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International audience; Aim: With the objective to investigate the mechanisms underlying absence of spontaneous axonal regeneration following spinal cord injury (SCI) we employ a multimodal label-free imaging approach to monitor glial scar in a mice SCI model. Method: To determine the relevant structural signature, and the nanobiomechanical behavior of healthy and injured spinal cord tissue we combine the non-linear, multiphoton microscopy (MPM) technique with force measurements via atomic force microscopy (AFM). The glial scar at different post lesion time-points is investigated with these two techniques to monitor structural and elasticity (Young modulus) changes of the tissue.Results: 2-photon excited fluorescence (2PEF) and second harmonic generation (SHG) signals of excised mice SC injured tissues were recorded in MPM at 72h, 1week and 6 weeks post-lesion. The MPM images revealed the apparition of a strong SHG signal at 1week post injury, due to the formation of fibrillar collagen fibers (collagen type I) by the injury site in the glial scar. At 6 weeks’post-injury, the SHG signal is more intense and a higher number of fibers are detected in average. We further assessed the preferential orientation of the collagen bundles performing polarization dependent measurements of the SHG signal. The AFM based force spectroscopy measurements have been performed at the same post-lesion time-points to map the elastic properties of the healthy (grey and white matters) and injured (lesion) parts in the spinal cord tissue. The results suggested an increase of the lesion area stiffness over time that could be correlated with the apparition of fibrillar collagen observed in MPM, indicating the presence of a fibrotic process seven days after injury, that develops in time. As tissue stiffness is a regulator of neuronal growth, such kind of measurements might help to understand why adult mammalian axons do not regenerate after an injury. Our next step is to investigate the effect of a treatment (pharmacological transient depletion of microglia/macrophage in mice that underwent SCI) on the structure and mechanical properties of the lesion site at 1 week and 6 weeks post injury.

International audience; Spinal cord injuries (SCI) affect between 2.5 and 4 million patients worldwide yielding major handicaps and inducing high economical costs. A scar, called glial scar and composed of two main cellular populations i.e. astrocytes and microglia, inhibits axonal regeneration by forming a physical and chemical barrier. Currently, there is no curative treatment on any symptoms associated with SCI. In this context, with the objective to investigate the mechanisms underlying absence of spontaneous axonal regeneration following SCI, we employ a multimodal label-free imaging approach to monitor glial scar in a mice SCI model. Method: To determine the relevant structural signature and the nanobiomechanical behavior of healthy and injured spinal cord tissue, we combine the non-linear, multiphoton microscopy (MPM) technique with force measurements via atomic force microscopy (AFM). The glial scar at different key post lesion time-points is investigated with these two techniques to monitor structural and elasticity (Young modulus) changes of the tissue.Results: 2-photon excited fluorescence (2PEF) and second harmonic generation (SHG) signals of excised mice SC injured tissues were recorded in MPM at 72h,1 week and 6 weeks post-lesion. The MPM images revealed the apparition of a strong SHG signal at 1week post injury, due to the formation of fibrillar collagen fibers (collagen type I) by the injury site in the glial scar. At 6 weeks post-injury, the SHG signal is more intense and a higher number of fibers are detected in average. We further assessed the preferential orientation of the collagen bundles performing polarization dependent measurements of the SHG signal. The AFM based force spectroscopy measurements have been performed at the same post-lesion time-points to map the elastic properties of the healthy (grey and white matters) and injured (lesion) parts in the spinal cord tissue. The results suggested an increase of the lesion area stiffness over time that could be correlated with the apparition of fibrillar collagen observed in MPM, indicating the presence of a fibrotic process seven days after injury, that develops in time. As tissue stiffness is a regulator of neuronal growth, such kind of measurements might help to understand why adult mammalian axons do not regenerate after an injury. Our next step is to investigate the effect of a treatment (pharmacological transient depletion of microglia/macrophage in mice that underwent SCI) on the structure and mechanical properties of the lesion site at 2 weeks and 6 weeks post injury.

