bounda ies; and GCL om he RNFL o he inne
nuclea laye bounda ies.
2.6.2. Pos mo em
Unde gene al anes hesia, he animals we e eu hanized wi h an in aca diac injec ion
o sodium hiopen al (25 mg/mL). Enuclea ed eyes used o his ology and immunohis-
ochemis y we e ixed in neu al bu e ed o malin (10%) and embedded in pa a in. A
o al o 18 REs belonging o 18 a s injec ed in he an e io chambe wi h Ms (20–10
µ
m
and 38–20
µ
m) and injec ed wi h a saline solu ion in he episcle al eins (6 eyes each) we e
analyzed. As con ols, he con ala e al noninjec ed le eyes we e used. Eyes embedded
in pa a in we e immed o each he op ic ne e head. A e ha , 5-
µ
m sec ions we e
depa a ined, ehyd a ed and washed using H
2
O
2
(10%) o 5 min (quenching) be o e
incuba ion (o e nigh a 4
◦
C) o he ollowing p ima y an ibodies: mouse an i-B n3a
(San a C uz Bio echnology Inc., Dallas, TX, USA) a 1:50 dilu ion; and abbi an i-glial
ib illa y acidic p o ein (GFAP) (DAKO, Ba h, Uni ed Kingdom) a 1:1000 dilu ion. Then,
sec ions we e incuba ed a oom empe a u e (90 min) wi h speci ic seconda y an ibodies:
bio inyla ed ho se an i-mouse a 1:50 dilu ion and bio inyla ed goa an i- abbi a 1:100
dilu ion (Vec o Labo a o ies, Bu lingame, CA, USA). A e ha , incuba ion was pe o med
wi h ABC-HRP (The mo Fishe Scien i ic, Wal ham, MA, USA) a 1:50 dilu ion a oom
empe a u e (90 min). In a nex s ep, sec ions we e washed be o e and a e e e y incuba-
ion in phospha e bu e ed saline solu ion inally, s aining o sec ions was pe o med wi h
diaminobenzidine (DAB) (3 min) and coun e s ained wi h Ha ys Hema oxylin (Sigma-
Ald ich Co p., S . Louis, MO, USA) a oom empe a u e (20 min). Ganglion cells we e
coun ed in adial sec ions o he e ina along 2 mm o a linea egion o he ganglion cell
Pha maceu ics 2021,13, 237 5 o 20
laye , co esponding o ou a eas, wo on each side o he op ic ne e head. Images we e
hen analyzed by an ope a o blinded o he ea men g oups.
To analyze he loca ion o he mic osphe es in he eye, pa a in sec ions s ained wi h
Hema oxylin/Eosin and wi h he luo escen dye BODIPY (In i ogen, Ca lsbad, CA,
USA) a 1:50 dilu ion we e used. Once loca ed, he na u e o he cells su ounding he
mic osphe es was analyzed by immunohis ochemis y using a an i-Mac2 (Ceda lane,
Bu ling on, NC, USA) a 1:50 dilu ion. A e incuba ion wi h bio ynila ed an i- a an ibody
(Abcam, Camb idge, UK) a 1:250 dilu ion and S ep a idin Alexa Fluo 568 (In i ogen,
Ca lsbad, CA, USA) a 1:100 dilu ion, nuclea coun e s aining was pe o med wi h Bis-
benzimide (Sigma-Ald ich, Mad id, Spain) a 1:100 dilu ion and mic oscopic analysis
was pe o med using a lase -scanning con ocal mic oscope (TCS SP2; Leica Mic osys ems
GmbH, Heidelbe g, Ge many). P ocedu al immunohis ochemis y con ols we e ca ied
ou by omi ing he p ima y an ibody in a sequen ial issue sec ion.
