Molecular Imaging of Retinal Endothelial Injury in Diabetic Animals

Transcription

1 Moleulr Imging of Retinl Endothelil Injury in Dieti Animls The Hrvrd ommunity hs mde this rtile openly ville. Plese shre how this ess enefits you. Your story mtters. Cittion Pulished Version Aessed Citle Link Terms of Use Frimmel, Sonj, Sousk Zndi, Dwei Sun, Zhongyu Zhng, Alexnder Shering, Mrk I. Melhorn, Shintro Nko, nd Ali Hfezi-Moghdm Moleulr Imging of Retinl Endothelil Injury in Dieti Animls. Journl of Ophthlmi & Vision Reserh 12 (2): doi: /jovr.jovr_243_16. doi: /jovr.jovr_243_16 My 3, :52:24 AM EDT This rtile ws downloded from Hrvrd University's DASH repository, nd is mde ville under the terms nd onditions pplile to Other Posted Mteril, s set forth t (Artile egins on next pge)

2 Originl Artile Moleulr Imging of Retinl Endothelil Injury in Dieti Animls Sonj Frimmel 1,2, MD; Sousk Zndi 1,2,3, MD; Dwei Sun 1,2,4, MD, PhD; Zhongyu Zhng 1,4, MD, PhD Alexnder Shering 1,2, PhD; Mrk I. Melhorn 1,2, PhD; Shintro Nko 1,2,5, MD, PhD Ali Hfezi Moghdm 1,2, MD, PhD 1 Moleulr Biomrkers Nno Imging Lortory, Brighm nd Women s Hospitl, Deprtment of Rdiology, Hrvrd Medil Shool, Boston, MA, USA 2 Angiogenesis Lortory, Msshusetts Eye nd Er Infirmry, Deprtment of Ophthlmology, Hrvrd Medil Shool, USA 3 Swiss Eye Institute nd Berner Augenklinik m Lindenhofspitl, Bern, Switzerlnd 4 Deprtment of Ophthlmology, The Seond Affilited Hospitl of Hrin Medil University, Chin 5 Deprtment of Ophthlmology, Grdute Shool of Medil Sienes, Kyushu University, Fukuok, Jpn Astrt Purpose: Dieti retinopthy is leding use of vision loss. There is gret need for erly dignosis prior to the ourrene of irreversile struturl dmges. Expression of endothelil dhesion moleules is oserved efore the onset of dieti vsulr dmge; however, to dte, these moleules nnot e visulized in vivo. Methods: To quntify the expression of endothelil surfe moleules, we generted imging proes tht ind to ICAM 1. The α ICAM 1 proes were hrterized vi flow ytometry under mirofluidi onditions. Proes were systemilly injeted into norml nd dieti rts, nd their dhesion in the retinl mirovessels ws visulized vi onfol snning lser ophthlmosopy. Histology ws performed to vlidte in vivo imging results. Vsulr pthologies were visulized using trypsin digested retinl preprtions. Results: The α ICAM 1 proes showed signifintly higher dhesion to retinl mirovessels in dieti rts thn in norml ontrols (P < 0.01), wheres inding of ontrol proes did not differ etween the two groups. Western lotting results showed higher ICAM 1 expression in retins of T1D nimls thn in norml ontrols. Retinl endothelil ICAM 1 expression ws oserved vi moleulr imging efore mrkers of struturl dmge, suh s periyte ghosts nd ellulr pillries. Conlusion: Results indite tht moleulr imging n e used to detet sutle hnges in the dieti retin prior to the ourrene of irreversile pthology. Thus, ICAM 1 ould serve s dignosti trget in ptients with dietes. This study provides proof of priniple for non invsive sulinil dignosis in experimentl dieti retinopthy. Further development of this tehnology ould improve mngement of dieti omplitions. Keywords: Biomrkers; Dieti Retinopthy; Erly Dignosis; ICAM 1 J Ophthlmi Vis Res 2017; 12(2): Correspondene to: Ali Hfezi Moghdm, MD, PhD. Brighm Women s Hospitl, Moleulr Biomrkers Nno Imging Lortory (MBNI), 75 Frnis St., TH315, Boston, Msshusetts 02115, USA. E-mil: hm@wh.hrvrd.edu Reeived: Aepted: Quik Response Code: Aess this rtile online Wesite: INTRODUCTION The prevlene of dietes is rpidly inresing worldwide. [1] The vsulr omplitions of dietes, This is n open ess rtile distriuted under the terms of the Cretive Commons Attriution NonCommeril ShreAlike 3.0 Liense, whih llows others to remix, twek, nd uild upon the work non ommerilly, s long s the uthor is redited nd the new retions re liensed under the identil terms. For reprints ontt: reprints@medknow.om DOI: /jovr.jovr_243_16 How to ite this rtile: Frimmel S, Zndi S, Sun D, Zhng Z, Shering A, Melhorn MI, et l. Moleulr Imging of Retinl Endothelil Injury in Dieti Animls. J Ophthlmi Vis Res 2017;12: Journl of Ophthlmi nd Vision Reserh Pulished y Wolters Kluwer - Medknow 175