International audience; Spinal cord injuries (SCI) affect between 2.5 and 4 million patients worldwide (40 000 in France). Handicaps induced by SCI range from minimal sensory motor deficits to complete tetraplegia. Currently, there is no curative treatment on any symptoms associated with SCI. A scar, called glial and composed mainly of astrocytes and microglia, appears at the the injury site and inhibits axonal regeneration. This mechanism is poorly understood. Better monitoring of animal models of SCI in particular through the development of bioimaging translational tools are therefore mandatory.Our main objectives are to pursue and extend a multimodal imaging approach combining multiphoton spectroscopy (MPM), Atomic Force Microscopy (AFM) and Broadband coherent anti-Stokes Raman Spectroscopy (BCARS), a novel technique [1] based on Raman spectroscopy with faster image acquisitions [2], to better understand mechanisms that underlie the glial scar formation and the subsequent absence of spontaneous axonal regeneration following SCI, as well as to evaluate outcomes of therapeutic strategies that favor axonal regeneration in mice and non-human primates. MPM, conceived to specifically reveal non centro-symmetric structures with second harmonic generation (SHG) [3], provided a first evidence of the presence of collagen fibers as a biomarker of the injury site. AFM showed significant differences in Young Modulus between healthy and injured areas. These results must be supplemented with a deeper collagen characterization and axons imaging using MPM, as well as a treatment monitoring by co-localizing drugs within healthy/diseased tissues using BCARS [4].[1]. C. H. Camp Jr, Y. J. Lee, J. M. Heddleston, C. M. Hartshorn, A. R. Hight Walker, J. N. Rich, J. D. Lathia, M. T. Cicerone, Nat. Photonics 2014, 8, 627.[2]. C. M. Hartshorn, Y. J. Lee, C. H. Camp Jr, Z. Liu, J. M. Heddleston, N. Canfield, T. A. Rhodes, A. R. Hight Walker, P. J. Marsac, M. T. Cicerone, Anal. Chem. 2013, 85, 8102[3]. L. Mostaço-Guidolin, N.L. Rosin, T.L. Hackett, Int. J. Mol. 2017, 18(8), 1772. [4]. C. Zhang, D. Zhang, J.-X. Cheng, Annu. Rev. Biomed. Eng. 2015, 17, 415. [4]. C. Zhang, D. Zhang, J.-X. Cheng, Annu. Rev. Biomed. Eng. 2015, 17, 415.

International audience; Aim: Our main objective is to implement a multimodal (bio)physical approach to decipher mechanisms that underlie absence of spontaneous axonal regeneration following spinal cord injury (SCI) in mice and nonhuman primates. Method: A multimodal imaging strategy is employed combining state-of-the-art non-linear microscopy techniques (multiphoton - MPM and BCARS microscopy) with force spectroscopy measurements via atomic force microscopy (AFM). This approach enables to assess the relevant structural, morphological and spectral signature as well as the nanobiomechanical behavior of healthy and injured spinal cord tissue at cellular level. Results: We have first imaged excised mice SC tissues in 2-photon excited fluorescence and second harmonic generation (SHG). These MPM studies revealed the apparition of a strong SHG signal due to the formation of collagen I fibers by the injury site in the glial scar. AFM based force spectroscopy measurements have been also performed to map the elastic properties of the healthy and injured spinal cord tissue. Therefore, MPM data could be correlated with Young modulus elasticity results, indicating the presence of a fibrotic process 7 days after injury. As tissue stiffness is a regulator of neuronal growth, such kind of measurements will enable to understand why adult mammalian axons do not regenerate spontaneously after injury. Our next step will be to use a novel, emerging imaging technology that is the broadband coherent anti-Stokes Raman scattering (BCARS) microscopy for fast, label-free, highly selective and specific screening of spinal cord injury and potentially of drugs uptake into excised tissues. Combination of BCARS and MPM will first enable to retrieve information on the biological state of the cells/tissues that may underly the absence of spontaneous axonal regeneration following SCI (Raman neuronal markers: myelin, acetylcholine neurotransmitter, glutamate; MPM markers: collagen, neural axons, mitochondria,…). Second, it will permit to further correlate modification of these structural, morphological and spectral signatures in the context of therapeutic strategies.

Summary: Anamniotes, rodents, and young humans maintain neural stem cells in the ependymal zone (EZ) around the central canal of the spinal cord, representing a possible endogenous source for repair in mammalian lesions. Cell diversity and genes specific for this region are ill defined. A cellular and molecular resource is provided here for the mouse and human EZ based on RNA profiling, immunostaining, and fluorescent transgenic mice. This uncovered the conserved expression of 1,200 genes including 120 transcription factors. Unexpectedly the EZ maintains an embryonic-like dorsal-ventral pattern of expression of spinal cord developmental transcription factors (ARX, FOXA2, MSX1, and PAX6). In mice, dorsal and ventral EZ cells express Vegfr3 and are derived from the embryonic roof and floor plates. The dorsal EZ expresses a high level of Bmp6 and Gdf10 genes and harbors a subpopulation of radial quiescent cells expressing MSX1 and ID4 transcription factors. : A niche of stem cells is present around the central canal of the adult spinal cord. A better description of cell diversity and genes expressed in this niche may help to use it to promote spinal cord regeneration after lesions. In this article, based on several techniques, Ghazale and colleagues provide a cellular and molecular resource for the adult human and mouse stem cell niches. Keywords: spinal cord, niche, neural stem cells, regionalization, ependyma, ependymal cells, radial glial cells, transcription factors, Msx1, roof plate, floor plate