A o al o 6 REs belonging o 6 a s injec ed in he an e io chambe wi h mic osphe es
(20–10
µ
m and 38–20
µ
m) and injec ed in he episcle al eins (2 eyes each) wi h a saline
solu ion we e analyzed using ansmission elec on mic oscopy. As con ols, he con ala -
e al noninjec ed le eyes we e used. Re inal agmen s we e ixed in 2.5% glu a aldehyde
and 2% pa a o maldehyde and hen pos ixed in 1% osmium e oxide, s ained in aqueous
u anyl ace a e, dehyd a ed and embedded in epoxy esin. Ul a hin sec ions (70 nm) we e
s ained using lead ci a e and examined using a ansmission elec on mic oscope (Jeol 1400,
Jeol L d., Tokyo, Japan).
Fo scanning elec on mic oscopy, a o al o 4 REs we e ixed in 2.5% glu a alde-
hyde and dissec ed. The i idoco neal angle was exposed o examine he p esence o
mic osphe es in he abecula meshwo k. A e dehyd a ion, samples we e c i ical-poin
d ied, moun ed on s ubs, coa ed wi h gold-palladium, and obse ed in a scanning elec-
on mic oscope (JEOL JSM-5410, Tokyo, Japan) a an accele a ion ol age o 20 kV using
Semapho e so wa e, Digi al Mic og aph 2.32, Ga an, Inc, Pleasan on, CA, USA (JEOL)
o image acquisi ion.
2.7. S a is ical Analysis
Da a we e eco ded in an Excel da abase and s a is ical analysis was pe o med
using SPSS so wa e e sion 20.0 (SPSS Inc., Chicago, IL, USA). The Kolmogo o –Smi no
es was used o assess sample dis ibu ion. Ne e heless, due o he non-pa ame ic
dis ibu ion o mos o he da a, he Mann–Whi ney U es was employed o e alua e
he di e ences be ween bo h coho s. A pai ed Wilcoxon es was also employed o
change compa ison eco ded in each eye o e he s udy pe iod. Values we e exp essed as
means (
±
s anda d de ia ions). Values o p< 0.05 we e conside ed o indica e s a is ical
signi icance. In o de o a oid a high alse-posi i e a e he Bon e oni co ec ion o
mul iple compa isons was also calcula ed. The le el o signi icance o each a iable was
es ablished acco ding o Bon e oni calcula ions (exp essed as #). S a is ical analysis o he
numbe o ganglion cells was conduc ed on he G aphPad P ism 5 so wa e (G aphPad
So wa e, Inc., La Jolla, CA, USA) using one-way ANOVA. The esul s a e shown as
mean ±SEM. Values o p< 0.05 we e conside ed o indica e s a is ical signi icance.
3. Resul s
3.1. P oduc ion Yield, Mean Pa icle Size and Pa icle Size Dis ibu ion
The PLGA Ms p epa ed showed a p oduc ion yield (PY) o 44.94
±
1.60% o he
38–20
µ
m size ange, while he 20–10
µ
m ac ion esul ed in a PY o 39.37
±
1.75%. In
bo h cases, a monodispe se pa icle size dis ibu ion was obse ed in he selec ed size
ange) (see Sec ion 3.2). The mean pa icle size o he 38–20
µ
m ac ion and he 20–10
µ
m
ac ion esul ed in 21.84 ±1.26 µm and 14.07 ±1.07 µm, espec i ely.

Pha maceu ics 2021,13, 237 6 o 20
3.2. Mo phological E alua ion
Acco ding o he SEM images, bo h size ac ions con ained sphe ical pa icles in he
mic o ange wi h nonpo ous smoo h su aces (Figu e 1A).
1
(A)
(B)
Figu e 1.
Visualiza ion o mic osphe e (Ms) Scanning Elec on Mic oscopy (SEM). (
A
) (SEM) images and poly (lac ic-co-
glycolic) acid (PLGA) mic osphe e pa icle size dis ibu ion, 38–20
µ
m Ms ( op); 20–10
µ
m Ms (bo om) and monodispe se
size dis ibu ion. Indi idual mic osphe es magni ica ion 2500
×
and g oup mic osphe es 700
×
(
B
) Mic osphe e dis ibu ion
in he an e io chambe o a a eye du ing ollow-up. Abb e ia ions: p: pupil; : ligh e lex; a: mic osphe es a in e io
i idoco neal angle; b: emnan s o mic osphe es; c: mic osphe e pelle .