3 whih develop over mny yers, re the mjor uses of moridity nd mortlity. A mirovsulr mnifesttion of dietes is dieti retinopthy (DR), leding use of vision loss. [2] Detetion during the sulinil period leds to higher hne of the prevention of disese progression to the linil stge. [3] An urgent need thus exists to develop strtegies to dignose DR prior to the ourrene of struturl dmge. Retinl vessels n e visulized through non invsive mens, suh s fundusopy, ngiogrphy, nd optil oherene tomogrphy (OCT); thus, in the pst dedes, severl studies hve ttempted to identify erly hnges serving s preditors of retinopthy, [3] fousing on morphologil hrteristis, suh s retinl vsulr dimensions or funtionl hrteristis, inluding lood flow rte, oxygention, or lekge of systemilly pplied dyes, suh s those used in fluoresein ngiogrphy. [3] However, n effetive iomrker for DR ws not suessfully identified. [3] Indeed, results of the Wisonsin Epidemiologi Study of Dieti Retinopthy (WESDR) [4] nd the New Jersey 725 study reveled no orreltion etween retinl vsulr lier nd the risk of DR. [5] It is unertin whether ontinued use of the existing rhiteturl nd funtionl vsulr mesurements with more rigor would provide relile iomrker for erly dignosis of DR. Therefore, we hve hosen tegorilly different strtegy to visulize moleulr iomrkers for DR. The urrent requirement for iomrker is ojetive mesurement s n inditor of pthogeni proesses. [3] For effetive nd timely prevention of retinopthy, iomrker should e non invsively detetle in the erly sulinil disese stge, suh s in the period efore pillries oliterte. Furthermore, the idel iomrker should e uslly linked to retinl pthology development. [6] In dietes, the mirovsulr endothelium is known to express ICAM 1. [7,8] Endothelil ICAM 1 inds to leukoyte β2 integrins nd results in leukoyte umultion in retinl mirovessels. Binding of the β2 integrins to the endothelil ICAM 1 in experimentl DR susequently leds to opening of the lood retinl rrier nd lekge. [9] Our urrent pproh is to trget retinl endothelil ICAM 1 s n inditor of erly pthology. We introdue new imging tehnique ple of deteting single moleules in the retin. [10] This ws omplished y generting imging proes trgeting speifi endothelil surfe moleules; [10-13] one in irultion, the proes intert with their trgets, loted on the intr luminl vessel surfes. [13] The inding intertions re then visulized using light sed imging, [10,11] whih provides quntittive, highly sensitive nd speifi expression nd loliztion informtion for the trgets. [10] Using this pproh, we hve previously shown tht VEGFR 2 is upregulted in the retinl vessels of dieti nimls. [14] The noninvsive nture of the desried imging pproh n e further pplied in longitudinl investigtions, whih is not highly fesile using urrently existing endpoint tehniques. [15] Considering tht leukoyte dhesion in retinl vessels preedes struturl dmge in DR, [16] we hypothesized tht mimiking immune ell reruitment using our ustom designed imging proes n revel the erliest signs of DR. In this study, we evluted endothelil ICAM 1 in norml nd dieti nimls s potentil inditor for endothelil injury. METHODS Streptozotoin indued Dietes All experiments were performed in ordne with the ARVO Sttement for the Use of Animls in Ophthlmi nd Vision Reserh. Mle Long Evns rts ( g, six to seven weeks old) were otined from Chrles River Lortory (Wilmington, MA). After 16 h of overnight fsting, dietes ws indued in eh niml vi intrperitonel injetion of 12 mg of streptozotoin (STZ, minimum 98%, HPLC, Sigm Aldrih, St. Louis, MO) diluted in 0.2 ml of itrte uffer (0.1 M, ph = 4.5). Control nimls reeived n intrperitonel injetion of vehile (itrte uffer). Animls with lood gluose levels greter thn 250 mg/dl t 24 h fter STZ injetion