Biomedicine & Pha maco he apy 132 (2020) 110811
A ailable online 15 Oc obe 2020
0753-3322/© 2020 Published by Else ie Masson SAS. This is an open access a icle unde he CC BY-NC-ND license
(h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
O iginal a icle
Cho oidal and e inal s uc u al, cellula and ascula changes in a a
model o Type 2 diabe es
An ´
onio Campos
a
,
b
,
c
,
d
,
e
,
1
, Jo˜
ao Ma ins
a
,
b
,
c
,
,
g
,
1
, Elisa J. Campos
a
,
b
,
c
,
h
,
*, Ru ino Sil a
a
,
h
,
i
,
An ´
onio F ancisco Amb ´
osio
a
,
b
,
c
,
h
,
*
a
Uni e si y o Coimb a, Coimb a Ins i u e o Clinical and Biomedical Resea ch (iCBR), Coimb a, Po ugal
b
Uni e si y o Coimb a, Cen e o Inno a i e Biomedicine and Bio echnology (CIBB), Coimb a, Po ugal
c
Clinical Academic Cen e o Coimb a (CACC), Coimb a, Po ugal
d
Depa men o Oph halmology, Cen o Hospi ala Lei ia E. P. E., Lei ia, Po ugal
e
ciTechCa e, Cen e o Inno a i e Ca e and Heal h Technology, Poly echnic Ins i u e o Lei ia, Lei ia, Po ugal
Uni e si y o Coimb a, Coimb a Ins i u e o Biomedical Imaging and T ansla ional Resea ch (CIBIT), Coimb a, Po ugal
g
Uni e si y o Coimb a, Ins i u o de Ciˆ
encias Nuclea es Aplicadas `
a Saúde (ICNAS), Coimb a, Po ugal
h
Associa ion o Inno a ion and Biomedical Resea ch on Ligh and Image (AIBILI), Coimb a, Po ugal
i
Depa men o Oph halmology, Cen o Hospi ala e Uni e si ´
a io de Coimb a (CHUC), Coimb a, Po ugal
ARTICLE INFO
Keywo ds:
Type 2 diabe es
Cho oid
Re ina
Cho oidal hickness
Mic oglia
VEGFR2
ABSTRACT
Inc easing e idence poin s o in lamma ion as a key ac o in he pa hogenesis o diabe ic e inopa hy (DR).
Cho oidal changes in diabe es ha e been epo ed and se e al a emp s we e made o alida e in i o cho oidal
hickness (CT) as a ma ke o e inopa hy. We aimed o s udy cho oidal and e inal changes associa ed wi h
e inopa hy in an animal model o spon aneous Type 2 diabe es, Go o-Kakizaki (GK) a s. Scle ocho oidal whole
moun s and c yosec ions we e p epa ed om 52-week-old GK and age-ma ched con ol Wis a Han a s. CT was
measu ed by op ical cohe ence omog aphy. Mic oglia eac i i y, pe icy e and endo helial cells dis ibu ion, and
immuno eac i i y o ascula endo helial g ow h ac o (VEGF) and VEGF ecep o 2 (VEGFR2) we e e alua ed
by immuno luo escence. Cho oidal essels we e isualized by di ec pe usion wi h 1,1’-dioc adecyl-3,3,3’,3’-
e ame hylindoca bocyanine pe chlo a e (Dil). Cho oidal ascula densi y was e alua ed by luo escence mi-
c oscopy. GK a s had inc eased CT (58.40 ±1.15
μ
m e sus 50.90 ±1.58
μ
m, p <0.001), educed ascula
densi y o he cho iocapilla is (CC) (p =0.045), inc eased Iba1
+
cells densi y in he ou e e ina (p =0.003) and
inc eased VEGFR2 immuno eac i i y in mos e inal laye s (p =0.021 o 0.037). Cho oidal mic oglial cells and
pe icy es showed pola i y in hei dis ibu ion, spa ing he inne mos cho oid. This cell- ee gap in he inne
cho oid was mo e p onounced in GK a s. In summa y, GK a s ha e inc eased CT wi h dec eased ascula
densi y in he inne mos cho oid, inc eased VEGFR2 immuno eac i i y in he e ina and inc eased Iba1
+
cells
densi y in he ou e e ina.
Abb e ia ions: AGE, ad anced glyca ion end-p oduc ; BRB, blood- e inal ba ie ; CC, cho iocapilla is; CT, cho oidal hickness; DAPI, 4’,6-diamidino-2-phenyl-
indole; Dil, 1,1’-dioc adecyl-3,3,3’,3’- e ame hylindoca bocyanine pe chlo a e; DME, diabe ic macula edema; eNOS, endo helial ni ic oxide syn hase; DR, diabe ic
e inopa hy; EDI, enhanced deep imaging; GCL, ganglion cell laye ; GK, Go o-Kakizaki; HbA1c, hemoglobin A1c; Iba1, ionized calcium binding adap e molecule 1;
INL, inne nuclea laye ; IPL, inne plexi o m laye ; MHC II, majo his ocompa ibili y complex II; NG2, neu al-glial an igen 2; OCT, op ical cohe ence omog aphy;
OCTA, op ical cohe ence omog aphy angiog aphy; OLETF, O suka long-E ans Tokushima a ; ONH, op ic ne e head; ONL, ou e nuclea laye ; OPL, ou e plexi o m
laye ; PBS, phospha e bu e saline; PFA, pa a o maldehyde; RAAS, enin-angio ensin-aldos e one sys em; RECA-1, a endo helial cell an igen 1; RPE, e inal
pigmen epi helium; SD-OCT, spec al domain op ical cohe ence omog aphy; SDT, spon aneously diabe ic To ii; SEM, s anda d e o o he mean; T1D, Type 1
diabe es; T2D, Type 2 diabe es; VEGF, ascula endo helial g ow h ac o ; VEGFR2, ascula endo helial g ow h ac o ecep o 2.
* Co esponding au ho s a : Uni e si y o Coimb a, Coimb a Ins i u e o Clinical and Biomedical Resea ch, Azinhaga de San a Comba, 3000-548, Coimb a,
Po ugal.
E-mail add esses: [email p o ec ed] (E.J. Campos), [email p o ec ed] (A.F. Amb ´
osio).
1
These au ho s con ibu ed equally o his wo k.