3.3. Oph halmological Clinical Signs
No in ec ion, in aocula in lamma ion (synechiae), ca a ac o ma ion, o e inal
de achmen we e ound in any OHT model. I is wo h men ioning ha he co neal su ace
was be e p ese ed in he Ms20/10 and Ms38/20 models han in he EPI model. As
can be obse ed in Figu e 1B, mic opa icles showed a endency o agg ega e and we e
localized a he in e io i idoco neal angle, some imes o ming a solid deposi ion (pelle ).
This a angemen allowed a clea isual axis and, subsequen ly, co ec OCT acquisi ion.
3.4. Localiza ion o Mic osphe es in Injec ed Eyes
A eigh weeks pos -injec ion, a mix u e o mic osphe es a di e en s ages o biodeg a-
da ion o med agg ega es a he i idoco neal angle, hampe ing he abecula meshwo k
(Figu e 2). The mic osphe es we e no colo ed in Hema oxilin/Eosin-s ained sec ions,
appea ing as anspa en s uc u es (Figu es 2C and 3A). Howe e , using he bo on-
dipy ome hene (BODIPY) luo escen dye, PLGA mic osphe es we e speci ically ma ked
in g een (Figu e 3B). Mic osphe e agg ega es we e s uck o he an e io su ace o he i is
(Figu es 2C and 3A). In con as , only a ew mic osphe es we e in con ac wi h he pos e io
epi helium (endo helium) o he co nea (Figu e 2C). Pigmen ed and unpigmen ed cells
appea inside agg ega es su ounding he anspa en mic osphe es (Figu e 2C). These
cells we e ma ked wi h he an i-Mac2 an ibody (Figu e 3C), indica ing ha hey belong o
Pha maceu ics 2021,13, 237 7 o 20
he mac ophagic cellula lineage [
31
]. SEM images co obo a e he p esence o agg ega es
o mic osphe es and mac ophages a he i idoco neal angle (Figu e 2E,F).
Figu e 2.
Visualiza ion o mic osphe es a he i idoco neal angle wi h Hema oxilin/Eosin and scan-
ning elec on mic oscopy. (
A
–
C
) Localiza ion o mic osphe es a he i idoco neal angle hampe ing
he abecula meshwo k. Hema oxilin/Eosin his ological sec ions a he le el o he op ic ne e.
A owhead: mic osphe e in con ac wi h he endo helium o he co nea. Scale ba s o sub igu e
(
B
) is 53.47
µ
m and o sub igu e (
C
) is 6.73
µ
m. (
D
–
F
) Dissec ion o he i idoco neal angle. The
co nea has been sec ioned and emo ed lea ing he an e io eye chambe isible. No ice he whi ish
appea ance o he mic osphe es. (E,F) Scanning elec on mic oscopy images. In (F) an agg ega e o
Pha maceu ics 2021,13, 237 8 o 20
mic osphe es is isible a high magni ica ion. This PLGA s uc u e is pa ially co e ed by wo
pseudopodia om a cell (colo ed in ed) compa ible wi h a mac ophage. Scale ba s o sub igu e
(
E
) is 5.4
µ
m and o sub igu e (
F
) is 1.66
µ
m. Abb e ia ions: ICA: i idoco neal angle; ON: op ic
ne e; Tm: abecula meshwo k; Co: co nea; I : i is; L: lens; Ma: mac ophage; CON: con ol eye;
Ms38/20: eye injec ed wi h Ms size 38–20 µm; Ms20/10: eye injec ed wi h Ms size 20–10 µm.
Figu e 3.