were onsidered dieti. Body weights nd lood gluose levels were regulrly mesured. Animls were mintined in n ir onditioned room under 12 h light/drk yle nd were given free ess to wter nd food. Preprtion of the Moleulr Imging Proes Croxylted mirospheres (MS, 1 nd 2 μm, Polysienes, In.; Wrrington, PA) were ovlently onjugted to Protein G s previously desried. [14] Conjugted proes were then inuted with nti Interellulr Adhesion Moleule 1 (α ICAM 1 ma, Snt Cruz Biotehnology, In., Snt Cruz, CA) or humn IgG (higg, ZYMED Lortories, Crlsd, CA) for 2 hours t room temperture (pproximtely 22 degrees Celsius) on rotry shker. Proes were then wshed with PBS ontining 0.1% BSA. Intertion nd Aumultion of the Moleulr Imging Proes In vivo To evlute the dhesion of the imging proes under physiologil flow onditions in the rt retin during dietes, high resolution imges of the fundus were ptured ontinuously using snning lser ophthlmosope (SLO; HRA2; Heidelerg Engineering, Dossenheim, Germny) oupled with omputer ssisted imge nlysis system. 176 Journl of Ophthlmi nd Vision Reserh Volume 12, Issue 2, April-June 2017

4 Rts were nesthetized with xylzine hydrohloride (10 mg/kg) nd ketmine hydrohloride (50 mg/kg). Pupils were dilted with 0.5% tropimide nd 2.5% phenylephrine hydrohloride. A ontt lens ws used to retin ornel lrity throughout the experiment. Proes were injeted into the femorl vein. Therefter, nimls were pled on pltform in front of the SLO. Movement of the proes through the vessels nd their intertions with the endothelium were ptured in fluoresent mode. An rgon lue lser with regulr emission filter for fluoresein ngiogrphy ws used s the illumintion soure, euse the spetrl properties of the imging proes re similr to those of sodium fluoresein. Imges were otined t 30º ngle t rte of 15 frmes per seond nd reorded on omputer for further nlysis. Videos were otined for 10 minutes fter proe injetions. The numer of umulted proes in the retin ws quntified s distint sttionry fluoresent mrks with very high ontrst ginst the non fluoresent kground. [10-12] The numer of the interting proes were ounted using Imge J softwre (1.41o, Jv 1.60_15; NIH). [17] Ex vivo Evlution of the Aumultion of the Imging Proes To diretly visulize the intertions etween the imging proes nd the retinl endothelium, retinl fltmounts were prepred. Rts were perfused 30 minutes fter proe injetion with PBS nd rhodmine leled onnvlin A letin (ConA, Vetor Lortories, Burlingme, CA). For upper ody perfusion, the hest vity ws opened; the ven v inferior nd the dominl ort were lmped, fter whih 24 guge needle ws inserted from the pex of the left ventrile into the ort. Dringe ws hieved y opening the right trium. Animls were perfused with 30 ml PBS to wsh out intrvsulr ontent nd unound proes, followed y wshing with 0.15 ml of ConA in 15 ml of PBS to stin the vsulr endothelium nd firmly dhering leukoytes nd gin with 15 ml of PBS to remove the exess ConA. Immeditely fter perfusion, eyes were enuleted, nd the retins were mirodisseted nd fltmounted using fluoresene nti fding medium (Vetshield, Vetor Lortories, Burlingme, CA). Tissues were then oserved under n epifluoresene mirosope (DM RXA; Lei, Deerfield, IL) with oth n FITC (exittion, 488 nm; detetion, nm) nd rhodmine filter (exittion, 543 nm; detetion, >560 nm). Imges were quired using high sensitivity digitl mer onneted to omputer ssisted imge nlysis system. Openl imge nlysis softwre (Improvision, Boston, MA) ws used to merge imges of the proes (green) with those of the retinl vessels (red). The numer of proes ws ounted using Imge J softwre. In eh preprtion, mirogrphs (10 ) from five different fields of view (opti dis, nslly, temporlly, superiorly, nd inferiorly) were otined, nd the numer of proes were ounted nd verged. Flow Cytometry The verge numer of α ICAM 1 mas on the surfes of the imging proes ws determined using flow ytometry. Non fluoresent mirospheres (Polysienes, In., Wrrington, PA) onjugted to α ICAM