Con en s lis s a ailable a ScienceDi ec
Biomedicine & Pha maco he apy
jou nal homepage: www.else ie .com/loca e/biopha
h ps://doi.o g/10.1016/j.biopha.2020.110811
Recei ed 6 July 2020; Recei ed in e ised o m 16 Sep embe 2020; Accep ed 25 Sep embe 2020
Biomedicine & Pha maco he apy 132 (2020) 110811
2
1. In oduc ion
Type 2 diabe es (T2D) accoun s o mo e han 90 % o all cases o
diabe es and is ela ed, mos ly, wi h age, seden a y li e and die o e load
[1]. Diabe ic e inopa hy (DR) and i s complica ions a e commonly
ea ed wi h an i- ascula endo helial g ow h ac o (VEGF) agen s [2]
and he g ea e ocus has been pu on he ole o VEGF on he pa ho-
genesis o DR [3,4]. VEGF-d i en inne blood- e inal ba ie (BRB)
b eakdown in he enule side o he supe icial e inal ascula u e has
been poin ed as he ea lies e en in DR [3] and i was ela ed o pe icy e
loss and hypoxemia [5]. Ne e heless, VEGF is no inc eased in he
i eous o all pa ien s wi h diabe ic macula edema (DME), while
p oin lamma o y ma ke s we e ound o be inc eased [6]. Acco dingly,
abou one hi d o pa ien s wi h DME ail o espond o an i-VEGF
he apy [7]. Those ac s poin ed DR o be an in lamma o y condi ion
and ha was con i med expe imen ally [8,9]. The e inal esiden inna e
immune sys em, which is p ima ily composed o issue- esiden mac-
ophage-like mic oglial cells, become ac i a ed and s a o p oduce
p oin lamma o y media o s [9]. The pe spec i e o DR as an in lam-
ma o y disease b ough new a en ion on p e ious wo ks desc ibing
cho oidal in lamma o y al e a ions in diabe es, named as ‘diabe ic
cho oidopa hy’, including B uch’s memb ane deposi s and inc eased
hickness, and cho iocapilla is (CC) d opou [10,11]. Since he ad en o
op ical cohe ence omog aphy (OCT), he cho oidal hickness (CT) has
been sough as a su oga e o cho oidal lux, diabe ic cho oidopa hy o
DR, bu he esul s a e con lic ing and disappoin ing [12–15,20]. Recen
wo ks did no un eil he con adic ions. The CT was ound o be
dec eased [16–21], inc eased [15,22], o unchanged [14,23] in T2D
pa ien s. Mo e ecen ly, i was epo ed ha he CT inc eased in he
ea ly s ages o DR, and u he dec eased wi h DR p og ession [24]. The
hinning o he cho oid wi h he an i-VEGF ea men o DME inc eased
he assump ion ha he cho oid hickens in DR and DME in an
VEGF-dependen exclusi e manne [13]. Ne e heless, he hinning o
he cho oid unde an i-VEGF ea men seems o be only a side e ec ,
wi h poo p ognos ic alue [25], ocusing back he a en ion on cellula
and molecula signa u es ha migh ake place in diabe ic cho oidop-
a hy. Indeed, he wa e homeos asis in he e ina and in he cho oid
depend on di e en cell ypes. Re inal wa e clea ance a he cho oid
le el depends on he onco ic p essu e unde nea h he e inal pigmen
epi helium (RPE), de eloped by he exis ence o VEGF-dependen po es
in he CC [26]. Howe e , an i-VEGFs we e ound o dec ease such po e
numbe [27]. The hickening-back o he cho oid in DME, when he
an i-VEGF e ec subsides, may no necessa ily be ela ed o he wa e
homeos asis in he e ina, bu o e e sible asocons ic ion [28,29].
Se e al animal species, mos ly oden s, ha e been used as alida ed
models o diabe es and o s udy he cellula and molecula aspec s o
he pa hogenesis o DR [30,31]. Animal models o T2D include: i) obese
animal models, such as Lep
ob/ob
mice, Lep
db/db
mice, Zucke diabe ic
a y a s, O suka long-E ans Tokushima a (OLETF) a s [31], as well as
high a eeding [32]; and ii) nonobese animal models, such as
Go o-Kakizaki (GK) a s and spon aneously diabe ic To ii (SDT) a s [30,
31]. The GK a s a e one o he mos use ul animal models o s udying
he pa hophysiologic p og ess o T2D and i s complica ions, namely
e inopa hy, as o me ly published a icles in ou ins i u e showed [33,
34]. GK a s a e a lean model o T2D, which is cha ac e ized by ea ly and
ela i ely s able mild hype glycemia, hype insulinemia, and insulin
esis ance [31]. Mode a e diabe ic condi ion allows o p olonged dis-
ease in GK a s, simila ly o wha is obse ed in humans. GK a s also
p esen some ea u es ound in pa ien s wi h DR and hus hey a e a
model o s udy he kine ics and e en s o T2D. Inc eased NO p oduc ion,
ea ly inne BRB b eakdown and mig a ion o ac i a ed mic oglial cells
om he e ina o he cho oid by anscy osis has been demons a ed in
GK a s [34–36], bu he b eakdown o he ou e BRB and B uch’s
memb ane pe meabili y in diabe es is no comple ely unde s ood ye
[37].
We in es iga ed he impac o T2D on he CT and cho oidal ascula
densi y, as well as on endo helial cells and pe icy es, mic oglial cell
eac i i y, VEGF and VEGF- ecep o 2 (VEGFR2) immuno eac i i y, in
he e ina and cho oid o GK (52 weeks old, 52 W) and age-ma ched
con ol Wis a Han a s.
2. Ma e ials and me hods
2.1. Animals
Male spon aneously diabe ic GK a s we e ob ained om he Facul y
o Medicine o Uni e si y o Coimb a b eeding colony es ablished in
1995 wi h b eeding couples om he colony a he Tohoku Uni e si y
school o Medicine (Sendai, Japan; cou esy o D . K. I. Susuki). Con ol
animals we e age-ma ched non-diabe ic male Wis a a s, ob ained om
ou local colony a he Coimb a Ins i u e o Clinical and Biomedical
Resea ch (iCBR). Animals we e housed in he ce i ied animal acili y a
iCBR, on a 12 h ligh /12 h da k cycle, a 22−23 ◦C, and 60 % ela i e
humidi y, and wi h ee access o wa e and o oden s anda d main-
enance chow (4RF21, Mucedola s. .l., Se imo Milanese, I aly) con-
aining 18.5 % o p o ein and 3.0 % o lipids. Animals u he go
sh edded pape and ca dboa d olls o nes building and shel e . All
p ocedu es we e app o ed by he Animal Wel a e Commi ee o he
Coimb a Ins i u e o Clinical and Biomedical Resea ch (iCBR), Facul y
o Medicine, Uni e si y o Coimb a. The animals ecei ed humane ca e
acco ding o he c i e ia ou lined in he Guide o he Ca e and he Use o
Labo a o y Animals p epa ed by EU Di ec i e 2010/63/EU o animal
expe imen s and wi h he Associa ion o Resea ch in Vision and
Oph halmology (ARVO) s a emen o animal use. A 52 W, he animals
(n =12, con ol g oup; n =16, GK g oup) we e weigh ed and glycemia
was measu ed using a glucome e (Ascensia ELITE™, Baye Co po a-
ion, Mishawaka, IN, USA).
2.2. Op ical cohe ence omog aphy
The animals we e anes he ized ia i.p. injec ion wi h ke amine 80
mg/kg +xylazine 5 mg/kg (80:5, o sho ; Imalgene 1000, Me ial,
Lyon, F ance, and Rompum®, Baye , Le e kusen, Ge many, espec-
i ely), and co nea anes hesia (4 mg/mL oxybup ocaine hyd ochlo ide;
Anes ocil®, Labo a ´
o io Edol, Ca naxide, Po ugal), pupils dila ion (1 %,
T opicil®, Labo a ´
o io Edol, Ca naxide, Po ugal) and co neal hyd a ion
(2 % Me hocel™, D´
a i II Fa macˆ
eu ica S.A., Ba ca ena, Po ugal) was
kep du ing p ocedu e.