Iden i ica ion o mic osphe es. (
A
) In Hema oxilin/Eosin-s ained pa a in sec ions, mic osphe es (*) appea ed
anspa en and colo less. They we e su ounded by cells, some imes con aining black pigmen . (
B
) Mic osphe es we e
speci ically ma ked by bo on-dipy ome hene (BODIPY) luo escen dyes. (
C
) Double immuno luo escence using an i-
Mac2 an ibody, a speci ic ma ke o mac ophages, showed ha mic osphe es we e su ounded by mac ophages. Nuclei
coun e s ained wi h Dapi. ICA: i idoco neal angle; I : i is; 38/20 and 20/10
µ
m mic osphe es. Scale ba : (
A
) 21.58
µ
m,
(B) 18.49 µm, (C) 18.49 µm.
Pha maceu ics 2021,13, 237 9 o 20
3.5. In aocula P essu e (IOP)
No di e ences we e ound among coho s a baseline in e ms o IOP alues. Repea ed
injec ion o PLGA Ms in o he an e io chambe p oduced con inuous ele a ion o IOP.
While OHT was de ec ed h ee weeks a e he i s injec ion in bo h mic osphe e models,
i was ound om he i s week onwa ds in he EPI model. The Ms38/20 model showed
mo e luc ua ions in IOP alues han he Ms20/10 model (Figu e 4A). High pe cen ages o
OHT eyes we e ound in all h ee models o e ime, al hough he EPI model showed he
highes pe cen ages a nea ly all imes examined (see Figu e 4B). Le eyes also showed an
inc easing end, albei e a ded (see Supplemen a y Ma e ials: Figu e S1).
Figu e 4.
Analysis o in aocula p essu e (IOP). (
A
) Righ eye IOP cu e in ocula hype ensi e
(OHT) models o e ollow-up. (
B
) Pe cen age o OHT igh eyes (>20 mmHg) in he h ee OHT
models o e ollow-up. Abb e ia ions: EPI: episcle al scle osis model; Ms38/20: 38/20 mic osphe e
model; Ms20/10: 20/10 mic osphe e model; RE: igh eye; IOP: in aocula p essu e; w: weeks, OHT:
ocula hype ension; (%): pe cen age; #: s a is ical signi icance p< 0.020, o Bon e oni co ec ion o
mul iple compa isons.
Pha maceu ics 2021,13, 237 16 o 20
E alua ion using OCT e ealed ha he inc ease in IOP esul ed in neu o e inal
loss bo h in he inne sec o s o he GCL and in he sec o s o he supe io –in e io axis
o he RNFL, as se e al au ho s ound [
15
,
17
,
28
], bu con a y o he indings o o he
au ho s [
8
,
18
] ha de ec ed pe iphe al loss. These disc epancies be ween s udies migh
be due o e inal g ow h o he eye a he expense o pe iphe al a eas [
39
]. A a e o RNFL
loss simila o ha epo ed by o he au ho s [
15
] was also ound, occu ing in bo h he
episcle al model and in he Ms20/10 model, which suppo s he ep oducibili y o ou
esul s and he simila i y be ween he models in his eigh -week s udy.
Recen s udies ha e demons a ed bila e al neu odegene a ion ollowing unila e al
induc ion o hype ension using di e en models such as episcle al ein scle osis, cau e iza-
ion o episcle al eins and o he abecula meshwo k, o an in e mi en loop [
3
,
17
,
18
,
40
].
I is gene ally accep ed ha glaucoma ous damage is a consequence o axonal degene a ion
ha leads o e inal ganglion cell dea h. Glial ac i a ion is also p esen in glaucoma [
41
].