or IgG were inuted with either FITC onjugted got α mouse A or its isotype ontrol. Mirospheres were entrifuged, wshed twie, nd resuspended in PBS. The fluoresene intensities of the mirospheres were mesured using FACSn (Coulter EPICS XL) equipped with the System Work II softwre. To quntify the numer of opies of the moleules onjugted on the surfe of the mirospheres, men fluoresene Intensity (MFI) ws plotted ginst lirtion urve onstruted s previously desried. [11] Adhesion of the Imging Proes to Immoilized ICAM 1 Under Flow Conditions Adhesion properties of the onjugted proes were exmined using the miro flow hmer ssy. [18] Glss mirohmers were oted with reominnt rt ICAM 1 (5 μg/ml; R nd D, 583 IC 050) t 4 C overnight. The next dy, the ICAM 1 oted mirofluidi hmers were onneted to ioomptile polyester tuings (Smll Prts In. #TGY 010 C) nd 1 ml syringe filled with α ICAM 1 proes or ontrol proes (1:200 dilution ompred to niml experiments). A syringe pump injeted α ICAM 1 or humn IgG onjugted proes into the flow hmers t 2 dynes/m 2 sher stress, whih were prepred s previously desried. [19] Video mirosopi imges of the flow through the hmers were otined for further nlysis. Western Blotting Animls were perfused with PBS nd eyes susequently enuleted. The retin ws refully isolted nd pled in 100 µl of lysis uffer (mmmlin ell lysis kit MCL 1, Sigm Chemil Co, St. Louis, MO) supplemented with protese inhiitors nd phosphtse inhiitors (Sigm) nd then sonited. The lyste ws entrifuged, nd the superntnt ws olleted. Smples ontining equl mounts of totl protein were seprted vi SDS PAGE (sodium dodeyl sulfte polyrylmide gel eletrophoresis) nd eletro lotted onto PVDF (polyvinylidene fluoride) memrnes (Life Tehnologies, Crlsd, CA). Non speifi inding ws loked with 5% skim milk. Afterwrds, memrnes were inuted with rit nti ICAM 1 polylonl ntiody (1:200 Snt Cruz Biotehnology, Snt Cruz, CA) or n nti Journl of Ophthlmi nd Vision Reserh Volume 12, Issue 2, April-June

5 β tuulin ma (1:2000; Am, Cmridge, MA) t 4 C overnight, followed y inution with horserdish peroxidse onjugted donkey or sheep nti rit or mouse IgG (1:2000; GE Helthre UK limited Bukinghmshire, UK). Signls were visulized sed on hemiluminesene (ECL kit; GE Helthre UK limited, Bukinghmshire, UK) ording to the mnufturer s protool. Trypsin Digestion nd Anlysis of Retinl Vsulture Trypsin digestion of retinl vessels is the stndrd tehnique urrently used to detet struturl pthologies in dietes. [19] The eyes of norml nd dieti rts were enuleted nd fixed in 10% neutrl uffered formlin. Eyes were pled in six well pltes in PBS. Retins were then disseted under surgil mirosope nd wshed 4 5 times using filtered wter to filitte seprtion of the neurl retinl lyers from the lood vessels. Susequently, retins were inuted in 0.1 M Tris uffer (ph 7.8, Sigm, T8193) ontining 3% trypsin (Difo 1:250, Amreso LLC, 0458) t 37 C for 90 min. Wter wshes were used to disintegrte the neuronl tissues nd deris from the vsulr network. The retinl vsulr network ws then fltmounted on slide nd stined with periodi id solution (PAS, Sigm, 3951), Shiff s regent (Sigm, ), nd hemtoxylin (Sigm, GHS316). Sttistil Anlysis Vlues were expressed s men ± SEM. Dt were nlyzed using Student s t test. Sttistil signifine ws onsidered t P < Unless speified otherwise, n indites the numer of independent niml experiments. RESULTS Dieti Retinl Leukoyte Aumultion nd ICAM 1 Expression Leukoyte umultion is key omponent of experimentl dieti retinopthy (DR). [20] To enumerte the firmly dhering leukoytes, histologil fltmounts from the retins of norml nd dieti nimls were prepred [Figure 1]. Retinl rteries nd veins of dieti nimls showed more pronouned leukoyte umultion ompred to those of norml ontrols [Figure 1]. Next, we stined for the leukoyte tivtion mrker CD18, lignd for endothelil ICAM 1. Firmly dhering leukoytes in the retinl vessels expressed d e Figure 1. Leukoyte umultion nd ICAM 1 expression in the dieti retin. () Leukoyte dhesion in retinl vessels of norml nd dieti rts. () Quntifition of leukoytes in retinl rteries nd veins (n = 6). () Immunohistohemistry of firmly dhering leukoytes (red) for CD18, lignd for the endothelil ICAM 1. (d) Western lot nlysis for ICAM 1 using retinl extrts of norml nd dieti rts (three weeks fter STZ injetion). (e) Western lotting results showing higher ICAM 1 expression in retinl tissues of dieti rts (n = 8 nimls) thn in those of norml ontrols (n = 7 nimls). 178 Journl of Ophthlmi nd Vision Reserh Volume 12, Issue 2, April-June 2017