International audience; Spinal cord injuries (SCI) are neuropathologies causing enormous physical and emotional anguish as well as irreversibly disabilities with great socio/economic burdens to our society. The availability of multiple mouse strains is important for studying the underlying pathophysiological response after SCI. Although strain differences have been shown to directly affect spontaneous functional recovery following incomplete SCI, its influence after complete lesion of the spinal cord is unclear. To study the influence of mouse strain on recovery after severe SCI, we first carried out behavioral analyses up to 6 weeks following complete transection of the spinal cord in mice with two different genetic backgrounds namely, C57BL/6 and Swiss Webster. Using immunohistochemistry, we then analyzed glial cell reactivity not only at different time-points after injury but also at different distances from the lesion epicenter. Behavioral assessments using CatWalk™ and open field analyses revealed increased mobility (measured using average speed) and differential forelimb gross sensory response in Swiss Webster compared to C57BL/6 mice after complete transection of the spinal cord. Comprehensive histological assessment revealed elevated microglia/macrophage reactivity and a moderate increase in astrogliosis in Swiss Webster that was associated with reduced microcavity formation and reduced lesion volume after spinal cord transection compared to C57BL/6 mice. Our results thus suggest that increased mobility correlates with enhanced gliosis and better tissue protection after complete transection of the spinal cord.

Central nervous system (CNS) injury has been observed to lead to microglia activation and monocytes infiltration at the lesion site. Ex vivo diffusion magnetic resonance imaging (diffusion MRI or DWI) allows detailed examination of CNS tissues, and recent advances in clearing procedures allow detailed imaging of fluorescent-labeled cells at high resolution. No study has yet combined ex vivo diffusion MRI and clearing procedures to establish a possible link between microglia/monocytes response and diffusion coefficient in the context of spinal cord injury (SCI). We carried out ex vivo MRI of the spinal cord at different time-points after spinal cord transection followed by tetrahydrofuran based clearing and examined the density and morphology of microglia/monocytes using two-photon microscopy. Quantitative analysis revealed an early marked increase in microglial/monocytes density that is associated with an increase in the extension of the lesion measured using diffusion MRI. Morphological examination of microglia/monocytes somata at the lesion site revealed a significant increase in their surface area and volume as early as 72 hours post-injury. Time-course analysis showed differential microglial/monocytes response rostral and caudal to the lesion site. Microglia/monocytes showed a decrease in reactivity over time caudal to the lesion site, but an increase was observed rostrally. Direct comparison of microglia/monocytes morphology, obtained through multiphoton, and the longitudinal apparent diffusion coefficient (ADC), measured with diffusion MRI, highlighted that axonal integrity does not correlate with the density of microglia/monocytes or their somata morphology. We emphasize that differential microglial/monocytes reactivity rostral and caudal to the lesion site may thus coincide, at least partially, with reported temporal differences in debris clearance. Our study demonstrates that the combination of ex vivo diffusion MRI and two-photon microscopy may be used to follow structural tissue alteration. Lesion extension coincides with microglia/monocytes density; however, a direct relationship between ADC and microglia/monocytes density and morphology was not observed. We highlighted a differential rostro-caudal microglia/monocytes reactivity that may correspond to a temporal difference in debris clearance and axonal integrity. Thus, potential therapeutic strategies targeting microglia/monocytes after SCI may need to be adjusted not only with the time after injury but also relative to the location to the lesion site.

