gms | German Medical Science

Objective: A high accumulation of microglia cells is characteristic of malignant gliomas. Several in vitro studies discovered a multiple of the physiological microglia density in this tumor entity. A change of microglia cell shape has been visualized histologically. Nevertheless there is no data that shows dynamic changes intravitally. The aim of our studies was to analyze microglia motility, phenotypic changes of microglia cells in gliomas and microglia interaction with tumor vasculature.

Method: GL261-glioma cells have been implanted stereotactically into brain parenchyma of CX3CR1-eGFP mice with GFP-labeled microglia. A chronic cranial window enabled repetitive long time observation intravitally. Microscopy was performed on day 5, 10, 12, 14, 16, 17 after implantation. With Rhodamine B-dextran injection tumor vasculature was visualized. We recorded 3-6 z-stacks and 1 movie on each day and animal (n=8). We measured the volume of microglia cells, tumor vessels and microglia-tumor vessel-co-localization. For quantification of cell motility we analyzed changes of microglia shape over time and observed typical behavior of cells in different degrees of activation. Finally we confirmed our intravital results by histological analysis.

Results: We observed a significant increase of microglia density from day 5 (8.1% ±1.2%) up to day 14 (14.9% ±0.9%). At the same time we illustrated an increase of tumor vessel volume from day 5 (8.3% ±1.4%) to day 14 (14.8% ±7%). From day 14 we determined a plateau without significant changes of microglia and tumor-vessel density. In comparison to that, microglia-tumor vessel co-localization is increasing gradually from day 5 (0.151% ±0.025%) to day 17 (1.0% ±0.12%). We detected a change of microglial cell morphology from a resting state with a small cell body and many dynamic processes to a hypertrophic fast moving cell type. Activated microglia shows an ameboid pattern of movement with large unbranched processes.

Conclusions: With 2-photon microscopy we were able to analyze microglia motility in an orthotopic glioma model. Intravital real time records show highly dynamic cell changes, that were not visualized before. Microglia enhancement proceeds simultaneous to tumor angiogenesis with high multiplication of intratumoral microglia-vessel interaction. Our results contribute to a more detailed understanding of intratumoral behavior of microglia cells and implicates a direct connection between tumor angiogenesis and microglia accumulation