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Insights into cell-to-cell and cell-to-blood-vessel communications in the brain: in vivo multiphoton microscopy

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Abstract

A complex and reciprocal communication of cells with each other and with relevant parts of the tissue stroma governs many biological processes in both health and disease. However, in the past, the study of these anatomical and molecular interactions has suffered from a lack of appropriate experimental models. An imaging methodology aimed at changing this should allow intravital display and quantification in an intact non-traumatized organ, imaging over a wide range of time spans including extended periods (i.e., months), many repetitive measurements of the same cell or area to permit the study of the cause and consequence of biological processes, the display of various cell types and their reciprocal interaction with each other in three dimensions, the co-registration of relevant physiological parameters and reporters for selected molecular pathways and as high as possible resolution to visualize sub-cellular structures such as organelles. Remarkably, intravital multiphoton microscopy (in vivo MPLSM) through a chronic cranial window allows us to do all these things, making the brain the inner organ of choice for this technology. Here, we give an overview of the application of in vivo MPLSM to study the choreography of cellular, vascular and molecular interactions in the healthy brain and in neurological diseases. We focus on brain tumor formation, progression and response to therapies. This review further aims at demonstrating that we stand at the beginning of full exploitation of the opportunities provided by this technology and gives clues to future directions that appear most promising.

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Correspondence to Frank Winkler.

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Timecourse (1 h 5 min) of the dynamic microglial reaction after laser-induced cell death of a single microglial cell; 40 subsequent z-stacks of 36 slices, scaling 1.5 μm, are shown in 3D view over time. M. Osswald, unpublished. Bar 15 μm. For video sequence, see Supplementary video 1. (AVI 1212 kb)

Three-dimensional reconstruction and simulation (Imaris Bitplane) of a melanoma brain metastasis (green cerebral blood vessels, red melanoma cells); 500×500×400 μm. M. Osswald, unpublished. For video sequence, see Supplementary video 3. (AVI 63296 kb)

Micro- and astroglial cells can be tracked over time (26 min). Whereas the astrocytes remain in a stable network with stable protrusions foming one part of the blood-brain barrier, the microglia patrol the shown brain region. M. Osswald, unpublished. Bar 15 μm. For video sequence, see Supplementary video 4. (AVI 1210 kb)

Imaging of tumor cell migration and proliferation in vivo. Lewis lung cancer cells were double-labeled with dsRed (cytoplasm) and GFP (nuclei, H2B). Because of the labeling of the nuclei, single cells can easily be tracked over time. A mitosis can also be seen. M. Osswald, unpublished. Bar 30 μm. (AVI 545 kb)

Glioma-blood-vessel interaction. Video (10 min) of a 3D stack showing a glioma cell (green), which ties up a twisted and angiogenic deformed blood vessel (red). The black discs that can be seen in the blood vessel are erythrocytes and show a highly variable intravascular velocity, depending on the vascular constriction of the tumor cell. M. Osswald, unpublished. Bar 15 μm. (AVI 1091 kb)

Supplementary video 1

Timecourse (1 h 5 min) of the dynamic microglial reaction after laser-induced cell death of a single microglial cell; 40 subsequent z-stacks of 36 slices, scaling 1.5 μm, are shown in 3D view over time. M. Osswald, unpublished. Bar 15 μm. For video sequence, see Supplementary video 1. (AVI 1212 kb)

Supplementary video 2

Three-dimensional reconstruction and simulation (Imaris Bitplane) of a melanoma brain metastasis (green cerebral blood vessels, red melanoma cells); 500×500×400 μm. M. Osswald, unpublished. For video sequence, see Supplementary video 3. (AVI 63296 kb)

Supplementary video 3

Micro- and astroglial cells can be tracked over time (26 min). Whereas the astrocytes remain in a stable network with stable protrusions foming one part of the blood-brain barrier, the microglia patrol the shown brain region. M. Osswald, unpublished. Bar 15 μm. For video sequence, see Supplementary video 4. (AVI 1210 kb)

Supplementary video 4

Astrocytic calcium imaging by cortical application of fluo-4 AM. Changes in intracellular [Ca] are reflected by the change in fluorescent intensity. Thin astrocytic processes can be seen during astrocytic calcium waves (the first three waves from the left evolving to the right side, one wave from the right evolving to the left side). M. Osswald, unpublished. (AVI 5033 kb)

Supplementary video 5

Imaging of tumor cell migration and proliferation in vivo. Lewis lung cancer cells were double-labeled with dsRed (cytoplasm) and GFP (nuclei, H2B). Because of the labeling of the nuclei, single cells can easily be tracked over time. A mitosis can also be seen. M. Osswald, unpublished. Bar 30 μm. (AVI 545 kb)

Supplementary video 6

Glioma-blood-vessel interaction. Video (10 min) of a 3D stack showing a glioma cell (green), which ties up a twisted and angiogenic deformed blood vessel (red). The black discs that can be seen in the blood vessel are erythrocytes and show a highly variable intravascular velocity, depending on the vascular constriction of the tumor cell. M. Osswald, unpublished. Bar 15 μm. (AVI 1091 kb)

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Osswald, M., Winkler, F. Insights into cell-to-cell and cell-to-blood-vessel communications in the brain: in vivo multiphoton microscopy. Cell Tissue Res 352, 149–159 (2013). https://doi.org/10.1007/s00441-013-1580-3

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Keywords

  • Multiphoton microscopy
  • Two-photon microscopy
  • Cancer
  • Neuroscience
  • Brain