6 CD18 [Figure 1]. Western lotting ws performed to quntify endothelil ICAM 1 expression in the dieti retins [Figure 1d]. ICAM 1 protein levels in the retinl tissues of three week dieti rts were signifintly higher thn those in norml rts [Figure 1e] Chrteriztion of Moleulr Imging Proes The numer of lignds on the surfe of the imging proes ws quntified following our previously estlished protool. [10,12] PE onjugted nti murine IgG nd isotype mthed ontrol IgG were used to stin the ntiodies on the imging proes [Figure 2]. Fluoresene intensities of the proes nd lirtion mirospheres with known site densities of PE onjugted IgG were mesured vi flow ytometry. [13] A lirtion urve ws generted (R² =0.99) nd used to determine the verge numer of α ICAM 1 ntiodies (n = ) on the proe surfe. To exmine the speifiity nd dhesion properties of our imging proes under ontrolled flow onditions, we performed in vitro flow studies following our previously desried protools [Figure 2]. [18] Reominnt ICAM 1 protein ws immoilized on the inner hmer surfes. Susequently, live video mirosopy of the flow of the imging proes through the hmers ws reorded for 6 min, nd numers of dhering proes were quntified every 30 s [Figure 2]. At 6 min fter the strt of imging, the ICAM 1 oted flow hmers showed signifintly more pronouned umultion of α ICAM 1 proes thn those of IgG onjugted ontrol proes under the sme onditions (P < 0.01), therey demonstrting the speifiity of the umulted proes for ICAM 1 [Figure 2d]. Moleulr Imging of Endothelil Injury in Dieti Animls To monitor endothelil injury in vivo, we used our new moleulr imging pproh [10,12] designed for erly dignosis [12] nd use in mehnisti studies to monitor the retinl mirovessels. [15] Imging proes were generted y onjugting the inding lignds of endothelil ICAM 1 onto the surfes of injetle fluoresent mirospheres. These α ICAM 1 or ontrol proes were then injeted into the loodstrems of norml nd dieti nimls. Aumultion of the firmly dhering proes in vivo ws visulized using SLO [Figure 3]. Firmly dhering proes were visile s right dots in the fundus imges of the exmined eyes [Figure 3]. The numer of α ICAM 1 nd IgG onjugted proes, whih were used s negtive ontrols, did not differ signifintly etween dieti nd norml rts. However, signifintly higher numer of α ICAM 1 proes dhered to the retinl vessels of the dieti rts thn those in norml ontrols [Figure 3]. A signifint differene in dhesion of α ICAM 1 proes ws lso oserved etween norml Figure 2. Chrteriztion of the moleulr imging proes. () Flow ytometry nlysis of non fluoresent α ICAM 1 proes leled with FITC onjugted ma or isotype ontrol nd IgG onjugted proes with isotype ontrol. () Shemti of the mirofluidi experiments. Reominnt ICAM 1 ws immoilized, nd proe intertions were visulized vi live mirosopy. () Representtive video mirogrphs showing the intertions of the imging proes with the immoilized ICAM 1 t 2 dynes/m 2. (d) Quntifition of proe dhesion illustrtes speifi dhesion of proes to immoilized ICAM 1. d Journl of Ophthlmi nd Vision Reserh Volume 12, Issue 2, April-June

