Franck Debarbieux

Quantitative analysis by in vivo imaging of the dynamics of vascular and axonal networks in injured mouse spinal cord

Understanding the endogenous repair capacity of spinal cord is pivotal to develop strategies to improve it. Here we design a paradigm of spinal cord lesion in the dorsal column using a 2-photon microscopy technique to dynamically and chronically monitor simultaneous changes of vascular and axonal networks in living mice up to 4 months postinjury. High-resolution images showed that early explorative sprouting of surviving injured axons resulted in extensive regrowth until and past the lesion site within 2 months. Blood vessel density was transiently up-regulated and most neurovascular interactions occurred within 2 weeks. Time-lapse analysis showed that neovessels exerted a potent growth stimulating action, but no guidance effect on neighboring sprouts, possibly because of their geometry and plasticity. Nevertheless, if reconnection depends on axon sprout density, stimulation of angiogenesis would probably be beneficial to repair. More generally, this imaging approach is showing promise to aid in monitoring brain diseases and the efficacy of potential treatments.

Long‐term in vivo imaging of normal and pathological mouse spinal cord with subcellular resolution using implanted glass windows

•  Chronic in vivo imaging of cellular interactions within the adult spinal cord with subcellular resolution is important for understanding cellular physiology and disease progression. •  Previous approaches for chronic in vivo spinal cord microscopy have required surgery for each imaging session. •  Here we describe a novel method for implanting glass windows over the exposed spinal cords of adult mice for repeated in vivo microscopy. •  We show that the windows remain clear for many months after implantation, do not damage axons or blood vessels, and are useful for studying cellular dynamics after spinal cord injury. •  Our method represents an original technical breakthrough for scientists involved in spinal cord research and in vivo imaging, and is a useful tool for studying cellular physiology and disease progression.

Chemical optimization of new ligands of the low-density lipoprotein receptor as potential vectors for central nervous system targeting.

Drug delivery to the central nervous system is hindered by the presence of physiological barriers such as the blood-brain barrier. To accomplish the task of nutrient transport, the brain endothelium is endowed with various transport systems, including receptor-mediated transcytosis (RMT). This system can be used to shuttle therapeutics into the central nervous system (CNS) in a noninvasive manner. Therefore, the low-density lipoprotein receptor (LDLR) is a relevant target for delivering drugs. From an initial phage display biopanning, a series of peptide ligands for the LDLR was optimized leading to size reduction and improved receptor binding affinity with the identification of peptide 22 and its analogues. Further real-time biphoton microscopy experiments on living mice demonstrated the ability of peptide 22 to efficiently and quickly cross CNS physiological barriers. This validation of peptide 22 led us to explore its binding on the extracellular LDLR domain from an NMR-oriented structural study and docking experiments.

Fibronectin expression in glioblastomas promotes cell cohesion, collective invasion of basement membrane in vitro and orthotopic tumor growth in mice

Glioblastoma multiforme (GBM) are highly invasive and angiogenic malignancies with a median survival time from diagnosis of <15 months. Previous work has revealed robust overexpression of fibronectin (FN) mRNA in GBM, although immunohistochemical staining of FN in these tumors is typically associated with the angiogenic vasculature. Here we sought to examine the expression of tumor cell FN and address its possible involvement in the invasive phenotype of GBM. We found that FN was expressed and assembled into fibrillar arrays in human tumors and in established GBM lines. Cultured cells spontaneously formed dense cellular networks and spheroid-like domes. Depletion of FN by targeted-short hairpin RNA expression disrupted matrix assembly and multicellular network organization by exerting profound effects on cell adhesion and motility. Although FN depletion enhanced persistent directional migration of single cells, it compromised collective invasion of spheroids through a laminin-rich matrix and sensitized cells to ionizing radiation. In orthotopic grafts, FN depletion significantly reduced tumor growth and angiogenesis. Together our results show that FN produced by the tumor cells has a role in GBM pathophysiology and they provide insights into the implications that targeting FN interactions may have for combating this dreaded disease.

Six-color intravital two-photon imaging of brain tumors and their dynamic microenvironment

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 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.

Long- and short-term intravital imaging reveals differential spatiotemporal recruitment and function of myelomonocytic cells after spinal cord injury.

•  Inflammatory cells such as myelomonocytic cells are key players in the progression and recovery from spinal cord injury (SCI). •  However, the precise spatiotemporal distributions and roles of different subpopulations of myelomonocytic cells remain unclear in part because their dynamics have not been examined in vivo. •  Using chronic in vivo two‐photon microscopy techniques in adult transgenic mice with SCI, we show that infiltrating and resident myelomonocytic cells have differential spatiotemporal distribution patterns to injury sites. •  We also show that infiltrating myelomonocytic cells are associated with the collapse of certain cut axon terminals thus potentially impeding recovery, whereas resident myelomonocytic cells clear axon debris, which may be important for axon regrowth and recovery. •  These results set a framework to understand the roles of different subpopulations of myelomonocytic cells in SCI, and may be important for the development of therapies that target specific immune cell populations at precise times post‐injury.

Dynamic Quantitative Intravital Imaging of Glioblastoma Progression Reveals a Lack of Correlation between Tumor Growth and Blood Vessel Density

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.

First K-Edge Imaging With a Micro-CT Based on the XPAD3 Hybrid Pixel Detector

We investigate the capability to perform K-edge imaging with the newly developed micro-CT PIXSCAN based on the XPAD3 hybrid pixel detector. The XPAD3 detector surface of 8 cm ×11 cm makes it possible to perform whole body mouse imaging. We present a proof of principle of K-edge imaging of mouse-size phantoms filled with Silver and Iodine solutions. Results are compared with standard X-ray absorption tomography for several solution densities.

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment. Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

A delay between motor cortex lesions and neuronal transplantation enhances graft integration and improves repair and recovery

We previously reported that embryonic motor cortical neurons transplanted immediately after lesions in the adult mouse motor cortex restored damaged motor cortical pathways. A critical barrier hindering the application of transplantation strategies for a wide range of traumatic injuries is the determination of a suitable time window for therapeutic intervention. Here, we report that a 1 week delay between the lesion and transplantation significantly enhances graft vascularization, survival, and proliferation of grafted cells. More importantly, the delay dramatically increases the density of projections developed by grafted neurons and improves functional repair and recovery as assessed by intravital dynamic imaging and behavioral tests. These findings open new avenues in cell transplantation strategies as they indicate successful brain repair may occur following delayed transplantation. SIGNIFICANCE STATEMENT Cell transplantation represents a promising therapy for cortical trauma. We previously reported that embryonic motor cortical neurons transplanted immediately after lesions in the adult mouse motor cortex restored damaged cortical pathways. A critical barrier hindering the application of transplantation strategies for a wide range of traumatic injuries is the determination of a suitable time window for therapeutic intervention. We demonstrate that a 1 week delay between the lesion and transplantation significantly enhances graft vascularization, survival, proliferation, and the density of the projections developed by grafted neurons. More importantly, the delay has a beneficial impact on functional repair and recovery. These results impact the effectiveness of transplantation strategies in a wide range of traumatic injuries for which therapeutic intervention is not immediately feasible.