The a ec a ion o mic oglia in neu odegene a ion and i s p og ession ha e been demon-
s a ed [
18
] in p e ious me hods o inducing hype ension. All o hem a e agg essi e and
p oduce sudden inc eases in IOP, which sugges he p esence o an immune eac ion o
a s esso . Howe e , i has also been ecen ly sugges ed ha la en in lamma ion would
wo sen e inal degene a ion [
42
], as we ound in ou esul s, which showed inc eased
gliosis e en in Ms models ha p oduced gen le inc eases in IOP. The p esen wo k would
be he i s s udy demons a ing ha a gen le and sus ained inc ease in IOP also esul ed
in con ala e al e inal damage, as shown by he neu o e inal dec ease e ealed by OCT
and he ac ha , despi e he dec ease in he numbe o RGC, no s a is ical di e ences we e
ound be ween he eyes (con ala e al eye and induced eye) (Figu e 7A). This could be
due o he use o he opposi e eye as a con ol in ou s udy. As we p e iously men ioned,
se e al au ho s ha e ecen ly demons a ed he in ol emen o he opposi e eye a e he
induc ion o ocula hype ension [
3
,
17
,
18
,
40
], so i is ecommended o ake his aspec in o
accoun o o use heal hy animals o compa ison in u u e s udies. O he au ho s ha e
al eady shown ha a small inc ease in IOP, ela i e o he con ala e al eye, could igge
he T-cell-media ed neu odegene a i e p ocess [
4
]. This damage has been de ec ed a e
24 h in he induced eye [
40
] and a e one week in he uninduced con ala e al eye [
17
]. In
his s udy, OCT showed neu o e inal loss om he i s in e im scan ( wo weeks), hough
i could possibly be de ec ed e en ea lie . As glial ac i a ion is now known o sp ead ia
he isual pa hway, degene a i e ac o s could, in u n, ac i a e he lamina and p elami-
na glia [
43
] o he con ala e al eye, p oducing e og ade damage and also c ea ing an
imbalance in p essu e in he op ic ne e head [
44
]. On he o he hand, in addi ion o his
damage o he neu o e inal s uc u e, a g adual inc ease in IOP was also de ec ed in he
uninduced con ala e al eye, as also ound by o he au ho s [18,45].
I is impo an o ema k ha injec ion o PLGA Ms in he an e io chambe is an easy
su gical echnique o gene a e OHT and, mo eo e , adequa ely main ains ocula s uc u es,
making his a simple, use ul and e icien hype ensi e model. In his s udy, he small
olume o only 2
µ
L was chosen o a oid a signi ican inc ease in he ini ial in aocula
p essu e and o acili a e a mo e p og essi e mechanical blockage o he pa icles in he si e
o injec ion. In a p e ious wo k, i was shown ha an injec ion olume o 2.5 mL o saline
solu ion gene a ed IOP luc ua ions abou a consis en baseline and also ha olumes up
o 7
µ
L could e en be adminis e ed since he alues o he mean IOP o he eyes injec ed
wi h saline did no depend on injec ion olume [6].
In he cu en s udy, he impo ance o he adminis a ion ou e and he na u e o
he de ice in he obse ed e ec has also been demons a ed. The in acame al ou e is
used o he adminis a ion o an ibio ics and ecen ly se e al in acame al implan s a e
unde aking clinical ials o a e al eady comme cialized [
46
]. In he case o implan s
and om a clinical poin o iew, one o he side e ec s o he in acame al implan a ion
is ela ed o he i idoco neal angle. In ac , he use o he apeu ic de ices such as he
case o Du ys a, an implan made om PLGA and bima op os (Alle gan, I ine, CA,
USA), is es ic ed in pa ien s wi h small angle o any issue ha could p omo e he deposi

Pha maceu ics 2021,13, 237 17 o 20
o he de ice in he in e io angle [
46
]. Taking in o accoun his men ioned side e ec ,
we used he mic osphe es o p omo e an obs uc ion o he abecula meshwo k which
in u n p omo es an inc ease o IOP. Fu he mo e, he agg ega ion o he adminis e ed
pa icles and hei high equency o injec ion (baseline, wo, ou and six weeks) allow
he mic osphe es o emain longe in he si e o injec ion. This p oduces a mechanical
blockage ha has been p e iously desc ibed in o he s udies by using non-biodeg adable
pa icles [
6
–
8
]. I is wo h men ioning ha in his case he injec ion o he mic opa icles is
no used o he apeu ic pu poses and also ha he agg ega ion a he i idoco neal angle
is one o he causes o he inc ease o IOP. By using o he adminis a ion ou es, mainly
in a i eal and pe iocula , PLGA mic osphe es ha e demons a ed hei po en ial o he
ea men o se e al ocula diseases due o he abili y o hese sys ems o deli e mul iple
ac i e subs ances di ec ly o he a ge si e du ing he long e m [
20
,
22
]. P e ious s udies
ha e demons a ed ha mic osphe es o he apeu ic pu poses we e well ole a ed a e
in a i eal injec ion [
20
,
35
,
47
,
48
]. The amoun o PLGA mic osphe es is also an impo an
ac o o be conside ed. O he s udies ha e shown ha a high quan i y o mic osphe es can
induce e inal damage, being well ole a ed i low amoun s o hese d ug deli e y sys ems
a e used [
49
]. In he p esen s udy mic osphe es we e injec ed e e y wo weeks esul ing
in a o al o 0.8 mg (high quan i y in a abecula meshwo k) a he end o he s udy.