7 nd six week dieti nimls injeted with α ICAM 1 proes [Figure 3d]. To exmine the effet of proe size on inding effiy, we used proes with 1 nd 2 µm dimeters. In dieti nimls, the smller proes hd higher inding rtio thn the ontrol proes [Figure 3e]. Ex vivo Visuliztion of Proe Aumultion in Retinl Miro Vessels To verify the intertion of the imging proes with the retinl endothelium, histologil fltmounts were prepred nd susequently exmined vi epifluoresene mirosopy [Figure 4]. Consistent with our in vivo findings, retinl vessels of norml nd dieti nimls showed no differene in the dhesion of IgG proes, whih were used s negtive ontrols. However, retinl mirovessels of dieti rts showed signifintly more pronouned dhesion of the α ICAM 1 proes ompred to those of norml rts [Figure 4]. Leukoytes onstitutively express ICAM 1, whih ws lso trgeted y the imging proes in the dieti retin. A frtion of the α ICAM 1 imging proes ws firmly ound to dhering leukoytes [Figure 4], onsistent with our previous reports using lipopolyshride indued model of ute inflmmtion. [10,12] The pility of moleulr imging to detet umulted leukoytes ould potentilly e used for quntifition of speifi immune responses in the retin. Imging proe umultion preedes struturl injury in the retin A key morphologil feture of the dieti retin is the formtion of ellulr, nonperfused pillries, [21] whih is used y miroenvironmentl dmge to the endothelium, mtrix, nd periyte. [21] Erly dignosis is required for therpeutilly effetive tretment efore irreversile struturl dmge ours. To investigte these struturl hnges, we performed trypsin digestion of the retins of norml nd dieti nimls. Retins of norml rts showed ptent pillries ontining endothelil ells nd surrounding periytes [Figure 5]. Similrly, no evident struturl pthologies, suh s periyte loss or ellulr pillries, were oserved in the three week dieti nimls [Figure 5]. In ontrst, oliterted ellulr pillries, lssi sign of DR pthology, ws frequently oserved in long term dieti nimls [Figure 5]. Thus, moleulr imging n detet endothelil injury in the dieti retin t n erly time point when no visile struturl pthology is evident. DISCUSSION The progression from physiologil to pthologil onditions in DR involves sequene of events initited y moleulr hnges, followed y ellulr d Figure 3. In vivo detetion of endothelil injury using moleulr imging. () Shemti of our in vivo moleulr imging pproh. () Representtive SLO mirogrphs from the retins of norml nd dieti nimls. White dots represent firmly dhering proes. () In vivo proe dhesion in norml nd three week dieti nimls (n = 5, **P < 0.01). (d) Moleulr imging of retinl endothelil ICAM 1 in 6 dieti nimls (n = 6, **P < 0.01). (e) Comprison etween the inding of two differently sized α ICAM 1 imging proes (1 nd 2 µm) in dieti retins (n = 5). e Figure 4. Ex vivo evlution of imging proe umultion. Retinl fltmounts were prepred from nimls perfused with rhodmine ConA (red). Imging proes tht resisted perfusion re visile s green spots in fluoresene mirosopy. () Representtive retinl mirogrphs show proe dhesion in retinl vessels of norml nd dieti rts. () Quntifition of α ICAM 1 proe dhesion in retinl vessels (n = 6 8, **P < 0.01). () Imging proes ound to firmly dhering leukoytes in retinl vessels. Mirogrph shows rhodmine stined leukoytes (lue rrows) nd α ICAM 1 proes (green/yellow). 180 Journl of Ophthlmi nd Vision Reserh Volume 12, Issue 2, April-June 2017

8 responses eventully leding to struturl dmge. Clinilly detetle struturl hnges, suh s ellulr pillries, mironeurysms, or neovsulriztion, severely ompromise disese prognosis. Therefore, detetion of the erly moleulr hnges in the retinl endothelium during dietes onset is ritil s it n idelly enle dequte tretment efore irreversile struturl dmge ours. An urgent need thus exists to identify iomrkers for erly DR nd to develop prtil strtegies for sulinil dignosis. [3] However, retinl endothelil injury in dieti ptients [3] nnot e visulized using existing tehniques, whih only detet struturl dmge. To ddress the unmet need for n erly dignosti tool for DR, we developed n imging tehnique mesuring in vivo mono moleulr intertions using ustom designed moleulr imging proes. [10,11,12,15] Our pproh is sed on the priniple tht one of the erliest responses of the retin to hyperglyemi is immune