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The extension of the lesion following spinal cord injury (SCI) poses a major challenge for regenerating axons, which must grow across several centimetres of damaged tissue in the absence of ordered guidance cues. Biofunctionalized electroconducting microfibres (MFs) that provide biochemical signals, as well as electrical and mechanical cues, offer a promising therapeutic approach to help axons overcome this blind journey. We used poly(3,4-ethylenedioxythiophene)-coated carbon MFs functionalized with cell adhesion molecules and growth factors to bridge the spinal cord after a partial unilateral dorsal quadrant lesion (PUDQL) in mice and followed cellular responses by intravital two-photon (2P) imaging through a spinal glass window. Thy1-CFP//LysM-EGFP//CD11c-EYFP triple transgenic reporter animals allowed real time simultaneous monitoring of axons, myeloid cells and microglial cells in the vicinity of the implanted MFs. MF biocompatibility was confirmed by the absence of inflammatory storm after implantation. We found that the sprouting of sensory axons was significantly accelerated by the implantation of functionalized MFs after PUDQL. Their implantation produced better axon alignment compared to random and misrouted axon regeneration that occurred in the absence of MF, with a most striking effect occurring two months after injury. Importantly, we observed differences in the intensity and composition of the innate immune response in comparison to PUDQL-only animals. A significant decrease of immune cell density was found in MF-implanted mice one month after lesion along with a higher ratio of monocyte-derived dendritic cells whose differentiation was accelerated. Therefore, functionalized carbon MFs promote the beneficial immune responses required for neural tissue repair, providing an encouraging strategy for SCI management.

Leukocyte infiltration into the central nervous system (CNS) is a key pathological feature in multiple sclerosis (MS) and the MS animal model experimental autoimmune encephalomyelitis (EAE). Recently, preventing leukocyte influx into the CNS of MS patients is the main target of MS therapies and insight into cell behaviour in the circulation is needed for further elucidation of such therapies. In this study, we aimed at in vivo visualization of monocytes in a time-dependent manner during EAE. Using intravital two-photon microscopy (IVM), we imaged CX3CR1gfp/gfp mice during EAE, visualizing CX3CR1-GFP+ monocytes and their dynamics in the spinal cord vasculature. Our observations showed that intraluminal crawling of CX3CR1-GFP+ monocytes increased even before the clinical onset of EAE due to immunization of the animals. Furthermore, intraluminal crawling remained elevated during ongoing clinical disease. Besides, the displacement of these cells was larger during the peak of EAE compared to the control animals. In addition, we showed that the enzyme tissue transglutaminase (TG2), which is present in CNS-infiltrated cells in MS patients, is likewise found in CX3CR1-GFP+ monocytes in the spinal cord lesions and at the luminal side of the vasculature during EAE. It might thereby contribute to adhesion and crawling of monocytes, facilitating extravasation into the CNS. Thus, we put forward that interference with monocyte adhesion, by e.g. inhibition of TG2, should be applied at a very early stage of EAE and possibly MS, to effectively combat subsequent pathology. Electronic supplementary material The online version of this article (doi:10.1007/s00726-016-2359-0) contains supplementary material, which is available to authorized users.

Myelin around axons is currently widely studied by structural analyses and large-scale imaging techniques, with the goal to decipher its critical role in neuronal protection. Although there is strong evidence that in myelin, lipid composition, and lipid membrane morphology are affected during the progression of neurodegenerative diseases, there is no quantitative method yet to report its ultrastructure in tissues at both molecular and macroscopic levels, in conditions potentially compatible with in vivo observations. In this work, we study and quantify the molecular order of lipids in myelin at subdiffraction scales, using label-free polarization-resolved coherent anti-Stokes Raman, which exploits coherent anti-Stokes Raman sensitivity to coupling between light polarization and oriented molecular vibrational bonds. Importantly, the method does not use any a priori parameters in the sample such as lipid type, orientational organization, and composition. We show that lipid molecular order of myelin in the mouse spinal cord is significantly reduced throughout the progression of experimental autoimmune encephalomyelitis, a model for multiple sclerosis, even in myelin regions that appear morphologically unaffected. This technique permits us to unravel molecular-scale perturbations of lipid layers at an early stage of the demyelination progression, whereas the membrane architecture at the mesoscopic scale (here ∼100 nm) seems much less affected. Such information cannot be brought by pure morphological observation and, to our knowledge, brings a new perspective to molecular-scale understanding of neurodegenerative diseases.