Based on he esul s ob ained in he p esen wo k we could p edic ha u u e
s udies wi h longe ollow-up imes and less neu odegene a i e damage (by modula-
ion/minimiza ion o einjec ion) would allow ge ing close o eal-wo ld glaucoma
models. Ch onic glaucoma animal models a e o g ea in e es o he s udy o he di e -
en phases and s ages o glaucoma, and o es new ea men s based on long- e m d ug
deli e y sys ems.
In conclusion, his s udy desc ibes a new glaucoma model using biodeg adable PLGA
mic osphe es injec ed in o he an e io chambe o he eye ha causes a p og essi e inc ease
in IOP and neu o e inal degene a ion like he ones obse ed in he episcle al ein model.
This s udy pa es he way o new models o neu o e inal degene a ion using a me hod ha
is simple o pe o m and mo e ep oducible han episcle al ein injec ion. This model is
also less agg essi e o he su ace o he animal’s eye, he eby making anspa en s uc u es
a ailable in ha eye. This ac allows epea ed e alua ion by nonin asi e echnology such
as OCT o moni o he de elopmen o he pa hology gene a ed and o quan i y in an
objec i e and nonha m ul way he e ec i eness o new he apies o ea glaucoma.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h ps://www.mdpi.com/1999-492
3/13/2/237/s1, Figu e S1: (A) Le eye IOP cu e in he OHT models du ing ollow-up. (B) Pe cen -
age o OHT le eyes (>20 mmHg) in he h ee OHT models du ing ollow-up. EPI: episcle al scle osis
model; Ms38/20: 38/20 mic osphe e model; Ms20/10: 20/10 mic osphe e model; LE: le eye; IOP:
in aocula p essu e; w: weeks; OHT: ocula hype ension; (%): pe cen age; *: s a is ical signi icance
p< 0.050; #: s a is ical signi icance p< 0.020 o Bon e oni co ec ion o mul iple compa isons,
Table S1: S uc u al neu o e inal analysis using OCT wi h he Ms38/20 model. Ms38/20: 38/20
mic osphe e model; RNFL: e ina ne e ibe laye ; GCL: ganglion cell laye complex; hickness in
mic ons (
µ
m); mean
±
SD (SD: s anda d de ia ion); *: p< 0.050 s a is ical signi icance; #: p< 0.020
s a is ical signi icance wi h Bon e oni co ec ion o mul iple compa isons. G ey cells show he wo
hinnes sec o s a e e y examina ion, Table S2: Le eye neu o e inal analysis using OCT in bo h
mic osphe e models a week 8. Ms38/20: 38/20 mic osphe e model, Ms20/10: 20/10 mic osphe e
model; RNFL: e ina ne e ibe laye ; GCL: ganglion cell laye complex; hickness in mic ons (
µ
m);
mean
±
SD (SD: s anda d de ia ion); *: p< 0.050 s a is ical signi icance; #: p< 0.020 s a is ical
signi icance wi h Bon e oni co ec ion o mul iple compa isons; w: week. G ey cells show he wo
hinnes sec o s a e e y examina ion, Table S3: Righ eye neu o e inal loss a e measu ed using OCT
in he OHT models. EPI: episcle al scle osis model; Ms38/20: 38/20 mic osphe e model; Ms20/10:
20/10 mic osphe e model; RNFL: e ina ne e ibe laye ; GCL: ganglion cell laye complex; RE:
igh eye; hickness in mic ons (
µ
m); w: week. G ey cells show he lowes measu emen s, Table S4:
Righ eye neu o e inal analysis ollow-up using OCT in bo h OHT models. EPI: episcle al scle osis
model; Ms20/10: 20/10 mic osphe e model; RNFL: e ina ne e ibe laye ; GCL: ganglion cell laye
Pha maceu ics 2021,13, 237 18 o 20
complex; hickness in mic ons (
µ
m); mean
±
SD (SD: s anda d de ia ion); % Ch: pe cen age change
in hickness loss; *: p< 0.050 s a is ical signi icance; #: p< 0.020 s a is ical signi icance wi h Bon e oni
co ec ion o mul iple compa isons; w: week. Cells colo ed g ey when EPI model showed hinne
sec o s and/o highe pe cen age loss compa ed o Ms20/10.