The CT was e alua ed in he animals using spec al domain op ical
cohe ence omog aphy (SD-OCT) a 52 W. The SD-OCT sys em is able o
cap u e 10,000–20,000 A scans pe second wi h an axial esolu ion o 2
μ
m and a ans e se esolu ion o 4
μ
m. OCT was pe o med in bo h eyes
o all animals a 52 W, jus be o e being eu hanized. The 830 nm SD-OCT
Imagine Sys em (Phoenix Mic on IV, Phoenix Resea ch Labs, Pleas-
an on, CA, USA) [38] was placed close o he eye such ha an in e ed
image was ob ained and he deepe s uc u es we e placed close o
ze o-delay [39]. Scans we e ob ained supe io o he op ic ne e
head/op ic disc (ONH), in bo h eyes o all animals, in he a ea wi hin
1–3 ONH diame e om he op ic disc. Fo each loca ion, he de ice
collec ed a se o 1024 as e scans along he scan leng h. Upon collec-
ion, a se o i e images was con e ed in o a single image o educe he
noise obse ed on indi idual images. The CT was measu ed using
InSigh image segmen a ion so wa e ( .1, Voxele on LLC - Image
analysis solu ions, Chabo D i e, CA, USA). An a e age o h ee inde-
penden sco es ob ained om 3 se s “ i e ames a e aged” was used as
he CT alue pe eye pe ime-poin .
2.3. Tissue p epa a ion o c yosec ions
The animals we e anes he ized, as desc ibed abo e, and in aca di-
ally pe used wi h 0.1 M phospha e bu e saline (PBS, 137 mM NaCl, 2.7
mM KCl, 1.8 mM KH
2
PO
4
, 10 mM Na
2
HPO
4
, pH 7.4), ollowed by 4 %
A. Campos e al.
Biomedicine & Pha maco he apy 132 (2020) 110811
3
pa a o maldehyde (PFA) in 0.1 M PBS, p e-wa med a 37 ◦C.
The enuclea ed eyes o GK (n =8) and age-ma ched con ol Wis a
Han (n =5) a s we e pos - ixed in 4% PFA o 1 h. The eyes we e
washed in successi e solu ions o PBS and imme sed in solu ions con-
aining 15 % and 30 % o suc ose in PBS, o 1 h in each solu ion. They
we e embedded in a 1:1 30 % suc ose and embedding esin (Shandon™
C yoma ix™, The mo Fishe Scien i ic, Wal ham, MA, USA) solu ion,
be o e eezing in d y ice. The samples we e s o ed a -80 ◦C, un il
u he use. Eyeball sec ions 14
μ
m- hick om bo h igh and le eyes
we e ob ained using a c yos a (Leica CM3050S, Nussloch, Ge many), a
-22 ◦C, and moun ed on adhesi e slides (Supe os Plus™, The mo
Fishe Scien i ic). A o al o ou sec ions (14
μ
m apa ) we e collec ed
pe slide.
2.4. Immuno luo escence
Eye sec ions we e labeled o assess he e inal and cho oidal s uc-
u e, ollowing a p ocedu e desc ibed p e iously [40]. The c yosec ions
we e ehyd a ed wice in PBS o 5 min, ollowed by blocking and
pe meabiliza ion o 1 h in 10 % goa se um and 0.5 % T i on X-100 in
PBS. The sec ions we e hen incuba ed o e nigh wi h p ima y an i-
bodies (Table S1) dilu ed in 0.5 % T i on X-100, a 4 ◦C. A e washing
wi h PBS, sec ions we e incuba ed wi h co esponding seconda y an i-
bodies (Table S2) dilu ed in 0.5 % T i on X-100, o 1 h. A e washing,
he sec ions we e incuba ed wi h 1:5,000 4’,6-dia-
midino-2-phenylindole (DAPI, In i ogen™), and co e slipped using
moun ing medium (Glyce gel, Dako, Ca pin e ia, CA, USA).
Digi al images we e cap u ed using an in e ed luo escence mic o-
scope (Axio Obse e .Z1, Zeiss, Ca l Zeiss Medi ec AG, Jena, Ge many)
and a lase scanning con ocal in e ed mic oscope (LSM 710 Axio
Obse e , Zeiss), using objec i es “Plan-Apoch oma ” 20×/0.8 (Zeiss).
Eigh bi images we e analyzed using he ImageJ so wa e ( e sion
1.48, Na ional Ins i u es o Heal h, USA) [41]. Iba1
+
and MHC II
+
cells
we e manually coun ed in he cho oid and e ina. Da a we e exp essed
as numbe o cell/mm o cho oidal o e inal leng h, espec i ely. Ra
endo helial cell an igen 1 (RECA-1) and he neu al-glial an igen 2 (NG2)
immuno eac i i ies we e sco ed in he cho oid as luo escence in ensi y
pe a ea selec ed ( e e ence a ea selec ed o 10,737.08 ±6,306.11
μ
m
2
),
while luo escen NG2
+
cells and RECA-1 ocal immunos aining we e
manually coun ed in he e ina. VEGF and VEGFR2 immuno eac i i y
was quan i ied as luo escence in ensi y/a ea o each laye analyzed.
All esul s we e exp essed as he mean coun o 12 slices pe eye ( igh
eye only).
2.5. Di ec labeling and isualiza ion o cho oidal essels in
scle ocho oidal whole moun s
In aca diac pe usion wi h PBS was pe o med in a s (n =7, con ol
g oup; n =8, GK g oup) unde anes hesia (i.p.; ke amine:xylazine
160:30). Dil (Ca . #D-282, In i ogen™, Ca lsbad, CA, USA) 0.120 mg/
mL in 1 % glucose in PBS was applied ia ca diac pe usion, ollowing
pe usion wi h 4 % PFA. The eyes we e enuclea ed, and cho oidoscle al
whole moun s we e p epa ed. The whole moun s we e ixed in 4 % PFA
o 15 min, washed wi h PBS, and blocked wi h 10 % goa se um in 0.3
% Tween in PBS o 1 h. Samples we e incuba ed wi h he p ima y an-
ibodies dilu ed in 3 % goa se um in PBS o 3 days a 4 ◦C (Table S1).
A e washing o e nigh wi h PBS, whole moun s we e incuba ed wi h
seconda y an ibodies (Table S2) o e nigh a 4 ◦C. A e washing,
samples we e la moun ed on o glass slides using moun ing medium.
The images we e ob ained by lase scanning con ocal mic oscope LSM
710 (Zeiss), using an objec i e EC “Plan-Neo luo ” 40×/1.30 Oil M27
(Zeiss). A se ies o z-s acks we e cap u ed om he RPE ou e su ace, o
he ou e cho oid. Each z-s ack consis ed o a dep h o op ical sec ions, 3
μ
m apa , along he z-axis. Iba1
+
and MHC II
+
cells we e classi ied as
ami ied o ound cells and we e manually coun ed in he cho oid in all
in-dep h planes o he slide (Fig. S1). The cho oidal ascula densi y
(de ined as he pe cen age o o al a ea co e ed by CC essels) [42] was
de e mined om independen z-s acks collec ed a ≤10
μ
m (CC) and >
10
μ
m ou wa ds om he RPE plane (medium and la ge essels) in a
selec ed a ea (213 ×213
μ
m, Fig. S2). Resul s we e exp essed as he
mean o 14 coun s pe eye.
