970 research outputs found

    Video Object Tracking in Neural Axons with Fluorescence Microscopy Images

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    Neurofilament is an important type of intercellular cargos transmitted in neural axons. Given fluorescence microscopy images, existing methods extract neurofilament movement patterns by manual tracking. In this paper, we describe two automated tracking methods for analyzing neurofilament movement based on two different techniques: constrained particle filtering and tracking-by-detection. First, we introduce the constrained particle filtering approach. In this approach, the orientation and position of a particle are constrained by the axon’s shape such that fewer particles are necessary for tracking neurofilament movement than object tracking techniques based on generic particle filtering. Secondly, a tracking-by-detection approach to neurofilament tracking is presented. For this approach, the axon is decomposed into blocks, and the blocks encompassing the moving neurofilaments are detected by graph labeling using Markov random field. Finally, we compare two tracking methods by performing tracking experiments on real time-lapse image sequences of neurofilament movement, and the experimental results show that both methods demonstrate good performance in comparison with the existing approaches, and the tracking accuracy of the tracing-by-detection approach is slightly better between the two

    Scanning laser optical tomography for in toto imaging of the murine cochlea

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    The mammalian cochlea is a complex macroscopic structure due to its helical shape and the microscopic arrangements of the individual layers of cells. To improve the outcomes of hearing restoration in deaf patients, it is important to understand the anatomic structure and composition of the cochlea ex vivo. Hitherto, only one histological technique based on confocal laser scanning microscopy and optical clearing has been developed for in toto optical imaging of the murine cochlea. However, with a growing size of the specimen, e.g., human cochlea, this technique reaches its limitations. Here, we demonstrate scanning laser optical tomography (SLOT) as a valuable imaging technique to visualize the murine cochlea in toto without any physical slicing. This technique can also be applied in larger specimens up to cm3 such as the human cochlea. Furthermore, immunolabeling allows visualization of inner hair cells (otoferlin) or spiral ganglion cells (neurofilament) within the whole cochlea. After image reconstruction, the 3D dataset was used for digital segmentation of the labeled region. As a result, quantitative analysis of position, length and curvature of the labeled region was possible. This is of high interest in order to understand the interaction of cochlear implants (CI) and cells in more detail. © 2017 Nolte et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.DFG/EXC/1077/1Ministry of Lower SaxonyVolkswagenStiftun

    The role of the Schwann cell in the induction of elongative central axonal growth

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    The factors underlying the failure of axon regeneration in the CNS are thought to comprise of both the lack of supportive factors as well as the presence of inhibitory ones. Transplantation work has shown that the PNS is able to provide some of the necessary criteria and with it an increased capacity to regenerate. Studies have further shown that the crucial ingredient to such peripheral grafts is the presence of Schwann cells (SCs), the major glial cell of the PNS. I have used an extrusion transplantation system, recently developed in this laboratory, to study the effects of a SC column placed into the origin of the septo-hippocampal cholinergic projection. These SC columns integrate with host glia with minimal tissue damage, form a tight and ordered column with aligned cellular processes, and are able to recruit modest numbers of axons. Immunostaining with a cholinergic axon marker suggests that these axons arise from the septal nuclei. Given the limited availability and yield of primary SCs that current preparation protocols offer, I have engineered neonatal SC lines by transfecting the SV40 large T antigen into a population of primary neonatal rat SCs. Characterisation of these cell lines, with the use of immunocytochemistry, Western blotting and RT-PCR, shows that they retain the immunophenotype of primary SCs in vitro, although in vivo studies have posed more difficult with the lack of a suitable marker. In addition, I have set up a retroviral transfection system with the use of a bicistronic vector containing the Green Fluorescent Protein. This would provide a means of unique and efficient labelling prior to transplantation, and moreover offer the potential for further transfections of an additional gene of interest within the same vector

    Intravenously delivered mesenchymal stem cell-derived exosomes target M2-type macrophages in the injured spinal cord

