Effects Of Hyperglycemia On Bone Marrow-derived Cells During Choroidal Neovascularization

Backgroud：One common pathological change of certain blindness-causing diseases,such as age-related macular degeneration (AMD), eye tissue cytoplasm disease andpathological myopia, is the choroid neovascularization (CNV) formation, which includesangiogenesis and vascularization. Angiogenesis refers to the formation of new bloodvessels by intrinsic vessel cells, while vascularization means the development of newblood vessels by differentiated bone marrow-derived cells (BMCs). Inflammation,hypoxia and oxidative stress can influence the occurrence and development of CNV.Diabetes is a common chronic metabolic disease and the persistent hyperglycemiaresults in the damage to multiple organs. In the eyes, the most familiar complication isdiabetic retinopathy (DR), with the basic pathological chage being as the microangiopathy.At the late stage of the disease, retinal neovascularization usally occurs. AMD is the mostcommen disorder characterized by CNV and is much similar to DR in the predisposing factors and pathological changes of the formation of neovascularization. Epidemiologicalstudies also indicated that diabetes might be another dangerous factor for the occuranceand development of AMD, especially for neovascular AMD.Our previous study demonstrated that hyperglycemia deteriorated CNV and promotedbone marrow-derived cells (BMCs) chemotaxis. However, this study was unable todynamically observe the effects of hyperglycemia on BMCs during the development ofCNV because of the limitation of the cytological and histological methods as well as theonly observing time point used in the study. Bioluminescence imaging (BLI) is anemerging molecular imaging technique with the advantage of noninvasive, high sensitivityand real-time dynamic characters. Although our group has started to study BMCs duringthe occurance and development of CNV using BLI, we have not used BLI to monitor thereal time chemotaxis of BMCs to CNV and the occurance and development of CNV inhyperglycemia.Objective： This study was designed to dynamically observe the influence ofhyperglycemia on BMCs during the development of CNV with BLI, histopathology andmolecular biology methods.Methods：BMCs obtained from luciferase-green and fluorescent protein (Fluc-GFP)double transgenic mice were injected into wild type C57BL/6J mice via tail vein to createthe chimera models with over85%chimerism. The mice were randomized into fivegroups: euglycemic chimeric mice (Normal) group, hyperglycemic chimeric mice (DM)group, euglycemic chimeric mice with CNV (Normal+CNV) group, hyperglycemicchimeric mice with CNV (DM+CNV) group and negative control group(wild-type C57mice received Co60irradiation but without BMCs transplantation). Streptozotocin(50mg/(kg·d)) was intraperitoneally injected daily for5days to establish the hyperglycemicmodels in the chimeric mice. When the blood glucose concentration was over15mmol/L,the animals were considered as diabetic mice. Euglycemic chimeric mice were not giveninjection. Laser photocoagulation with532nm wave length was used to induce CNV. Themodels of each group were dealt with the methods as followed:(1) BLI signal ofBMCsFluc+GFP+was in vivo dynamically examined by IVIS Kinetics system on day1,3,5, 7,14,21and28after CNV modeling in five groups.(2) The mice in Normal+CNV groupand DM+CNV group were sacrificed on day7after laser treatment, and the choroidmembranes were made into homogenate. The luciferase activities in the eyes weremeasured and the numbers of infiltrated BMCs were reflected by Relative Light Units offirely luciferase (RLU1), Relative Light Units of renilla luciferase (RLU2) and their ratio(RIOT).(3) On1,3,5,7,14,21and28days after laser treatment, the mice weresacrificed, choroidal and retinal were isolated for histopathological examination. Thelength and thickness of CNV in Normal+CNV group and DM+CNV group were measured.(4) At the seventh day after laser treatment, choroidal flatmout were used to measure thesize of CNV between Normal+CNV group and DM+CNV group.(5) The mice inNormal+CNV group and DM+CNV group were sacrificed on day1,3,5,7,14,21and28after laser treatment, and RPE-choroidal-sclera complex were collected. Vascularendothelia growth factor (VEGF) and stromal cell derived factor-1(SDF-1) expressionwere analyzed by Western blot.Results：(1) Flow cytometry analysis suggested the mean chimerism degree of thechimeric mice was (88.85±2.46)%on day28after BMCs transplantation, and the meanblood glucose concentration was (17.88±0.86) mmol/L.(2)①Light signals appeared1day and reached its peak7days after CNV modeling in Normal+CNV group, and thengradually decreased. The light signals in Normal group were stable. The light signals ofNormal+CNV group were significantly higher than Normal group on day1,3,5,7,14,21and28after CNV modeling (t=19.545,14.572,23.172,11.329,16.439,17.382,7.616,P<0.05).②The light signals in DM+CNV group indicated that BMCs began to infiltrateinto eyes on day1after laser and reached its peak on day7. Then the light signalsgradually decreased later. The light signals in DM group were stable and weaker thanDM+CNV group on1,3,5,7,14,21and28days after CNV modeling (t=15.366,11.701,17.933,23.356,16.607,13.933,11.406, P<0.05).③Light signals appeared on day1andreached its peak on day7after CNV modeling in Normal+CNV group and DM+CNVgroup. The Light signals were stronger in the DM+CNV group than Normal+CNV groupon day5,7,14and21after choroid neovascularization modeling (t=3.869,3.351,6.142, 3.021, P<0.05).(3) At the seventh day after laser treatment, the mice were sacrificed andluciferase activities were measured, RLU1and RIOT in DM+CNV group were higher thanNormal+CNV group(t=28.88,P<0.05; t=8.448,P<0.05).(4) Histopathological examinationrevealed that CNV broke through the Bruch membrane toward subretinas on1,3,5,7,14,21and28days. The length of CNV in DM+CNV group were markedly different fromNormal+CNV group on days5,7,14and21(t=7.513,11.224,9.509,9.732, P<0.05).However, at the different time points, there were no significant differences of the CNVthickness were observed between the two groups (t=0.865,0.301,0.691,0.503,0.164,0.402,1.105, P>0.05).(5) On day7after laser treatment, the area of CNV in DM+CNVgroup (32218.00±1687.16) μm2was nearly1.5-fold than Normal+CNV group(20688.67±1488.05) μm2through choroidal flatmout (t=4.820, P<0.05).(6) Theexpression of VEGF and SDF-1were higher in DM+CNV group than Normal+CNVgroup by means of Western blot (P<0.05).Conclusions：Based on our previous study, the present study further demonstrated,using BLI, histological and molecular biological methods, that CNV peaked7days afterthe initiatioan of CNV. Hyperglycaemia can promote the severity of CNV directly andindirectly by enhancing more BMCs to CNV. These effects are related with the level oflocal VEGF and SDF-1proteins.