In Vivo Bioluminescence Imaging Of Transplanted Bone Marrow–derived Cells In Choroidal Neovascularization

Background The development of choroidal neovascularization (CNV) in patientswith age-related macular degeneration (AMD) often leads to a significant decrease invisual function. Both angiogenesis and vasculogenesis are involved in the process of CNV.Several mechanisms for example, the presence of senescence changes in the RPE–Bruch’smembrane–photoreceptor complex, the accumulation of oxidative damage, and thedevelopment of ischemia of the outer retina have been proposed to explain thepathogenesis of CNV among others.Our group found that BMCs plays an important role in the formation of CNV bytransplanting unpurified bone marrow derived cells from GFP+/+transgenic mice to wildtype adult C47mice to develop GFP chimeric mice. We also proved that BMCsdeferentiating into multiple cell-types such as vascular smooth muscle cells (VSMCs), vascular endothelial cells (VECs), macrophage, epithelial cell and fibroblast. Not only dothese cells involved in the formation of CNV, immunofluorescent staining showed thatsome BMCs in CNV were VEGF or bFGF positive. Previously reported age-relatedchanges in stem cells include loss of differenciation potencial, loss of proliferationpotential, increases in senescent cell numbers and loss of in vivo tissue formation. Theseprevious studies have addressed the effects of aging on different stem cells have providedconflicting results. Bioluminescent imaging (BLI) is extremely useful for longitudinalassessment of cell fate in vivo and progressively becoming more widely applied becauseof its low cost, high throughput, and relative ease of operation in visualizing a widevariety of in vivo cellular events.Objectives This study aims to evaluate the effectiveness of in vivo BLI as a tool forobserving the dynamic conduct of BMCs in CNV, and pave the way for using BLI toinvestigate the underlying mechanism of CNV. We also sought to determine whether ifBMCs derived from old donor mice can convey increased severity into young recipients.Methods (1) Chimeric mice were developed by transplanting unpurified bonemarrow-derived cells from C57BL/6J mice expressing Fluc and green fluorescent protein(GFP) reporter genes to wild-type adult C57mice, after the bone marrow transplantation,we chose the qualified mice and divided them into two groups. The test group underwentlaser rupture of Bruch membrane to induce CNV while Control group not. Two groupmice then placed in a prone position in the IVIS Kinetics system, and imaged on day1,3,7,14,21,28. Peak signals of regions of interest (ROIs) were evaluated by Living ImagingVersion4.0software.(2) After lethal irradiation, young (4months) recipient mice receivedBMCs from aged donor mice(18months) expressing Fluc and GFP were the test group,and young (4months) recipient mice received bone marrow transplantation from young (4months) donors were the control group. One month after reconstitution we performedLaser-induced CNV. Two group mice then placed in a prone position in the IVIS Kineticssystem, and imaged on day1,3,7,14,21,28. Peak signals of ROIs were evaluated byLiving Imaging Version4.0software.(3) Two weeks later, histopathologic study wereperformed to measure the CNV severity. Results (1) Quantitative analysis of BLI data showed that one day after the test groupunderwent CNV, the radiance (photos/second/cm2/sr) of BMCs in the ROIs showed highsignal. The radiance increased fastest from day1to day3and showed robust signal in theROIs on day7. The minimum of the signal was on day14and then slightly increased butremain stable after day28. the radiance (photos/second/cm2/sr) of BMCs in the ROIs ofthe control group slightly changed and obviously lower than the test group (P<0.05).(2)Young mice that received BMCs transplants from aged donors demonstrated more severeCNV, including increased diameter and thickness compared to the control group (P<0.05).Quantitative analysis of BLI data showed that young mice that received BMCs transplantsfrom aged donors showed stronger signal strength than young recipient mice receivedbone marrow transplantation from young donors in first14days after modeling. After that14days, there were no significant differences between tow groups (P＞0.05). The BLIsignal of young mice that received BMCs transplants from aged donors changing fasterthan the control group among the first14days.Conclusion BLI can be noninvasive and useful to observe in vivo behavior of BMCsin CNV area, together with histology data can help to understand the underlyingmechanisms of CNV.