2.6. S a is ical analysis
The s a is ical analysis was pe o med using SPSS (Ve sion 25.0, IBM
Co p., A monk, NY, USA). The Shapi o-Wilk es was used o assess he
no mali y o da a (p >0.05). The no mally dis ibu ed da a we e e al-
ua ed conce ning he homogenei y o a iance, using he Le ene’s es (p
>0.05). The independen - es was used o compa e he means be ween
wo expe imen al g oups o he same a iable. S a is ical signi icance
was de ined as p <0.05. Values we e p esen ed as mean ±s anda d
e o o he mean (SEM).
3. Resul s
3.1. Diabe ic animals exhibi dec eased body weigh and hype glycemia
A o al o 28 animals (52 W) we e en olled in he s udy (16 GK and
12 age-ma ched con ol Wis a Han a s). The weigh o GK a s was
signi ican ly lowe han ha o age-ma ched con ol a s (416.31 ±7.0 g
and 462.5 ±9.24 g, espec i ely, p <0.001) and glycemia was signi i-
can ly highe (227.75 ±12.1 mg/dL e sus 107.0 ±0.94 mg/dL, p <
0.001, Fig. S3).
3.2. Inc eased cho oidal hickness in GK a s
CT was assessed in bo h eyes o all GK (n =32 eyes) and age-ma ched
con ol Wis a Han (n =24 eyes) a s a 52 W, using OCT. CT was highe
in GK a s (58.40 ±1.15
μ
m e sus 50.90 ±1.58
μ
m, p <0.001, Fig. 1A
and B).
3.3. Cho oidal blood ascula densi y is educed in he inne cho oid in
GK a s
Fi een animals (8 GK and 7 age-ma ched con ol Wis a Han a s)
we e assigned o pe usion wi h Dil and scle ocho oidal whole moun s
we e p epa ed and assessed by con ocal mic oscopy.
The cho oidal ascula densi y in he inne mos cho oid/ CC (≤10
μ
m) was signi ican ly dec eased in GK a s (p =0.045, Fig. 2A and C and
Table S3). Cho oidal Iba1
+
and MHC II
+
cell numbe was no signi i-
can ly di e en be ween GK and age-ma ched con ol a s (Fig. 2B and D
and Table S4).
In e es ingly, Iba1
+
cells and MHC II
+
cells opog aphic disposi ion
in he cho oid spa e he inne mos cho oid, whe e hey a e no ably a e
o absen , being p e e en ially p esen in he middle and ou e cho oidal
s oma, ei he in GK o age-ma ched con ol a s (Videos S1-6).
3.4. Inc eased immuno eac i i y o mic oglial cell ma ke s in he ou e
e ina o GK a s
Iba1
+
cells inc eased in he e ina o GK a s, being s a is ically
signi ican ly inc eased in he ou e plexi o m laye (OPL, (11.000) =
-3.872, p =0.003). Iba1
+
(p =0.195) and MHC II
+
cells densi y was no
s a is ically signi ican ly di e en in he cho oid o GK a s ( (11.000) =
2.190, p =0.051, Fig. 3 and Table S5). In e es ingly, Iba1
+
cells we e
dis ibu ed h oughou he e ina o GK a s pa alleling he opog aphic
dis ibu ion o he 3 ascula plexuses o he e ina. Iba1
+
cell ex ensions
om he inne plexi o m laye (IPL) o he OPL esemble he commu-
nica ions be ween he deep and middle ascula plexuses o he e ina,
sugges ing ha mig a ing glial cells owa ds he ou e e ina may use he
communica ing in e -plexuses e inal capilla ies as a sca old (see
Fig. 3A, bo om le panel). Communica ions be ween plexus we e
A. Campos e al.
Biomedicine & Pha maco he apy 132 (2020) 110811
4
e idenced.
3.5. Pe icy es a e a e in he inne mos cho oid o GK a s
The e we e no s a is ically signi ican di e ences in he immuno e-
ac i i y o NG2 and RECA-1 be ween GK and age-ma ched con ol
Wis a Han a s, wha e e he loca ion conside ed (Fig. 4 and Table S6).
Ne e heless, he e was a end o inc eased NG2 immunos aining in he
cho oid o GK a s. Combined immuno eac i i y o RECA-1 and NG2
e idenced a a e ac ion o pe icy es in he inne mos cho oid o GK a s.
RECA-1 co-localized wi h he CC laye , jus unde nea h and in close
con inui y wi h he RPE cell plane (Fig. 4A, le panels). NG2 immuno-
eac i i y was absen in some a eas o he CC plane, lea ing luo escen -
ee gaps be ween he RPE and he inne cho oid, d awing a ypical
jagged pa e n, co esponding o he absence o pe icy es/mu al cells in
he inne mos cho oid (Fig. 4A, middle panels, blue a ows). This ‘po-
la i y’ in cho oidal ascula egula o y cells disposi ion e idenced by
NG2, co esponding o a a e ac ion o pe icy es in he inne mos
cho oid, was mo e e idenced in GK a s. The h ee e inal plexuses
(supe icial, in he e inal ne e ibe and ganglion cell (GCL) laye s;
middle, in he IPL and deep, in he OPL) we e isualized by RECA-1
immunolabeling (Fig. 4A, op panels, le and igh ).
3.6. VEGFR2 immuno eac i i y was signi ican ly inc eased in GK a s
VEGF immuno eac i i y was mo e in ense in he le el o he OLM,
RPE and cho oid, while VEGFR2 immuno eac i i y was negligible a
hose loca ions. VEGFR2 immuno eac i i y was mo e in ense in he
inne e ina. Highe VEGFR2 immuno eac i i y was obse ed in GK a s,
signi ican ly in he IPL, INL, OPL, ONL and OLM, when compa ing wi h
age-ma ched con ol a s (Fig. 5 and Table S7).
4. Discussion
CT has been sea ched as a su oga e o DR, DME, cho oidal lux and
diabe ic cho oidopa hy in T2D pa ien s, bu con adic o y esul s ha e
been epo ed [12–15,20]. Al hough GK a s a e no bluep in s o he
diseased T2D humans, hey p o ide excellen insigh s in o he pa ho-
genesis o T2D [43]. Fu he mo e, a s a e ecognized as he p eeminen
model o s udying he cho oid [44,45]. We ound ha CT was signi i-
can ly inc eased in GK a s when compa ing wi h age-ma ched con ol
Wis a Han a s. To ou knowledge, his is he i s ime ha he CT is
assessed in i o in a a model o T2D.