International audience; Neurons have inherent competence to regrow following injury, although not spontaneously. Spinal cord injury (SCI) induces a pronounced neuroinflammation driven by resident microglia and infiltrating peripheral macrophages. Microglia are the first reactive glial population after SCI and participate in recruitment of monocyte-derived macrophages to the lesion site. Both positive and negative influence of microglia and macrophages on axonal regeneration had been reported after SCI, raising the issue whether their response depends on time post-lesion or different lesion severity. We analyzed molecular alterations in microglia at several time-points after different SCI severities using RNA-sequencing. We demonstrate that activation of microglia is time-dependent post-injury but is independent of lesion severity. Early transcriptomic response of microglia after SCI involves proliferation and neuroprotection, which is then switched to neuroinflammation at later stages. Moreover, SCI induces an autologous microglial expression of astrocytic markers with over 6% of microglia expressing glial fibrillary acidic protein and vimentin from as early as 72 h post-lesion and up to 6 weeks after injury. We also identified the potential involvement of DNA damage and in particular tumor suppressor genebreast cancer susceptibility gene 1(Brca1) in microglia after SCI. Finally, we established that BRCA1 protein is specifically expressed in non-human primate spinal microglia and is upregulated after SCI. Our data provide the first transcriptomic analysis of microglia at multiple stages after different SCI severities. Injury-induced microglia expression of astrocytic markers at RNA and protein levels demonstrates novel insights into microglia plasticity. Finally, increased microglia expression of BRCA1 in rodents and non-human primate model of SCI, suggests the involvement of oncogenic proteins after CNS lesion.

In the last decade, the number of emerging Flaviviruses described worldwide has increased considerably. Among them Zika virus (ZIKV) and Usutu virus (USUV) are African mosquitoborne viruses that recently emerged. Recently, ZIKV has been intensely studied due to major outbreaks associated with neonatal death and birth defects, as well as neurological symptoms. USUV pathogenesis remains largely unexplored, despite significant human and veterinary associated disorders. Circulation of USUV in Africa was documented more than 50 years ago, and it emerged in Europe two decades ago, causing massive bird mortality. More recently, USUV has been described to be associated with neurological disorders in humans such as encephalitis and meningoencephalitis, highlighting USUV as a potential health threat. The aim of this study was to evaluate the ability of USUV to infect neuronal cells. Our results indicate that USUV efficiently infects neurons, astrocytes, microglia and IPSc-derived human neuronal stem cells. When compared to ZIKV, USUV led to a higher infection rate, viral production, as well as stronger cell death and anti-viral response. Our results highlight the need to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence.

International audience

Background: There is growing evidence that microglia are key players in the pathological process of amyotrophic lateral sclerosis (ALS). It is suggested that microglia have a dual role in motoneurone degeneration through the release of both neuroprotective and neurotoxic factors. Results: To identify candidate genes that may be involved in ALS pathology we have analysed at early symptomatic age (P90), the molecular signature of microglia from the lumbar region of the spinal cord of hSOD1(G93A) mice, the most widely used animal model of ALS. We first identified unique hSOD1(G93A) microglia transcriptomic profile that, in addition to more classical processes such as chemotaxis and immune response, pointed toward the potential involvement of the tumour suppressor gene breast cancer susceptibility gene 1 (Brca1). Secondly, comparison with our previous data on hSOD1(G93A) motoneurone gene profile substantiated the putative contribution of Brca1 in ALS. Finally, we established that Brca1 protein is specifically expressed in human spinal microglia and is up-regulated in ALS patients. Conclusions: Overall, our data provide new insights into the pathogenic concept of a non-cell-autonomous disease and the involvement of microglia in ALS. Importantly, the identification of Brca1 as a novel microglial marker and as possible contributor in both human and animal model of ALS may represent a valid therapeutic target. Moreover, our data points toward novel research strategies such as investigating the role of oncogenic proteins in neurodegenerative diseases. We are grateful to ALS patients and their relatives that donate their tissues. We acknowledge the New York Brain Bank-The Taub Institute, Columbia University (NYBB). The hybridoma CD11b antibody developed by Timothy A. Springer was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology, Iowa city, IA 52242. We thank the iGE3Genomics Platform, University of Geneva Switzerland for their assistance in transcriptomic and qPCR analysis. This work was supported by the Spanish Government, Plan Nacional de I+D+I 2008-2011 and ISCIII-Subdireccion General de Evaluacion y Fomento de la investigacion (PI10/00709) [to FEP], the Government of the Basque Country grant (Proyectos de Investigacion Sanitaria and Fondo Comun de Cooperacion Aquitania-Euskadi) [to FEP], the "Fondation pour la Recherche Medicale" [to FEP] and the French Government, ANR-FNS grant, GliALS (No ANR-14-CE36-0009-01) [to FEP], the patient organisations "Demain Debout Aquitaine" [to YNG and HNN] and "Verticale" [to FEP and HNN].