ell umultion in the retinl vsulture. Leukoyte umultion is used y the expression of dhesion moleules, suh s ICAM 1, in the retinl endothelium of dieti nimls. Higher ICAM 1 expression hs een oserved in the eyes of dieti individuls, mking ICAM 1 linilly relevnt trget. [7] To visulize ICAM 1, we developed imging proes tht mimi erly leukoyte endothelil intertions nd whih n e deteted using fluoresent signls [Figure 6]. Our urrent pproh is idel for visuliztion of retinl endothelil moleules, sine the struture of the eye provides unique pltform for light sed moleulr imging. The high sensitivity nd speifiity of the moleulr imging proes mke them highly suitle for the detetion of low grde inflmmtion, s oserved in DR. [10,11] Using this non invsive pproh, we demonstrted elevted ICAM 1 expression levels in the retinl vessels of T1D nimls. This novel pproh represents gret dvnement tht n e pplied to humns. Clinil use of this tehnique is fesile s the imging proes n e onstruted entirely from FDA pproved omponents sfe for humn use, suh s iodegrdle polymers inluding poly(epsilon proltone) (PCL) nd poly (D, L ltide o glyolide) (PLGA), [22,23] oth of whih re pproved for linil use in trgeted drug delivery systems. The FDA pproved dyes indoynine green (ICG) nd fluoresein [24] hve estlished sfety profiles nd n thus e utilized s fluorophores for enpsultion in the imging proes. We demonstrte tht non invsive moleulr imging n distinguish etween norml nd dieti retins t n erly time point when struturl dmge is not yet evident. In this study, ICAM 1 ws seleted s the imging trget euse it medites leukoyte umultion in the retin. [7,8] However, other endothelil surfe moleules, suh s growth ftor reeptors nd glyolyx omponents, n e trgeted using the sme priniples. We reently demonstrted the in vivo upregultion of VEGFR 2 in oth the retinl mirovessels of dieti nimls nd humn eyes. [14] The high sensitivity nd speifiity of the moleulr proes, [10] the quntittive nture of the deteted signls, [11] nd the utomted proessing [12] mkes this tehnique potentilly powerful tool for monitoring erly events in dieti retinl mirongiopthy tht n meet the need for n erly dignosti tool for DR detetion. Moleulr imging of retinl endothelil ICAM 1 ould provide n erly wrning signl efore linil symptoms develop, nd effetive tretments t this erly stge ould prevent disese progression. Figure 5. Erly detetion y moleulr imging preedes struturl dmge. Retins of norml nd dieti nimls were trypsin digested to visulize vsulr hnges in the dieti retin. PAS nd hemtoxylin stined fltmounts of trypsin digested norml retins show ptent retinl pillries, whih re omprised of endothelil ells nd re surrounded y periytes (). At three weeks of dietes, retins of dieti nimls show no signs of struturl dmge (). In ontrst, long term dieti nimls (six months) disply oliterted ellulr pillries (rrow) (). Br, 50 µm. Figure 6. Sensitive detetion of endothelil injury y mimiking leukoyte funtion. Under norml onditions, leukoytes freely flow in the loodstrem nd do not intert with helthy endotheli (upper left). Dieti endotheli express ICAM 1, whih medites firm leukoyte dhesion (upper right). Imging proes tht trget endothelil ICAM 1 pply this priniple to detet erly vsulr hnges in vivo. α ICAM 1 proes tht trget endothelil ICAM 1 show signifintly higher intertion with dieti endotheli (lower right) thn in those of norml nimls (lower left). Journl of Ophthlmi nd Vision Reserh Volume 12, Issue 2, April-June