The ascula densi y was educed in he inne cho oid o GK a s,
co esponding o he CC. This obse a ion is in acco dance wi h CC
degene a ion obse ed in T2D humans, ei he by pos mo em specimens
o by OCT-angiog aphy (OCTA) [11,46]. OCTA bina iza ion echnique
in human diabe ics showed esul s o dec eased low o deple ion a he
CC le el ha ag ee wi h ou esul s in he animal [47]. The indings o no
signi ican di e ence in he ou e cho oid’s ascula densi y combined
wi h a signi ican ly highe CT in GK a s, enhances he ole o he ou e
cho oid laye , he sup acho oid, as an impo an con ibu o o o al CT
and may explain why a ailable da a on human CT a e so con adic o y
[13–15,48,49]. The sup acho oid is he cho oidal laye mos p one o
change, bu i is he mos di icul o e alua e co ec ly when no using
high de ini ion de ices such as he Swep Sou ce OCT and he high
esolu ion mode enhanced deep imaging (EDI) o he SD-Spec alis OCT
[12,25]. Un o una ely, de ices based on ed blood cells’ mo emen as
he OCTA a e no expec ed o help much in his issue ei he [50].
The no mal dis ibu ion o in lamma o y cells in he cho oidal
s oma, spa ing he inne mos cho oid, sugges s ha unde in lamma-
o y s ess a d ama ic inc ease in he numbe o in lamma o y cells may
esul in packing o cells a his a ea, leading o dis u bances o he
pho o ecep o s/RPE/B uch’s memb ane/CC ape o e inal uni (S1-3
Videos) [51]. Ne e heless, we did no ind in lamma o y cells o be
signi ican ly inc eased in he cho oid o GK a s. I is possible ha his
inding co ela es wi h he low le el o diabe es in his animal model.
Ac ually, we ound inc eased in lamma o y cells in he cho oid o a mo e
agg essi e diabe ic animal model, s ep ozo ocin-induced Type 1 dia-
be es (T1D) [52]. The numbe o in lamma o y cells we e inc eased in
he e ina, as p e iously desc ibed, mainly in he OPL [9,36]. In e es -
ingly, Iba1
+
cells disposi ion pa allels he h ee e inal plexuses’, and
he mig a ion o cells om he inne o he ou e e ina esembles he
capilla y communica ions be ween plexuses, sugges ing ha essels a e
a sca old o cell mig a ion.
The VEGF le els we e no signi ican ly inc eased in he e ina, RPE o
cho oid, in GK a s. VEGFR2 exp ession was negligible in he RPE o
cho oid bo h in GK and age-ma ched con ol Wis a Han a s.
Fig. 1. Cho oidal hickness (CT) o GK and age-ma ched
con ol Wis a Han a s (52 W). (A) Images esul ing om
i e ames a e aged, collec ed as in e ed images using OCT
as e scans ob ained in 1024 con inuous poin s, by
app oaching he de ice o he ze o delay line. Ho izon al as e
scan line encompasses an a ea wi hin 1 o 3 disc diame e s
om he op ic ne e head. Cho oidal laye ob ained by au o-
ma ic segmen a ion was manually co ec ed. A mean o h ee
independen sco es ob ained om 3 di e en loca ed se s “ i e
ames a e aged” we e used as he CT alue pe eye pe ime-
poin . (B) CT alues om all eyes o GK (n =32) and age-
ma ched con ol (n =24) a s. Da a a e exp essed as mean ±
SEM. Scale ba : 100
μ
m. Signi icance be ween g oups was
de e mined using he independen - es : ***p <0.001. GCL,
ganglion cell laye ; INL, inne nuclea laye ; ONL, ou e nu-
clea laye ; RPE, e inal pigmen epi helium.
A. Campos e al.
Biomedicine & Pha maco he apy 132 (2020) 110811
5
Con e sely, VEGFR2 immuno eac i i y peaked in he e inal ne e ibe
laye o bo h GK and con ol a s, and i was inc eased h oughou he
e ina o GK a s. The p ecise cellula backg ound o his inc eased
exp ession is no clea , bu he combina ion o maximum exp ession in
he inne mos e ina and exp ession h oughou he e ina esembles he
dis ibu ion o Mülle cells and neu ons wi hin he e ina. Ne e heless,
a p e ious epo in an animal model o T1D showed VEGFR2 o be
exp essed mainly in he capilla ies o he e ina and, in opposi ion o ou
indings, in he CC [53]. Con e sely, o he da a co ela ed VEGFR2 mo e
s ikingly wi h neu ons and Mülle cells a he han wi h endo helial
cells [54]. In e es ingly, he VEGFR2 exp ession by neu ons and Mülle
cells combined wi h he ac ion o MHC II
+
cells, was ela ed wi h
capilla y e ical sp ou ing and he o ma ion o he deep e inal plexus,
an example o neu o ascula c oss alk o coupling. The ela ionship
be ween inc eased VEGFR2 and no mal VEGF immuno eac i i y is no
clea , i any a all. I ha esul s om a milde diabe es in ou model,
VEGFR2 would be among he i s molecula bioma ke s o change in
DR. Howe e , his da um needs o be con i med in u u e s udies.
Disposi ion o pe icy es in he CC was p e iously desc ibed in mice o
be a i s scle al side only (pola ized dis ibu ion) and ocal pe icy e
deple ion has been ela ed o ascula emodeling [55]. We ound dis-
ibu ion o NG2
+
cells in he inne mos cho oid o d aw a jagged
pa e n, when compa ing wi h RECA-1 endo helial immunos aining.
These ocal ‘gaps’, sugges ing he exis ing o cell- ee spaces in he
inne mos cho oid, may be ela ed o ascula emodeling o he CC and
no necessa ily o pe manen al e a ions, and we e mo e p onounced in
T2D. In e es ingly, his disposi ion is sha ed by s omal Iba1
+
cells in he
same loca ion (S1−6 Videos). Reduc ion o he ascula componen o
he diabe ic CC epo ed in OCTA s udies did no sepa a e pe manen
degene a i e om ansien and e e sible changes, mi o ing inc eased
CC emodeling in diabe es epo ed in pos mo em s udies in humans
and in a s [11,49,50,56]. Inc eased emodeling o he CC, p obably
media ed by abluminal pe icy es con ac ing he endo helium as
peg-in-socke o ongue-in-socke p ocesses, may be an adap a ion o he
po e kine ics, wa e load, ionic and pH al e a ions, VEGF le els and he
p esence o in lamma o y media o s and cells in he diabe ic cho oid
[55,57]. OCTA, being able o esol e he mic o ascula ne wo k o he
e ina and nonpe usion a eas a he CC, does no e alua e hi he o
Fig. 2. Vascula and cell p o iles o he cho oid o GK and age-ma ched con ol Wis a Han a s (52 W). (A) Rep esen a i e images o ascula densi y in he
inne cho oid (≤10
μ
m). (B) Cho oidal Iba1
+
and MHC II
+
cells. Rami ied cells (g een a ows) and ound cells ( ed a ows) we e highligh ed. (C) Quan i ica ion o
he cho oidal ascula densi y in he inne and ou e cho oid using he ‘image >adjus > h eshold’ window ool o ImageJ o ob ain he pe cen age o ascula
co e age, ob ained om z-s acks collec ed a ≤10
μ
m o >10
μ
m om he ou e RPE plane, espec i ely. (D) Iba1
+
and MHC II
+
cell numbe in he cho oid in all in-
dep h z-s acks. Images we e collec ed wi h Zeiss EC Plan-Neo luo 40x oil objec i e lens, NA 1.3. Quan i a i e analyses we e pe o med based on 14 independen
coun s pe eye in each and all in-dep h z-s acks pe specimen. Da a a e exp essed as mean ±SEM (n =7, con ol g oup; n =8, GK g oup). Scale ba : 50
μ
m. Sig-
ni icance be ween g oups was de e mined using he independen - es : *p <0.05, **p <0.01. (Fo in e p e a ion o he e e ences o colo in his igu e legend, he
eade is e e ed o he web e sion o his a icle).