Au ho Con ibu ions: Concep ualiza ion, M.J.R., I.B.-O., J.G.-F., L.P., E.G.-M. and R.H.-V.; me hod-
ology, D.G.-H., M.J.R., M.S., T.M.-R., S.M.-M., I.B.-O., A.B., J.R., L.P., E.G.-M. and R.H.-V.; alida ion,
D.G.-H., M.J.R., I.B.-O., L.P., E.G.-M. and R.H.-V.; o mal analysis, D.G.-H., M.J.R., M.S., T.M.-R.,
S.M.-M., A.B. and J.R.; in es iga ion, D.G.-H., M.J.R., M.S., T.M.-R., S.M.-M., I.B.-O., A.B., J.R., J.G.-F.,
L.P., E.G.-M. and R.H.-V.; esou ces, L.P., E.G.-M. and R.H.-V.; da a cu a ion, D.G.-H., M.J.R., E.G.-M.
and R.H.-V.; w i ing—o iginal d a p epa a ion, D.G.-H. and M.J.R.; w i ing— e iew and edi ing,
D.G.-H., M.J.R., I.B.-O., L.P., E.G.-M. and R.H.-V.; isualiza ion, L.P., E.G.-M. and R.H.-V.; supe i-
sion, I.B.-O., L.P., E.G.-M. and R.H.-V.; p ojec adminis a ion, R.H.-V.; unding acquisi ion, M.J.R.,
E.G.-M., I.B.-O., J.G.-F., L.P. and R.H.-V. All au ho s ha e ead and ag eed o he published e sion o
he manusc ip .
Funding:
This s udy was suppo ed by Rio Ho ega Resea ch G an M17/00213, PI17/01726,
PI17/01946 (Ca los III Heal h Ins i u e), and by MAT2017-83858-C2-1 and MAT2017-83858-C2-2
MINECO/AEI/FEDER, Resea ch G oup UCM 920415, ISCIII-FEDER “Una mane a de hace Eu opa”
RETICS O a ed, RD16/0008/0004, RD16/0008/0009, and RD16/0008/029. D.G.H. acknowledges o
UCM-San ande ellowship (CT17/17-CT17-18).
Ins i u ional Re iew Boa d S a emen :
This s udy was app o ed be o ehand by he E hics Commi -
ee o Animal Resea ch o Za agoza Uni e si y (PI34/17, 27 June 2017) and was ca ied ou in s ic
acco dance wi h he Associa ion o Resea ch in Vision and Oph halmology’s S a emen o he Use
o Animals.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen :
The da a p esen ed in his s udy a e a ailable on eques om he
co esponding au ho .
Acknowledgmen s:
The au ho s would like o acknowledge he con ibu ion o he Se icio Gene al
de Apoyo a la In es igación (SAI) o he Uni e si y o Za agoza and he echnical SEM assis ance o
he Cen o de Mic oscopía Elec ónica Luis B u (CAI) o he Complu ense Uni e si y o Mad id and
o hank ZocoEs udio (zocoes udio.com) o he in og aphics designed o his a icle.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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