Abstract
Purpose: :
Construction of chimeric GFP+–mice has become a powerful tool in the field of stem cell–participation and –homing in various models of ocular pathology. This study evaluates the course of retinal pathology following lethal irradiation for bone marrow depletion and substitution.
Methods: :
Lethal irradiation in C57/Bl6 mice was conducted with a low voltage radiation unit (U=120kV, I=25mA). Applied radiation was varied between 8 and 13 Gy. Subsequently, mice were transplanted with 1.5 million cells of whole bone marrow derived from mice expressing GFP under a b–actin promoter. GFP–transformation rates were investigated by FACS analysis. Mice were sacrificed at various timepoints between one and ten months after irradiation. In order to localize and quantify bone marrow derived cells within the retina in relationship to the retinal vasculature, transcardial perfusion with rhodamine–coupled concanavalin A lectin was performed. Extravasal cells were counted via an image analysis software. For colocalization studies flatmounts were stained with F4/80 and CD11c antibodies (for labelling of macrophages and dendritic cells, respectively) and evaluated using confocal and fluorescence microscopy.
Results: :
Lethal irradiation was achieved at a total dose of 11 Gy, higher doses were lethal notwithstanding successful transplantation procedure. FACS–analysis revealed average transfomation rates of 75% with a trend towards higher rates with increasing age of recipient mice, ranging as high as 90%. Perfused flatmounts 4 weeks after transplantation were virtually free of extravasal GFP+–cells, whereas in the 4–months group cluster cell infiltrations, preferentially in the peripheral retina, became apparent. Cell morphology appeared from oval to cells with few extensions to dendritiform cells. In the 10–month group a variable cell number was found, and cells with a typical macrophage–like morphology were noted. Colocalization staining showed GFP+– as well as GFP––macrophages and dendritic cells.
Conclusions: :
Following lethal irradiation, bone marrow–derived leukocytes seem to infiltrate the retina, commencing at about 4 months after irradiation. With regard to the observed radiation–related inflammatory reaction after long–term transplantation, this model may be unsuitable for investigation of prolonged retinal diseases.
Keywords: inflammation • radiation damage: light/UV