A. Campos e al.

Biomedicine & Pha maco he apy 132 (2020) 110811
6
de ailed mo phological ascula pa e ns o he CC, since being as hin as
10−20
μ
m, i is no esol ed by a sys em ha has a maximum la e al
esolu ion wi hin ha ange (~15−20
μ
m), [58] and, as a o emen-
ioned, i is no sui able o e alua ed he whole CT.
Inc eased cho oidal hickness in GK a s migh be ela ed o wa e
homeos asis and egula ion o he ex acellula luid, since he cho oidal
ascula lux has been epo ed o dec ease in diabe es [59,60]. This
issue is no comple ed unde s ood ye , bu as he wa e pa hway ac oss
he e ina ollows a di ec ion om he i eous o he cho oid, one may
assume ha inc eased VEGF le els and inc eased capilla y pe meabili y
in he e ina, may inc ease he ex acellula luid in he e ina inc easing
he wa e load on he cho oid ia a well- unc ioning RPE [38].
Cho oidal-dependen e inal d ying is modula ed by po e egula ion o
he CC, which a e mainly acing he RPE side o he CC and a e
VEGF-dependen [57,61]. This wa e o e load is e y well pic u ed in
OCT when he e is impai men o he RPE in DME and sub e inal luid
accumula es, as a consequence o impai men o he ac i e ionic ans-
po by he RPE [62]. This may explain in pa why he cho oid hins
wi h an i-VEGF adminis a ion by in a i eal injec ions, as a conse-
quence o imp o ed inne BRB homeos asis and dec eased wa e load on
he cho oid [25]. This e ina-cen e ed line o hough may be en o ced
by ealizing ha unde an i-VEGF adminis a ion po e dis ibu ion in
he CC is educed, hus dec easing he cho oidal-dependen e inal
d ying mechanism [27]. Mo eo e , he es o a ion o ac i e ionic
anspo by he RPE, wi h esolu ion o sub e inal luid ia wa e low
in o he cho oid, would be expec ed o hicken and no o hin he
cho oid, as ac ually happens [62].
A mos likely explana ion o CT dec ease wi h an i-VEGFs is a
e e sible asocons ic ion o he cho oidal essels, as demons a ed in
he e inal essels [28,29], unde pe icy e egula ion [55,63]. In he
cho oid, he blood lows in a e ies and eins in he same di ec ion,
con a iwise o mos o gans. I is possible ha some egula ion o his
ascula pa hway, namely by pe icy es o in lamma o y cells, om he
sho pos e io cilia y a e ies o he o ex eins occu s a di e en
le els, including he CC [64]. Pe i ascula mu al cells in he middle and
ou e cho oid we e p e iously epo ed o show con ac ile p ope ies
ha we e ela ed o aso egula o y unc ions [55]. Mo eo e , pe icy e
loss has been epo ed in GK a s [65], media ed by hypoxia and/o
in lamma ion, ia ascula endo helial ecep o 1 (VEGFR1, Fl -1), ni ic
oxide (NO) and eac i e oxygen species (ROS) [63].
In addi ion, VEGF in e ac s wi h se e al asoac i e agen s. NO plays
a c i ical ole in many VEGF ac ions [66]. Endo helial ni ic oxide syn-
hase (eNOS), which is exp essed in he CC, egula es he cho oidal
ascula u e, since p oduc ion o NO by his enzyme, causes asodila ion
[57]. Since he e a e no ue lympha ic essels in he cho oid, a signi -
ican amoun o ex acellula luid is eabso bed ia he lamellae o he
sup acho oid ha a e unde non- ascula smoo h muscle cells egula-
ion and a e esponsi e o mul iple in lamma o y cy okines [12].
Inc eased NO may be in ol ed in dec eased ex acellula luid eab-
so p ion in he sup acho oid. Apa om asodila ion, NO induces
inc eased pe meabili y o p e- e minal cho oidal a e ies o
pos -capilla ies eins, as well [66], despi e he educ ion o cho oidal
Fig. 3. Mic oglial cells in he e ina and cho oid o GK and
age-ma ched con ol Wis a Han a s (52 W). (A) Rep e-
sen a i e eye c oss-sec ions immunolabeled agains Iba1 (le
panels), MHC II (middle panels) and me ge ( igh panels).
Iba1
+
cells a e loca ed in he supe icial and plexi o m laye s o
he e ina, mainly. Iba1
+
cells loca ed in he OPL o he GK
coho only (g een a ows). In GK a s, Iba1
+
cells mig a e om
he IPL o he OPL, c ossing he INL ( ed a ow). (B) Quan i-
ica ion o Iba1
+
and MHC II
+
cell densi y o GK (n =8) and
age-ma ched con ol (n =5) a s based on 12 independen
specimen coun s pe eye. Coun ing was done o he igh eye
only, in all animals. Da a a e exp essed as mean ±SEM. Scale
ba : 100
μ
m. Signi icance be ween g oups was de e mined
using he independen - es : **p <0.01. GCL, ganglion cell
laye ; IPL, inne plexi o m laye ; INL, inne nuclea laye ; OPL,
ou e plexi o m laye ; ONL, ou e nuclea laye ; RPE, e inal
pigmen epi helium. (Fo in e p e a ion o he e e ences o
colo in his igu e legend, he eade is e e ed o he web
e sion o his a icle).
A. Campos e al.
Biomedicine & Pha maco he apy 132 (2020) 110811
7
Fig. 4. Localiza ion o mu al and
endo helial cells in he e ina and
cho oid o GK and age-ma ched
con ol Wis a Han a s (52 W).
(A) Rep esen a i e eye c oss-sec ions
immunolabeled agains RECA-1 (le
panels), NG2 (middle panels) and
me ge ( igh panels). The 3 plexuses
o he e ina a e e idenced by RECA-1
immunos aining (whi e a ows).
Communica ions be ween (i) he su-
pe icial and middle plexuses o he
e ina ( ed a ow), (ii) he middle and
deep plexuses and (iii) he deep and
supe icial plexuses (g een a ow),
a e isible. RECA-1 immuno eac i i y
ela es o he p esence o endo helial
cells and luo escence o he CC
endo helial cells is con inuous wi h
he RPE cell plane (le panels).
Con e sely, NG2 immunos aining o
pe icy e/mu al cells lea es ocal gaps
be ween he RPE and he inne
cho oid, d awing a jagged pa e n,
mo e p onounced in GK a s (blue
a ows). (B) Quan i ica ion o RECA-1
and NG2 immuno eac i i y in he
e ina and cho oid o GK (n =8) and
age-ma ched con ol (n =5) a s.
