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In vivo bioluminescence imaging of hyperglycemia exacerbating stem cells on choroidal neovascularization in mice
Xiang Gao1, Yu Wang1, Hui-Yuan Hou1
1Department of Ophthalmology, Xijing Hospital, Eye Institute of Chinese PLA, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
Aim:
To investigate the influence of hyperglycemia on the severity of choroidal neovascularization (CNV), especially the involvement of bone marrow-derived cells (BMCs) and underlying mechanisms.
Methods:
BMCs from firefly luciferase (Fluc)/green fluorescent protein (GFP) double transgenic mice were transplanted into C57BL/6J wide-type mice. The recipient mice were injected intraperitoneally with streptozotocin (STZ) daily for 5 consecutive days to induce diabetes mellitus (DM), followed by CNV laser photocoagulation. The BMCs recruitment in CNV exposed to hyperglycemia was firstly examined in Fluc/GFP chimeric mice by in vivo optical bioluminescence imaging (BLI) and in vitro Fluc assays. The CNV severity was evaluated by H&E staining and choroidal flatmount. The expression of vascular endothelial growth factor (VEGF) and stromal cell derived factor-1 (SDF-1) was detected by Western Blot.
Results:
BLI showed that the BMCs exerted dynamic effects in CNV model in Fluc/GFP chimeric mice exposed to hyperglycemia. The signal intensity of transplanted Fluc(+)GFP(+) BMCs in the DM chimeric mice was significantly higher than that in the control chimeric mice with CNV induction at days 5, 7, 14 and 21 (121861.67±9948.81 vs 144998.33±13787.13 photons/second/cm(2)/sr for control and DM mice, P 5d<0.05; 178791.67±30350.8 vs 240166.67±22605.3, P 7d<0.05; 124176.67±16253.52 vs 196376.67±18556.79, P 14d<0.05; 97951.60±10343.09 vs 119510.00±14383.76, P 21d<0.05), which was consistent with in vitro Fluc assay at day 7 [relative light units of Fluc (RLU1)], 215.00±52.05 vs 707.33±88.65, P<0.05; RLU1/ relative light units of renilla luciferase (RLU2), 0.90±0.17 vs 1.83±0.17, P<0.05]. The CNVs in the DM mice were wider than those in the control group at days 5, 7, 14 and 21 (147.83±17.36 vs 220.33±20.17 µm, P 5d<0.05; 212.17±24.63 vs 326.83±19.49, P 7d<0.05; 163.17±18.24 vs 265.17±20.55, P 14d<0.05; 132.00±10.88 vs 205.33±12.98, P 21d<0.05). The average area of CNV in the DM group was larger at 7d (20688.67±3644.96 vs 32218.00±4132.69 µm(2), P<0.05). The expression of VEGF and SDF-1 was enhanced in the DM mice.
Conclusion:
Hyperglycemia promots the vasculogenesis of CNV, especially the contribution of BMCs, which might be triggered by VEGF and SDF-1 production.
