In both multiple sclerosis and its model experimental autoimmune encephalomyelitis (EAE), the extent of resident microglia activation and infiltration of monocyte-derived cells to the CNS is positively correlated to tissue damage. To address the phenotype characterization of different cell subsets, their spatio-temporal distributions and contributions to disease development we induced EAE in Thy1-CFP//LysM-EGFP//CD11c-EYFP reporter mice. We combined high content flow cytometry, immunofluorescence and two-photon imaging in live mice and identified a stepwise program of inflammatory cells accumulation. First on day 10 after induction, EGFP⁺ neutrophils and monocytes invade the spinal cord parenchyma through the meninges rather than by extravasion. This event occurs just before axonal losses in the white matter. Once in the parenchyma, monocytes mature into EGFP⁺/EYFP⁺ monocyte-derived dendritic cells (moDCs) whose density is maximal on day 17 when the axonal degradation and clinical signs stabilize. Meanwhile, microglia is progressively activated in the grey matter and subsequently recruited to plaques to phagocyte axon debris. LysM-EGFP//CD11c-EYFP mice appear as a powerful tool to differentiate moDCs from macrophages and to study the dynamics of immune cell maturation and phenotypic evolution in EAE.

Inflammatory cells, an integral component of tumor evolution, are present in Glioblastomas multiforme (GBM). To address the cellular basis and dynamics of the inflammatory microenvironment in GBM, we established an orthotopic syngenic model by grafting GL261-DsRed cells in immunocompetent transgenic LysM-EGFP//CD11c-EYFP reporter mice. We combined dynamic spectral two-photon imaging with multiparametric cytometry and multicolor immunostaining to characterize spatio-temporal distribution, morphology and activity of microglia and blood-derived infiltrating myeloid cells in live mice. Early stages of tumor development were dominated by microglial EYFP⁺ cells invading the tumor, followed by massive recruitment of circulating LysM-EGFP⁺ cells. Fluorescent invading cells were conventional XCR1⁺ and monocyte-derived dendritic cells distributed in subpopulations of different maturation stages, located in different areas relative to the tumor core. The lethal stage of the disease was characterized by the progressive accumulation of EGFP⁺/EYFP⁺ monocyte-derived dendritic cells. This local phenotypic regulation of monocyte subtypes marked a transition in the immune response.

The majority of intravital studies on brain tumor in living animal so far rely on dual color imaging. We describe here a multiphoton imaging protocol to dynamically characterize the interactions between six cellular components in a living mouse. We applied this methodology to a clinically relevant glioblastoma multiforme (GBM) model designed in reporter mice with targeted cell populations labeled by fluorescent proteins of different colors. This model permitted us to make non-invasive longitudinal and multi-scale observations of cell-to-cell interactions. We provide examples of such 5D (x,y,z,t,color) images acquired on a daily basis from volumes of interest, covering most of the mouse parietal cortex at subcellular resolution. Spectral deconvolution allowed us to accurately separate of each cell population as well as some components of the extracellular matrix. The technique represents a powerful tool for investigating how tumor progression is influenced by the interactions of tumor cells with host cells and the extracellular matrix micro-environment. It will be especially valuable for evaluating neuro-oncological drug efficacy and target specificity. The imaging protocol provided here can be easily translated to other mouse models of neuropathologies, and should also be of fundamental interest for investigations in other areas of systems biology.

The spatiotemporal and longitudinal monitoring of cellular processes occurring in tumors is critical for oncological research. We focused on glioblastoma multiforme (GBM), an untreatable highly vascularized brain tumor whose progression is thought to critically depend on the oxygen and metabolites supplied by blood vessels. We optimized protocols for orthotopic GBM grafting in mice that were able to recapitulate the biophysical constraints normally governing tumor progression and were suitable for intravital multiphoton microscopy. We repeatedly imaged tumor cells and blood vessels during GBM development. We established methods for quantitative correlative analyses of dynamic imaging data over wide fields in order to cover the entire tumor. We searched whether correlations existed between blood vessel density, tumor cell density and proliferation in control tumors. Extensive vascular remodeling and the formation of new vessels accompanied U87 tumor cell growth, but no strong correlation was found between local cell density and the extent of local blood vessel density irrespective of the tumor area or time points. The technique moreover proves useful for comparative analysis of mice subjected either to Bevacizumab anti-angiogenic treatment that targets VEGF or to AMD3100, an antagonist of CXCR4 receptor. Bevacizumab treatment massively reduced tumoral vessel densities but only transiently reduced U87 tumor growth rate. Again, there was no correlation between local blood vessel density and local cell density. Moreover, Bev applied only prior to tumor implantation inhibited tumor growth to the same extent as post-grafting treatment. AMD3100 achieved a potent inhibition of tumor growth without significant reduction in blood vessel density. These results indicate that in the brain, in this model, tumor growth can be sustained without an increase in blood vessel density and suggest that GBM growth is rather governed by stromal properties.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the...

Demyelination and axon degeneration are the central events in all neurodegenerative diseases, including multiple sclerosis. Intoxication of oligodendrocytes with lysophosphatidylcholine (LPC) is often used as a selective model of focal and reversible demyelination thought to have no incidence for neurons. To characterize the cascade of cellular events involved in LPC induced demyelination, we have combined intravital Coherent anti-stoke Raman scattering microscopy with intravital two-photon fluorescence microscopy in multicolor transgenic reporter mice. Moreover, taking advantage of a unique technique of spinal glass window implantation, we here provide the first longitudinal description of cell dynamics in the same volume of interest over weeks after insults. We have detected several patterns of axon-myelin interactions and classified them in early and advanced events. Unexpectedly, we have found that oligodendrocyte damages in fact lead to axon degeneration within 2 days after LPC incubation and this degeneration is amplified after the recruitment of the peripheral inflammatory cells at day 4. Beyond day 7, the recovery of axon number and myelin takes 3 more weeks post lesion and involves a new wave of innate immune cells at day 14. Therefore, recurrent imaging over several weeks suggests an important role of peripheral immune cells in regulating both the axonal and oligodendroglial fates, and thereby the remyelination status. Better understanding the recruitment of peripheral immune cells during demyelinating events should help to improve diagnosis and therapy.