Development of new preclinical imaging modalities to study the dynamics of cellular interactions in rodent models of CNS pathology. – PathoVisu3Dyn

In order to be able to concomittently visualize the nervous cells, the immune cells as well as the blood-vessels in murine models of pathologies of the central nervous system, we set up two in vivo imaging strategies with various scales of space resolution: tomodensitometry (TDM) with X-ray and 2-photons (2P) spectral microscopy. 2-P in vivo combined with the use of transgenic mice whose various cellular populations are marked with different fluorophores/colors, makes it possible to carry out an unlimited number of observations of the same field of vision. This opens up the way for a quantitative and correlative analysis of the distribution as well as the cellular interactions in a truly physiological environment. In collaboration with physicists, we validated a technique of spectral microtomodensitometry (spectral micro-CT scanner), based on a new generation of detectors with hybrid pixels (HPD), and made the proof of the interest of our patent on composite pixels to perform multicolor imaging on living animal. Although its resolution is lesser than microscopy 2P, it presents the complementary advantage to allow to explore the body in entirety and to be directly transferable to clinical applications.

We (1) produced the fluorescent transgenic mice and established three models of pathologies (glioblastoma, lesion of spinal-cord, multiple sclerosis) compatible with our imaging modalities (2) adapted the biphotonic microscope and the methodology for a micrometric repositioning of the mouse. We are able to simultaneously follow 5 cellular populations on the same animal during several months.
(3) described in space and time the interactions of neural cells with angiogenesis in the context of the glioblastomas and spinal cord lesions. In an unexpected way, the tumoral growth is not directly correlated with the density of blood-vessels and the transitory effect of anti-angiogenic therapies is likely due to their actions on the microenvironment. In the spinal cord lesions, the regeneration of the axons always occurs in the vicinity of angiogenic vessels.
4) established collaborations with clinicians and immunologists interested by the new capabilities of our imaging techniques. The patent on Imaging by X-rays with polychromatic source of which I am Co-inventor is exploited by the ImXPAD company.

In line with the results that we obtained, my interest focusses more specifically on the interactions betwenn nervous, vascular and immune systems. The specific objective is to identify the phenotype of the cell populations playing a pivotal role in amplification or remission of functional deficits associated to our models of pathologies in order to propose means to manipulate these population pharmacologically in predefined time-windows. Finally, the technological and methodological outcomes of this project are prone to knowledge transfer toward industry. This transfer has already been started through strong interactions with R&D departments of major companies in microscopy and laser science, through the expression of collaborative interest by pharmaceutical biotech or through the creation of the start-up company ImXPAD.

16 international publications internationales among which:
Fenrich KK, Weber P, Hocine M, Zalc M, Rougon G, Debarbieux F. (2012) Long-term in vivo imaging of normal and pathological mouse spinal cord with subcellular resolution using implanted glass windows. J Physiol.; 590:3665-75.
Fenrich K.K., Weber P., Rougon G, Debarbieux F. (2013) Long and short term intravital imaging reveals differential spatiotemporal recruitment and function of myelomonocytic cells after spinal cord injury J Physiol. 591: 4895-4902
Ricard C., Stanchi F., Rodriguez T., Amoureux M.C., Rougon G., Debarbieux F. (2013) Dynamic quantitative intravital imaging of glioblastoma progression reveals a lack of correlation between tumor growth and blood vessel density PLoS One 8(9):e72655;
Cassol Brunner F., Dupont M., Meessen C., Bouriser Y., Ouamara H., Bonissent A., Kronland-Martinet C., Clemens J.C., Debarbieux F., Morel C. (2013) First K-edge imaging with a micro-CT based on the XPAD3 hybrid pixel detector. , IEEE Trans. Nucl. Sci. 60 (1), pp.103-108 ISSN: 0018-9499
Ricard C., Debarbieux F. (2014) Six-color intravital two-photon imaging of brain tumors and their dynamic microenvironment. Frontiers in Cellular Neuroscience. ISSN: 1662-5102

This is a multi-disciplinary project focussed on the experimental and clinical problems associated with damage (spinal cord injury) and tumors (glioblastoma) in the central nervous system (CNS). A strong emphasis is placed on the development and the validation of two complementary advanced imaging modalities, namely two photon (2P) in vivo imaging and X-ray computed tomography (CT-scan), combined to the use of transgenic mouse lines engineered to express fluorescent cell populations and contrast agents of distinct spectroscopic signatures.

Context:
I recently set up 2P microscopy at IBDML to perform non invasive and repeated imaging in the CNS of living transgenic fluorescent mice and developed two models of CNS pathologies whose evolution could be followed optically over several months on the same animal: a microlesion of DRG axons in spinal cord of green fluorescent Thy1-GFP-M mice and an orthotopic glioblastoma (GBM) model where green GFP transfected human U87 GBM cells are grafted into the cortex. For both models, I showed that vasculature could be highlighted by intravenous injection of red dextrans, allowing to simultaneously image interaction kinetics between tumor cells (Stanchi et al., 2009 and unpublished) or severed axons degeneration/regeneration (Dray et al., 2009). To overcome 2P technical limitations, I participated together with Centre de Physique des Particules (CPPM) pysicists to the development and validation of a prototype of X-ray CT-scanner based on hybrid pixel detectors (Delpierre et al., 2007, Debarbieux et al., 2010).

Project:
(1) One of the goal of the present project is to up-grade our existing imaging systems and protocols towards multicolor approaches. For 2P, by crossing available monocolor mouse lines we will generate and image “multicolor” homozygous mice. These will have blue neurons/green macrophages/red vessels to study the spinal cord injury model or blue vessels/green tumors cells/red immune cells to study the GBM model. For CT scan, we will take advantage of hybrid pixel detector ability to discriminate photons based on their energy. We will first establish Digitial Subtraction Angiography with iodine then select a set of contrast agents whose spectroscopic signatures are sufficiently distinct to allow discrimination when simultaneously present in a mouse body.

(2) A second major goal, which can be attained independently of the previous one, is to assess quantitatively over time and space the interactions occurring between lesioned neurons or tumor cells and the vascular and immune systems. For the spinal cord, we will complement our observations by describing the dynamics of spontaneous cellular interactions between severed axons, angiogenic blood vessels and macrophages over several weeks. For GBM, we will describe in real time how vascular remodeling support tumor cell proliferation and migration and how immune system contributes to slow the disease progression until it gets defeated. The exact same regions in the same animals will be explored using both imaging modalities. Furthermore, the CT scan contrasts will give clues on areas of the body that cannot be explored by 2P microscopy.

(3) Angiogenesis and inflammation could have detrimental or beneficial effects depending of the pathology considered or its state of evolution. The third goal is to use the technology, animal models developed and knowledge acquired in the previous goals to examine in both pathology models the effects of pharmacological approaches preventing or enhancing angiogenesis and inflammation. For angiogenesis, we will either deliver Vascular Endothelial Factor (VEGF) or VEGF blocking antibody. For inflammation, we will trigger it with LPS or administrate anti-inflammatory drugs. These experiments should be seen as a first step toward the qualification of combined therapy targeting both angiogenesis and inflammation for SCI and GBM patients.