Choroidal and retinal structural, cellular and vascular changes in a rat model of Type 2 diabetes

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. 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