Malignant glioma—induced neuronal cell death in an organotypic glioma invasion model

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✓ Rapid growth and diffuse brain infiltration are hallmarks of malignant gliomas. The underlying molecular pathomechanisms of these tumors, however, remain to be determined. The authors present a novel glioma invasion model that allows researchers to monitor consecutively tumor cell proliferation and migration in an organotypic brain environment. Enhanced green fluorescent protein—labeled F98 rat glioma cells were implanted into slice cultures obtained from a rat hippocampus, and tumor growth was microscopically documented up to 20 days in vitro. Invasion along radially oriented migratory streams could be observed 5 days after implantation of rat F98, human U87MG, and mouse GL261 glioma cells, whereas human Be(2)c neuroblastoma cells and mouse HT22 hippocampal neurons failed to invade the brain parenchyma. Following implantation of F98 glioma cells into the entorhinal cortex, cell death was observed within the infiltrated brain parenchyma as well as in the neuroanatomically connected dentate gyrus. Application of the N-methyl-D-aspartate receptor antagonist MK801 to the culture medium significantly reduced neuronal degeneration in the dentate gyrus, whereas the a-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antagonist GYKI 52466 inhibited peritumoral cytotoxicity. This new model allows researchers to address in a systematic manner the molecular pathways of brain invasion as well as specific tumor—host interactions such as necrosis.

Article Information

Address reprint requests to: Ilker Y. Eyüpoglu, M.D., Department of Neurosurgery, University of Erlangen—Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany. email: eyupoglu@gmx.net.

© AANS, except where prohibited by US copyright law.

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    Example of the OGIM. A: Rat entorhinohippocampal slice cultured for 6 days. B: Same slice shown in A after implantation of approximately 5000 EGFP-transfected F98 glioma cells. Bar = 100 µm; DG = dentate gyrus; EC = entorhinal cortex; NC = neocortex.

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    Implantation of neoplastic and nonneoplastic cells into organotypic brain slice cultures. A–E: Rat F98 glioma infiltration areas (green fluorescence) have been quantified 1, 3, 5, 10, and 20 days postimplantation (DAI). Only fluorescence images are depicted, brightfield views of the slice culture are not shown (compare with Fig. 1). Arrows in B indicate the bulk tumor mass at 3 days postimplantation. A fluorescent corona emerges at 5 days postimplantation (arrowheads in C), indicating glioma cell invasion into the adjacent brain parenchyma. F: Time-dependent F98 tumor growth is quantified and calculated as a percentage of the EGFP-positive area at 1 day postimplantation (defined as 100%). Data are given as means ± standard deviations from nine slice culture experiments in each group (the same applies also for panels I, L, O, and R). G–I: Data from similar experiments performed using transplantation of human U87MG glioma cells. J–L: Data from similar experiments performed using mouse GL261 glioma cells. In this experiment, mouse slice cultures were used. M–O: Implantation of human Be(2)c neuroblastoma cells into the entorhinal cortex. Note the lack of any tumor corona at 5 days postimplantation. P–R: Implantation of HT22 neuronal cells into mouse entorhinohippocampal slice cultures. Bar = 100 µm (A–E, G, H, J, K, M, N, P, and Q).

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    Photomicrographs documenting the histopathological analysis of glioma-implanted organotypic slice cultures. A–C: The F89-implanted slice cultures were immersion fixed at different time periods and sectioned at a 14-µm thickness by using a cryostat. Fluorescence microscopic images of a representative study are shown; eight additional experiments yielded similar results. Arrows demarcate the sharp tumor border at 3 days postimplantation. At 5 days postimplantation, the glioma cells have migrated into the adjacent brain parenchyma along radially oriented trails (see also 10 days postimplantation). Arrowheads in B, C, F, and G mark green fluorescent glioma cells outside the tumor bulk. Confocal laser scanning microscopy (D) and routinely stained H & E sections (E) at 5 days postimplantation confirm centrifugal migration streams as well as invasion into the depth of the slice culture. F–I: Data from similar experiments performed using various neoplastic and nonneoplastic cell lines (see also Fig. 2). Bar = 12.5 µm (A–C and E–I) and 15 µm (D).

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    Glutamate receptor antagonist treatment in OGIM. A: Neurotoxic cell damage is visualized by an incubation of brain slice cultures with PI in an isotonic solution. Only subtle degeneration can be identified after 11 days in culture. Pictures of a representative study are shown; eight additional experiments yielded similar results. B: Following EGFP-labeled F98 glioma transplantation, significant PI uptake can be observed in the adjacent brain parenchyma (arrows). In addition, projection areas of the perforant pathway into the hippocampus and dentate gyrus are also compromised (arrowheads). C: The F98 glioma cells were implanted into the neocortex. Significant PI uptake can be observed in adjacent brain parenchyma (arrows), whereas no PI uptake is detectable in the entorhinal cortex and dentate gyrus compared with time-matched controls (A). D: Treatment with the NMDA receptor antagonist MK801 diminishes glioma-induced cell death in the dentate gyrus, but not in the vicinity of tumor infiltration. E: In contrast, treatment with the AMPA receptor antagonist GYKI 52466 diminishes glioma-induced cell death only in the entorhinal cortex and not in the hippocampus and dentate gyrus. F: Schematic drawing illustrating neuroanatomical connections in the organotypic brain slice culture assay. Red lines represent the lateral track of the perforant path; green lines represent the medial track. CA1, CA3 = pyramidal cell layers of the hippocampus; gcl = granule cell layer of the dentate gyrus. D: Bar graphs summarizing the PI uptake in the entorhinal and the dentate gyrus in slice cultures with or without F98 glioma implantation (all cell slice cohorts were kept 11 days in culture). Data are given as means ± standard deviations from nine slice cultures in each group. * p < 0.01; Student t-test. Scale bar = 100 µm (A–E).

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