RECA-1 and NG2 immuno eac i i ies
we e sco ed as luo escence in ensi y
pe a ea selec ed in he cho oid
( e e ence a ea selec ed o 10,737.08
±6,306.11
μ
m
2
), while NG2
+
cells
and RECA-1 ocal immunos aining
we e manually coun ed in he e ina.
Coun ing was done o he igh eye
only, in 12 independen specimen
coun s pe eye. Da a a e exp essed as
mean ±SEM. Scale ba : 100
μ
m. GCL,
ganglion cell laye ; IPL, inne plexi-
o m laye ; INL, inne nuclea laye ;
OPL, ou e plexi o m laye ; ONL,
ou e nuclea laye ; RPE, e inal
pigmen epi helium. (Fo in e p e a-
ion o he e e ences o colo in his
igu e legend, he eade is e e ed o
he web e sion o his a icle).
A. Campos e al.
Biomedicine & Pha maco he apy 132 (2020) 110811
8
Fig. 5. Immuno eac i i y o VEGF and VEGFR2 o GK and age-ma ched con ol Wis a Han a s (52 W). (A) Rep esen a i e eye c oss-sec ions immunolabeled
agains VEGF (le panels), VEGFR2 (middle panels) and me ge ( igh panels). VEGF immuno eac i i y sp eads h oughou he e ina, inc easing in he OPL, OLM,
RPE and cho oid. VEGFR2 immuno eac i i y is highe in he inne mos e ina ( e inal ne e ibe laye ) and e y low o absen in he RPE and cho oid. VEGFR2
immuno eac i i y is s ill isible as a ain colo a ion in he e inal laye s o he han he e inal ne e ibe laye o GK a s only (whi e a ows). (B) Quan i ica ion o
he VEGF and VEGFR2 immuno eac i i y in he e ina and cho oid o GK a s (n =8) and age-ma ched con ols (n =5) based on 12 independen specimen coun s pe
eye. VEGF and VEGFR2 immuno eac i i ies we e quan i ied as luo escence in ensi y/a ea pe laye . Coun ing was done o he igh eye only, in all animals. Da a
a e exp essed as mean ±SEM. Scale ba : 100
μ
m. Signi icance be ween g oups was de e mined using he independen - es : *p <0.05. GCL, ganglion cell laye ; IPL,
inne plexi o m laye ; INL, inne nuclea laye ; OPL, ou e plexi o m laye ; ONL, ou e nuclea laye ; OLM, ou e limi ing memb ane; RPE, e inal
pigmen epi helium.
A. Campos e al.
Biomedicine & Pha maco he apy 132 (2020) 110811
9
lux in diabe es [60]. An i-VEGFs coun e ac all hese ac ions. Fu he -
mo e, an i-VEGFs coun e ac he enin-angio ensin-aldos e one sys em
(RAAS) - VEGF c oss alk in he cho oid [67], known o ha e an impo -
an ole in he inc eased luid o e load p esen in he pachycho oid
synd omes and p ima y aldos e onism [68,69]. RAAS componen s p e-
sen in e inal cells, in he RPE and he cho oid, we e shown o be
in ol ed in he genesis o DME h ough he p oli e a ion and ac i a ion
o glial cells, exp essing angio ensin ecep o 1, mine aloco icoid e-
cep o and aldos e one syn hase. These in lamma o y e en s caused by
inc eased RAAS ac i i y we e ampli ied by he ad anced glyca ion
end-p oduc s (AGEs), esul ing in he elease o in lamma o y cy okines,
leading o leukos asis and apop osis o e inal pe icy es. Fu he mo e,
RAAS may di ec ly impai he wa e balance o he e ina, ac ing di ec ly
on Mülle cells, and in a i eal injec ion o aldos e one causes
inc eased CT and leakage om cho oidal essels [70].
The assump ion ha he CT is inc eased in diabe es and ha i de-
c eases wi h an i-VEGFs o a somewhe e baseline alue is a om being
consensual [12]. Mo eo e , HbA1c le els a e known o be co ela ed
wi h VEGF le els and in lamma ion [4,71], bu high HbA1c le els ha e
been su p isingly co ela ed wi h cho oidal hinning, and no wi h he
expec ed cho oidal hickening [21]. These con adic ions add ess he
need o be cau ious when ex apola ing he esul s o ou s udy o human
subjec s. I would be wo h o in es iga e whe he he CT inc eases in all
obese animal models o T2D. Since GK a s a e a non-obese model o
T2D, one canno exclude ha his pa icula ea u e is cha ac e is ic o
he animal model and no o he disease i sel . I is possible ha he
inc eased CT ound may be cons i u i e o he animal model, exp essing
a mo e asodila ed basal s a us o he aso egula o y mechanisms, a
ea u e seen among human subjec s as well, exp essed by CT
in e -indi idual a iabili y [25]. The e o e, i would be use ul ha in
u u e s udies o he animal models o T2D would be used o compa e
wi h hese esul s.
Un o una ely, hi he o OCTA does no de ec s uc u es wi h no
signi ican e y h ocy e mo emen , such as he sup acho oid. The e o e,
i does no add much in he e alua ion o he whole CT [50]. This is why
he bina iza ion echnique applied o ull CT was also applied o EDI
OCT, showing no change in CT in diabe es, despi e a educ ion o he
sub o eal cho oidal ascula index [72].
5. Conclusions
In conclusion, we ound an inc ease in CT, a dec ease in inne
cho oidal ascula densi y, inc eased Iba1
+
cells densi y in he ou e
e ina, and inc eased VEGFR2 immuno eac i i y in he e ina, in GK
a s. Disposi ion o pe icy es and Iba1
+
cells in he cho oidal s oma
spa e he inne mos cho oid, ei he in GK o age-ma ched con ol Wis a
Han a s.
Funding
This wo k was suppo ed by he Po uguese Founda ion o Science
and Technology (UID/NEU/04539/2013, UID/NEU/04539/2019,
UIDB/04539/2020 and UIDP/04539/2020), COMPETE-FEDER (POCI-
01-0145-FEDER-007440), Cen o 2020 Regional Ope a ional P o-
g amme (CENTRO-01-0145-FEDER-000008: B ainHeal h 2020) and
No a is. JM was inancially suppo ed by an un es ic ed g an om
No a is. The unding o ganiza ions had no ole in he design o conduc
o his esea ch.
Decla a ion o Compe ing In e es
AC pa icipa es in ad iso y boa ds o No a is. RS is a membe o
he Ad iso y Boa ds o Baye , Alcon, Alime a, Alle gan, No a is, and
Thea.
Appendix A. Supplemen a y da a
Supplemen a y ma e ial ela ed o his a icle can be ound, in he
online e sion, a doi:h ps://doi.o g/10.1016/j.biopha.2020.110811.
Re e ences
[1] K.M. Bulla d, C.C. Cowie, S.E. Lessem, S.H. Saydah, A. Menke, L.S. Geiss, T.
J. O cha d, D.B. Rolka, G. Impe a o e, P e alence o diagnosed diabe es in adul s
by diabe es ype - Uni ed S a es, 2016, MMWR Mo b. Mo al. Wkly. Rep. 67 (2018)
359–361, h ps://doi.o g/10.15585/mmw .mm6712a2.