After spinal cord injury (SCI), resident and peripheral myelomonocytic cells are recruited to the injury site and play a role in injury progression. These cells are important for clearing cellular debris, and can modulate the retraction and growth of axons in vitro. However, their precise spatiotemporal recruitment dynamics is unknown, and their respective roles after SCI remain heavily debated. Using chronic, quantitative intravital two-photon microscopy of adult mice with SCI, here we show that infiltrating lysozyme M (LysM(+)) and resident CD11c(+) myelomonocytic cells have distinct spatiotemporal recruitment profiles, and exhibit changes in morphology, motility, phagocytic activity and axon interaction patterns over time. This study provides the first in vivo description of the influx of inflammatory and resident myelomonocytic cells into the injured spinal cord and their interactions with cut axons, and underscores the importance of precise timing and targeting of specific cell populations in developing therapies for SCI.

Myelin around axons is currently widely studied by structural analyses and large scale imaging techniques, with the goal to decipher its critical role in neuronal protection. While there is strong evidence that in myelin, lipid composition and lipid membrane morphology are affected during the progression of neurodegenerative diseases, there is no quantitative method yet to report its ultrastructure in tissues at both molecular and macroscopic levels, in conditions potentially compatible within vivoobservations. In this work, we study and quantify molecular order of lipids in myelin at sub-diffraction scales, using label-free polarization resolved Coherent Anti Stokes Raman (PR-CARS), which exploits CARS sensitivity to coupling between light polarization and oriented molecular vibrational bonds. Importantly, the method does not use anya prioriparameters in the sample such as lipid type, orientational organization and composition. We show that lipid molecular order of myelin in the mouse spinal cord is significantly reduced throughout the progression of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, even in myelin regions that appear morphologically unaffected. This technique permits to unravel molecular-scale perturbations of lipid layers at early stage of the demyelination progression, while the membrane architecture at the mesoscopic scale (here about 100 nm) seems much less affected. Such information cannot be brought by pure morphological observation and opens new prospectives towards molecular-scale understanding of neurodegenerative diseases.

International audience; Purpose of review: Highly coordinated cellular interactions occur in the healthy or pathologic adult rodent central nervous system (CNS). Until recently, technical challenges have restricted the analysis of these events to largely static modes of study such as immuno-fluorescence and electron microscopy on fixed tissues. The development of intravital imaging with subcellular resolution is required to probe the dynamics of these events in their natural context, the living brain. Recent findings: This review focuses on the recently developed live non-linear optical imaging modalities, the core principles involved, the identified technical challenges that limit their use and the scope of their applications. We highlight some practical applications for these modalities with a specific attention given to Experimental Autoimmune Encephalomyelitis (EAE), a rodent model of a chronic inflammatory disease of the CNS characterized by the formation of disseminated demyelinating lesions accompanied by axonal degeneration. Summary: We conclude that label-free nonlinear optical imaging combined to two photon imaging will continue to contribute richly to comprehend brain function and pathogenesis and to develop effective therapeutic strategies.

International audience; Neuroinflammation demands a comprehensive appraisal in situ to gain in-depth knowledge on the roles of particular cells and molecules and their potential roles in therapy. Because of the lack of appropriate tools, direct visualization of cells has been poorly investigated up to the present. In this context, reporter mice expressing cell-specific fluorescent proteins, combined with multiphoton microscopy, provide a window into cellular processes in living animals. In addition, the ability to collect multiple fluorescent colors from the same sample makes in vivo microscopy uniquely useful for characterizing many parameters from the same area, supporting powerful correlative analyses. Here, we present an overview of the advantages and limitations of this approach, with the purpose of providing insight into the neuroinflammation field. We also provide a review of existing fluorescent mouse models and describe how these models have been used in studies of neuroinflammation. Finally, the potential for developing advanced genetic tools and imaging resources is discussed. ⃝C 2016 by John Wiley & Sons, Inc.

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.

Key points • 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. Abstract Repeated in vivo two-photon imaging of adult mammalian spinal cords, with subcellular resolution, would be crucial for understanding cellular mechanisms under normal and pathological conditions. Current methods are limited because they require surgery for each imaging session. Here we report a simple glass window methodology avoiding repeated surgical procedures and subsequent inflammation. We applied this strategy to follow axon integrity and the inflammatory response over months by multicolour imaging of adult transgenic mice. We found that glass windows have no significant effect on axon number or structure, cause a transient inflammatory response, and dramatically increase the throughput of in vivo spinal imaging. Moreover, we used this technique to track retraction/degeneration and regeneration of cut axons after a ‘pin-prick’ spinal cord injury with high temporal fidelity. We showed that regenerating axons can cross an injury site within 4 days and that their terminals undergo dramatic morphological changes for weeks after injury. Overall the technique can potentially be adapted to evaluate cellular functions and therapeutic strategies in the normal and diseased spinal cord.