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    In a previous report we showed that intravenous infusion of bone marrow-derived mesenchymal stem cells (MSCs) improved functional recovery after contusive spinal cord injury (SCI) in the non-immunosuppressed rat, although the MSCs themselves were not detected at the spinal cord injury (SCI) site [1]. Rather, the MSCs lodged transiently in the lungs for about two days post-infusion. Preliminary studies and a recent report [2] suggest that the effects of intravenous (IV) infusion of MSCs could be mimicked by IV infusion of exosomes isolated from conditioned media of MSC cultures (MSCexos). In this study, we assessed the possible mechanism of MSCexos action on SCI by investigating the tissue distribution and cellular targeting of DiR fluorescent labeled MSCexos at 3 hours and 24 hours after IV infusion in rats with SCI. The IV delivered MSCexos were detected in contused regions of the spinal cord, but not in the noninjured region of the spinal cord, and were also detected in the spleen, which was notably reduced in weight in the SCI rat, compared to control animals. DiR "hotspots" were specifically associated with CD206-expressing M2 macrophages in the spinal cord and this was confirmed by co-localization with anti-CD63 antibodies labeling a tetraspanin characteristically expressed on exosomes. Our findings that MSCexos specifically target M2-type macrophages at the site of SCI, support the idea that extracellular vesicles, released by MSCs, may mediate at least some of the therapeutic effects of IV MSC administration

    Graphene promotes axon elongation through local stall of Nerve Growth Factor signaling endosomes

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    Several works reported increased differentiation of neuronal cells grown on graphene; however, the molecular mechanism driving axon elongation on this material has remained elusive. Here, we study the axonal transport of nerve growth factor (NGF), the neurotrophin supporting development of peripheral neurons, as a key player in the time course of axonal elongation of dorsal root ganglion neurons on graphene. We find that graphene drastically reduces the number of retrogradely transported NGF vesicles in favor of a stalled population in the first two days of culture, in which the boost of axon elongation is observed. This correlates with a mutual charge redistribution, observed via Raman spectroscopy and electrophysiological recordings. Furthermore, ultrastructural analysis indicates a reduced microtubule distance and an elongated axonal topology. Thus, both electrophysiological and structural effects can account for graphene action on neuron development. Unraveling the molecular players underneath this interplay may open new avenues for axon regeneration applications

    Time-Lapse Imaging of the Dynamics of CNS Glial-Axonal Interactions In Vitro and Ex Vivo

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    Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualize the various stages of myelination.We took two approaches to follow myelination over time: i) time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii) ex vivo imaging of the spinal cord of shiverer (Mbp mutant) mice, transplanted with GFP-labelled murine neurospheres. We demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, we provide evidence from time-lapse fluorescence microscopy and confocal microscopy that the oligodendrocytes' cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs, which then extend longitudinally along the axons. We predict from this model that these spiral cuffs must extend over each other first before extending to form internodes of myelin.These experiments show the feasibility of visualizing the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualization of an entire internodal length of myelin and the ex vivo approach validating the in vitro data

    Effect of nitric oxide and inflammatory mediators on axonal transport

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    In dieser Arbeit wurde der Einfluss der Aktivierung von Microgliazellen auf den Transport von Organellen, Vorläufervesikel und Mitochondrien in Axonen untersucht. Mikrogliazellen sind mitverantwortlich für die Immunreaktionen im Gehirn und setzen dabei Stickstoffmonooxid und entzündliche Mediatoren frei Durch Kombination von biophysikalischen und molekularbiologischen Techniken konnte gezeigt werden, dass die Aktivierung von Microgliazellen eine Erhöhung der Anzahl an unbeweglichen Organellen in der Nähe von Axonen zur Folge hat. Der entzündliche Mediator Tumornekrosefaktor, der von aktivierten Microgliazellen freigesetzt wird, wirkte direkt auf Axone und inhibierte den Transport von synaptischen Proteinen und Mitochondrien. Außerdem konnte gezeigt werden, dass die Phosphorylierung der c-Jun- Kinase eine wichtige Funktion in diesem Prozess einnimmt. Mit Hilfe von biophysikalischen Verfahren wurde gezeigt, dass sich nach Phosphorylierung von c-Jun-Kinase das Kinesin-Motorprotein KIF5B vom Tubulin losgelöst hat. Die Ergebnisse lassen vermuten, dass entzündliche Mediatoren über die c-Jun-Kinase den axonalen Transport von synaptischen Proteinen und Mitochondrien inhibieren
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