[2] J.G. G oss, A.R. Glassman, D. Liu, J.K. Sun, A.N. An oszyk, C.W. Bake , N.
M. B essle , M.J. Elman, F.L. Fe is 3 d, T.W. Ga dne , L.M. Jampol, D.F. Ma in,
M. Melia, C.R. S ockdale, R.W. Beck, N. Diabe ic Re inopa hy Clinical Resea ch,
Fi e-yea ou comes o pan e inal pho ocoagula ion s in a i eous anibizumab
o p oli e a i e diabe ic e inopa hy: a andomized clinical ial, JAMA
Oph halmol. 136 (2018) 1138–1148, h ps://doi.o g/10.1001/
jamaoph halmol.2018.3255.
[3] T. Qaum, Q. Xu, A.M. Joussen, M.W. Clemens, W. Qin, K. Miyamo o, H. Hassessian,
S.J. Wiegand, J. Rudge, G.D. Yancopoulos, A.P. Adamis, VEGF-ini ia ed blood-
e inal ba ie b eakdown in ea ly diabe es, In es . Oph halmol. Vis. Sci. 42 (2001)
2408–2413.
[4] N. Baha i and, N. Za ghami, F. Panahi, M.Y. Dokh Gha a i, A. Mahda i Fa d,
A. Mohaje i, Rela ionship be ween i eous and se um ascula endo helial g ow h
ac o le els, con ol o diabe es and mic oalbuminu ia in p oli e a i e diabe ic
e inopa hy, Clin. Oph halmol. 6 (2012) 185–191, h ps://doi.o g/10.2147/OPTH.
S27423.
[5] H.P. Hammes, J. Lin, O. Renne , M. Shani, A. Lundq is , C. Be shol z, M. B ownlee,
U. Deu sch, Pe icy es and he pa hogenesis o diabe ic e inopa hy, Diabe es 51
(2002) 3107–3112, h ps://doi.o g/10.2337/diabe es.51.10.3107.
[6] H. Funa su, H. Yamashi a, T. Ikeda, T. Mimu a, S. Eguchi, S. Ho i, Vi eous le els o
in e leukin-6 and ascula endo helial g ow h ac o a e ela ed o diabe ic
macula edema, Oph halmology 110 (2003) 1690–1696, h ps://doi.o g/10.1016/
S0161-6420(03)00568-2.
[7] V.H. Gonzalez, J. Campbell, N.M. Holekamp, S. Kiss, A. Loewens ein, A.
J. Augus in, J. Ma, A.C. Ho, V. Pa el, S.M. Whi cup, P.U. Dugel, Ea ly and long-
e m esponses o an i- ascula endo helial g ow h ac o he apy in diabe ic
macula edema: analysis o p o ocol I da a, Am. J. Oph halmol. 172 (2016) 72–79,
h ps://doi.o g/10.1016/j.ajo.2016.09.012.
[8] A. Rubsam, S. Pa ikh, P.E. Fo , Role o in lamma ion in diabe ic e inopa hy, In .
J. Mol. Sci. 19 (2018), h ps://doi.o g/10.3390/ijms19040942.
[9] F.S. So en ino, M. Allkabes, G. Salsini, C. Boni azzi, P. Pe i, The impo ance o
glial cells in he homeos asis o he e inal mic oen i onmen and hei pi o al ole
in he cou se o diabe ic e inopa hy, Li e Sci. 162 (2016) 54–59, h ps://doi.o g/
10.1016/j.l s.2016.08.001.
[10] G.A. Lu y, E ec s o diabe es on he eye, In es . Oph halmol. Vis. Sci. 54 (2013),
h ps://doi.o g/10.1167/io s.13-12979. ORSF81-87.
[11] A.A. Hidaya , B.S. Fine, Diabe ic cho oidopa hy. Ligh and elec on mic oscopic
obse a ions o se en cases, Oph halmology 92 (1985) 512–522.
[12] A. Campos, E.J. Campos, J. Ma ins, A.F. Amb osio, R. Sil a, Viewing he cho oid:
whe e we s and, challenges and con adic ions in diabe ic e inopa hy and diabe ic
macula oedema, Ac a Oph halmol. 95 (2017) 446–459, h ps://doi.o g/10.1111/
aos.13210.
[13] H. Endo, S. Kase, M. Takahashi, M. Sai o, M. Yokoi, C. Sugawa a, S. Ka su a,
S. Ishida, M. Kase, Rela ionship be ween diabe ic macula edema and cho oidal
laye hickness, PLoS One 15 (2020), e0226630, h ps://doi.o g/10.1371/jou nal.
pone.0226630.
[14] D.M.F. Mohamed, N.A. Hassan, A.A. Osman, M.H. Osman, Sub o eal cho oidal
hickness in diabe ic macula edema, Clin. Oph halmol. 13 (2019) 921–925,
h ps://doi.o g/10.2147/OPTH.S207376.
[15] C. Gup a, R. Tan, C. Mish a, N. Khandelwal, R. Raman, R. Kim, R. Ag awal, P. Sen,
Cho oidal s uc u al analysis in eyes wi h diabe ic e inopa hy and diabe ic
macula edema—A no el OCT based imaging bioma ke , PLoS One 13 (2018),
e0207435, h ps://doi.o g/10.1371/jou nal.pone.0207435.
[16] B. Abadia, I. Su˜
nen, P. Cal o, F. Ba ol, G. Ve des, A. Fe e as, Cho oidal hickness
measu ed using swep -sou ce op ical cohe ence omog aphy is educed in pa ien s
wi h ype 2 diabe es, PLoS One 13 (2018), e0191977, h ps://doi.o g/10.1371/
jou nal.pone.0191977.
[17] V. Ambiya, A. Kuma , V.K. Ba anwal, G. Kapoo , A. A o a, N. Kal a, J. Sha ma,
Change in sub o eal cho oidal hickness in diabe es and in a ious g ades o
diabe ic e inopa hy, In . J. Re ina Vi eous 4 (2018) 34, h ps://doi.o g/10.1186/
s40942-018-0136-9.
[18] A. El Ho i, Co neal endo helial cell changes ollowing Fem osecond lase - assis ed
ca a ac su ge y e sus phacoemulsi ica ion, Del a J. Oph halmol. 19 (2018)
53–57, h ps://doi.o g/10.4103/jc s.Jc s_6_19.
[19] K. El Ghonemy, G.Z. Rajab, A.M. Ib ahim, I.M. Goha , Compa ison be ween
cho oidal hickness in pa ien s wi h diabe ic e inopa hy and no mal indi iduals
using enhanced-dep h imaging spec al-domain op ical cohe ence omog aphy,
Del a J Oph halmol. 19 (2018) 53–57. h p://www.djo.eg.ne / ex .asp?2018/19/
1/53/224572.
[20] S. Na nawa e, P. Bawankule, D. Raje, M. Chak abo y, Cho oidal hickness in ype
2 diabe ic pa ien s wi h a ious s ages o diabe ic macula edema and e inopa hy:
a p ospec i e s udy om cen al India, J. Clin. Oph halmol. Res. 7 (2019) 12–17,
h ps://doi.o g/10.4103/jco .jco _124_17.
A. Campos e al.