The CHiPSpeCT consortium aims at building a large multi-modules CdTe based photon counting detector for hard X-ray applications. For this purpose, we tested nine XPAD3.2 single chip hybrids in various configurations ( i.e. Ohmic vs. Schottky contacts or electrons vs. holes collection mode) in order to select the most performing and best suited configuration for our experimental requirements. Measurements have been done using both X-ray synchrotron beams and 241 Am source. Preliminary results on the image quality, calibration, stability, homogeneity and linearity of the different types of detectors are presented.

International audience; 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.

Simultaneous imaging of different cell types and structures in the mouse central nervous system (CNS) by intravital two-photon microscopy requires the characterization of fluorophores and advances in approaches to visualize them. We describe the use of a two-photon infrared illumination generated by an optical parametric oscillator (OPO) on quantum-dots 655 (QD655) nanocrystals to improve resolution of the vasculature deeper in the mouse brain both in healthy and pathological conditions. Moreover, QD655 signal can be unmixed from the DsRed2, CFP, EGFP and EYFP fluorescent proteins, which enhances the panel of multi-parametric correlative investigations both in the cortex and the spinal cord.

International audience; 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.

International audience; Hybrid pixel detectors represent a new generation of digital X-ray detectors for which photon counting replaces charge integration as used within standard digital radiography systems. This novel approach involves several advantages, among which the absence of dark noise and the ability to set an energy threshold for the detected photons are of uppermost importance for the development of new X-ray imaging approaches that will exploit spectral information on the detected X-rays. We investigate the capability to perform K-edge imaging by using the newly developed XPAD3 hybrid pixel detector with 500 kpixels of 130 x 130 μm2. After proper pixel calibration, K-edge imaging is obtained by changing the pixel threshold around the K-shell binding energy of selected contrast agents. K-edge tomography of silver and iodine is compared with standard X-ray absorption CT and further investigated with Monte Carlo simulation

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.

International audience; PIXSCAN is the first micro-CT prototype based on XPAD hybrid pixel detectors whose properties (high signal to noise ratio and high detection efficiency) allow imaging at low irradiation dose. We have tested the impact of repeated imaging sessions with PIXSCAN on living mice. Mice were subjected on average to 10 imaging sessions over two weeks without any detectable sign of X-ray injuries as assessed by spontaneous activity in the cage, examination of hair and skin, or comparison of lung absorbing properties for X-rays. PIXSCAN was therefore used to non-invasively monitor the progression of lung metastasis in a murine model of cancer. Aim of was two-fold: i) to provide imaging assistance to decide timing of animal sacrifice and subsequent histological characterization of the tumors; ii) to optimize imaging protocol to allow direct evaluation of new therapeutic agents to stop cancer progression. Comparing in vivo tomographic reconstruction of freely breathing mice with post mortem examination of their lungs, we have shown that identification and localization of millimetric tumors was compatible with our low dose X-ray imaging protocol, allowing determination of tumor growth kinetics. In this context, interest of respiratory gating will be discussed.

We characterize the imaging performance of the micro computed tomography (micro-CT) prototype PIXSCAN equipped with an x-ray photon counting camera based on XPAD3/Si hybrid pixel detectors. The camera, which is composed of eight distinctive horizontal modules, permits the performance of whole-body mouse scans. Photon counting supplied by hybrid pixels guarantees acquisitions at a Poisson noise level exclusively determined by the detected photon statistics. First, we characterize the performance of the imaging system while assessing its linearity, noise, spatial resolution and low contrast detectability estimated from scans of appropriate phantoms. Then, we show CT images of mice data acquired either in vivo or post mortem, without or with an injection of iodine as a contrast agent. Although hybrid pixels guarantee a minimal noise to the images, the limited detection efficiency of Si sensors impairs detected photon statistics and therefore the improvement in contrast-to-noise ratio for standard tomographic imaging. Indeed, the most innovative potential of the PIXSCAN prototype is its capacity to perform spectral tomographic imaging and fast dynamic imaging. The first is thanks to the possibility of setting an energy threshold on the detected photons, and the second is thanks to its fast acquisition rate (till 500 images/s). We present some examples of these imaging methods applied to mice and discuss their main limiting factors.