9 Aknowledgments We thnk Ree C. Grlnd for editoril help in the preprtion of this mnusript. This work ws supported y the NIDDK Dietes Complition wrd # , the NIH Impt Awrd (DK ), nd JDRF Innovtion wrd to A.H.M., Dr. Ernst und Anit Buer Stiftung (S.F.) nd Deutsher Akdemisher Austush Dienst (S.F. nd M.I.M). Finnil Support nd Sponsorship Nil. Conflits of Interest There re no onflits of interest. REFERENCES 1. Misr R, Ptel T, Koth P, Rji A, Gnd O, Bnerji M, et l. Prevlene of dietes, metoli syndrome, nd rdiovsulr risk ftors in US Asin Indins: Results from ntionl study. J Dietes Complitions 2010;24: Mohmed Q, Gillies MC, Wong TY. Mngement of dieti retinopthy: A systemti review. JAMA 2007;298: Ikrm MK, Cheung CY, Lorenzi M, Klein R, Jones TL, Wong TY, et l. Retinl vsulr lier s iomrker for dietes mirovsulr omplitions. Dietes Cre 2013;36: Klein R, Klein BE, Moss SE, Wong TY, Hurd L, Cruikshnks KJ, et l. The reltion of retinl vessel lier to the inidene nd progression of dieti retinopthy: XIX: The Wisonsin Epidemiologi Study of Dieti Retinopthy. Arh Ophthlmol 2004;122: Roy MS, Klein R, Jnl MN. Retinl venulr dimeter s n erly inditor of progression to prolifertive dieti retinopthy with nd without high risk hrteristis in Afrin Amerins with type 1 dietes mellitus. Arh Ophthlmol 2011;129: Steyererg EW, Penin MJ, Lingsm HF, Kttn MW, Vikers AJ, Vn Clster B. Assessing the inrementl vlue of dignosti nd prognosti mrkers: A review nd illustrtion. Eur J Clin Invest 2012;42: MLeod DS, Lefer DJ, Merges C, Lutty GA. Enhned expression of intrellulr dhesion moleule 1 nd P seletin in the dieti humn retin nd horoid. Am J Pthol 1995;147: Miymoto K, Khosrof S, Bursell SE, Rohn R, Murt T, Clermont AC, et l. Prevention of leukostsis nd vsulr lekge in streptozotoin indued dieti retinopthy vi interellulr dhesion moleule 1 inhiition. Pro Ntl Ad Si U S A 1999;96: Skondr D, Nod K, Almulki L, Tyyri F, Frimmel S, Nkzw T, et l. Chrteriztion of zuroidin s permeility ftor in the retin: Involvement in VEGF indued nd erly dieti lood retinl rrier rekdown. Invest Ophthlmol Vis Si 2008;49: Sun D, Nko S, Xie F, Zndi S, Shering A, Hfezi Moghdm A. Superior sensitivity of novel moleulr imging proe: Simultneously trgeting two types of endothelil injury mrkers. Fse J 2010;24: Miyhr S, Almulki L, Nod K, Nkzw T, Histomi T, Nko S, et l. In vivo imging of endothelil injury in horiopillris during endotoxin indued uveitis. FASEB J 2008;22: Xie F, Sun D, Shering A, Nko S, Zndi S, Liu P, Hfezi Moghdm A. Novel moleulr imging pproh for sulinil detetion of iritis nd evlution of therpeuti suess. Am J Pthol 2010;177: Hfezi Moghdm A, Thoms KL, Prorok AJ, Huo Y, Ley K. L seletin shedding regultes leukoyte reruitment. J Exp Med 2001;193: Sun D, Nko S, Xie F, Zndi S, Bgheri A, Knvi MR, et l. Moleulr imging revels elevted VEGFR 2 expression in retinl pillries in dietes: A novel iomrker for erly dignosis. FASEB J 2014;28:3942: Grlnd RC, Sun D, Zndi S, Xie F, Fez S, Tyyri F, et l. Noninvsive moleulr imging revels role of PAF in leukoyte endothelil intertion in LPS indued oulr vsulr injury. FASEB J 2011;25: Arit R, Ht Y, Nko S, Kit T, Miur M, Kwhr S, et l. Rho kinse inhiition y fsudil meliortes dietes indued mirovsulr dmge. Dietes 2009;58: Armoff MD, Mgelhes PJ, Rm SJ. Imge proessing with ImgeJ. Biophotonis Int 2004;11: Hfezi Moghdm A, Thoms KL, Cornelssen C. A novel mouse driven ex vivo flow hmer for the study of leukoyte nd pltelet funtion. Am J Physiol Cell Physiol 2004;286:C Chou JC, Rollins SD, Fwzi AA. Trypsin digest protool to nlyze the retinl vsulture of mouse model. J Vis Exp 2013:e Nod K, Nko S, Zndi S, Sun D, Hyes KC, Hfezi Moghdm A. Retinopthy in novel model of metoli syndrome nd type 2 dietes: New insight on the inflmmtory prdigm. FASEB J 2014:28: Hmmes HP, Feng Y, Pfister F, Brownlee M. Dieti retinopthy: Trgeting vsoregression. Dietes 2011;60: Iyer AK, Gnt S, Amiji MM. Polymeri nnoprtiles s trget speifi delivery systems. In: Hndook of Mterils for Nnomediine. Vol. 1. Pn Stnford Pulishing; pp Iftimi N, Amiji M, Milne L, Oldenurg A. Nnotehnology pprohes for ontrst enhnement in optil imging nd disese trgeted therpy. In: Iftimi N, Brugge W, Hmmer DX, editors. Advnes in Optil Imging for Clinil Mediine. Wiley Pulishing; 2011: Merin J, Grvier J, Nvrro F, Texier I. Fluoresent nnoproes dedited to in vivo imging: From prelinil vlidtions to linil trnsltion. Moleules 2012;17: Journl of Ophthlmi nd Vision Reserh Volume 12, Issue 2, April-June 2017