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      MyelinJ: an ImageJ macro for high throughput analysis of myelinating cultures

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          Abstract

          Summary

          MyelinJ is a free user friendly ImageJ macro for high throughput analysis of fluorescent micrographs such as 2D-myelinating cultures and statistical analysis using R. MyelinJ can analyse single images or complex experiments with multiple conditions, where the ggpubr package in R is automatically used for statistical analysis and the production of publication quality graphs. The main outputs are percentage (%) neurite density and % myelination. % neurite density is calculated using the normalize local contrast algorithm, followed by thresholding, to adjust for differences in intensity. For % myelination the myelin sheaths are selected using the Frangi vesselness algorithm, in conjunction with a grey scale morphology filter and the removal of cell bodies using a high intensity mask. MyelinJ uses a simple graphical user interface and user name system for reproducibility and sharing that will be useful to the wider scientific community that study 2D-myelination in vitro.

          Availability and implementation

          MyelinJ is freely available at https://github.com/BarnettLab/MyelinJ. For statistical analysis the freely available R and the ggpubr package are also required. MyelinJ has a user guide ( Supplementary Material) and has been tested on both Windows (Windows 10) and Mac (High Sierra) operating systems.

          Supplementary information

          Supplementary data are available at Bioinformatics online.

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          Most cited references11

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          Multiple sclerosis review.

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            In vitro myelin formation using embryonic stem cells.

            Myelination in the central nervous system is the process by which oligodendrocytes form myelin sheaths around the axons of neurons. Myelination enables neurons to transmit information more quickly and more efficiently and allows for more complex brain functions; yet, remarkably, the underlying mechanism by which myelination occurs is still not fully understood. A reliable in vitro assay is essential to dissect oligodendrocyte and myelin biology. Hence, we developed a protocol to generate myelinating oligodendrocytes from mouse embryonic stem cells and established a myelin formation assay with embryonic stem cell-derived neurons in microfluidic devices. Myelin formation was quantified using a custom semi-automated method that is suitable for larger scale analysis. Finally, early myelination was followed in real time over several days and the results have led us to propose a new model for myelin formation.
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              Astrocytes, but not olfactory ensheathing cells or Schwann cells, promote myelination of CNS axons in vitro.

              We have examined the interaction between olfactory ensheathing cells (OECs), Schwann cells (SC), oligodendrocytes, and CNS axons using cultures generated from embryonic rat spinal cord. Oligodendrocyte process extension and myelination in these cultures was poor if the cells were plated on OECs or SCs. Myelin internodes and nodes of Ranvier formed frequently if these cultures were plated onto monolayers of neurosphere-derived astrocytes (NsAs). In the myelinated fibers generated on NsAs, Nav channels, caspr, and neurofascin molecules were correctly assembled at the nodes of Ranvier. The density of neurites, survival, and antigenic differentiation of oligodendrocytes was similar on OEC and NsAs monolayers. However, on OEC monolayers, despite a transient increase in the number of endogenous oligodendrocytes, there was a decrease in oligodendrocyte process extension and axonal ensheathment when compared with cultures plated on NsAs monolayers. To determine if these changes were due to axonal or glial factors, spinal cord oligodendrocytes were plated onto monolayers of OECs, NsAs, and poly-L-lysine in the absence of neurons. In these cultures, process extension and myelin-like membrane formation by oligodendrocytes was improved on monolayers of OEC. This suggests that inhibition of process extension is mediated via cross-talk between OECs and neurites. In cultures containing axons plated on OEC monolayers, oligodendrocyte process formation, axonal ensheathment, and myelination occurred albeit lower if the cultures were supplemented with NsAs conditioned medium. These data suggest OECs can permit neurite extension and oligodendrocyte proliferation, but lack secreted factor(s) and possible cell-cell contact that is necessary for oligodendrocyte process extension and myelination.
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                Author and article information

                Contributors
                Role: Associate Editor
                Journal
                Bioinformatics
                Bioinformatics
                bioinformatics
                Bioinformatics
                Oxford University Press
                1367-4803
                1367-4811
                01 November 2019
                16 May 2019
                16 May 2019
                : 35
                : 21
                : 4528-4530
                Affiliations
                Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow , Glasgow, UK
                Author notes
                To whom correspondence should be addressed. E-mail: Susan.Barnett@ 123456glasgow.ac.uk
                Article
                btz403
                10.1093/bioinformatics/btz403
                6821319
                31095292
                0e3df5ce-f4ea-4afa-bdb9-a46e05686eaa
                © The Author(s) 2019. Published by Oxford University Press.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 06 February 2019
                : 08 April 2019
                : 07 May 2019
                Page count
                Pages: 3
                Funding
                Funded by: MRC 10.13039/501100000265
                Award ID: MR/K501335/1
                Funded by: BBSRC 10.13039/501100000268
                Award ID: BB/J013854/1
                Funded by: Wellcome Trust 10.13039/100004440
                Award ID: 202789/Z/16/Z
                Funded by: Medical Research Scotland 10.13039/501100000294
                Award ID: 56
                Categories
                Applications Notes
                Bioimage Informatics

                Bioinformatics & Computational biology
                Bioinformatics & Computational biology

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