The authors would like to thank the Kucenas Lab for valuable discussions and Quorum Technologies, Inc. for superb technical support. The work was supported by the UVa Fund for Excellence in Science and Technology (FEST) (S.K.).

1. Preparation and Mounting of Zebrafish Embryos for Ablation and Live Imaging
<ol>
	<li>Prepare a stock of 0.8% low melt agarose in egg water. Aliquot into 13X 100 mm disposable culture tubes and store at 4 &deg;C until needed.</li>
	<li>Cross adult zebrafish containing stably integrated transgenes to fluorescently label motor neurons and glial cell types of interest. Collect zebrafish embryos in egg water and place in 28.5 &deg;C incubator for correct staging later 3.</li>
	<li>At approximately 24 hr post ...

<ol>
	<li>Geuna, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3:+Histology+of+the+peripheral+nerve+and+changes+occurring+during+nerve+regeneration.">Chapter 3: Histology of the peripheral nerve and changes occurring during nerve regeneration.</a> Int. Rev. Neurobiol. 87, 27-46 (2009).</li><li>Hirata, K., Kawabuchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+phagocytosis+by+macrophages+and+nonmacrophages+during+Wallerian+degeneration.">Myelin phagocytosis by macrophages and nonmacrophages during Wallerian degeneration.</a> Microsc. Res. Tech. 57, 541-547 (2002).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+Embryonic+Development+of+the+Zebrafish.">Stages of Embryonic Development of the Zebrafish.</a> Developmental Dynamics. 203, 253-310 (1995).</li><li>Martin, S. M., O'Brien, G. S., Portera-Cailliau, C., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wallerian+degeneration+of+zebrafish+trigeminal+axons+in+the+skin+is+required+for+regeneration+and+developmental+pruning.">Wallerian degeneration of zebrafish trigeminal axons in the skin is required for regeneration and developmental pruning.</a> Development. 137, 3985-3994 (2010).</li><li>O'Brien, G. S., Rieger, S., Martin, S. M., Cavanaugh, A. M., Portera-Cailliau, C., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+axotomy+and+time-lapse+confocal+imaging+in+live+zebrafish+embryos.">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos.</a> J. Vis. Exp. (24), e1129 (2009).</li><li>Parrinello, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EphB+signaling+directs+peripheral+nerve+regeneration+through+Sox2-dependent+Schwann+cell+sorting.">EphB signaling directs peripheral nerve regeneration through Sox2-dependent Schwann cell sorting.</a> Cell. 143, 145-155 (2010).</li><li>Rosenberg, A. F., Wolman, M. A., Franzini-Armstrong, C., Granato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+nerve-macrophage+interactions+following+peripheral+nerve+injury.">In vivo nerve-macrophage interactions following peripheral nerve injury.</a> J. Neurosci. 32, 3898-3909 (2012).</li><li>Stoll, G., Muller, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+injury,+axonal+degeneration+and+neural+regeneration:+basic+insights.">Nerve injury, axonal degeneration and neural regeneration: basic insights.</a> Brain Pathol. 9, 313-325 (1999).</li><li>Villegas, R., Martin, S. M., O'Donnell, K., Carrillo, S., Sagasti, A., Allende, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+degeneration+and+regeneration+in+developing+zebrafish+peripheral+axons+reveals+a+requirement+for+extrinsic+cell+types.">Dynamics of degeneration and regeneration in developing zebrafish peripheral axons reveals a requirement for extrinsic cell types.</a> Neural Development. 7, 19 (2012).</li><li>Waller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+the+section+of+the+glossopharyngeal+and+hypoglossal+nerves+of+the+frog,+and+observations+of+the+alterations+pro-+duced+thereby+in+the+structure+of+their+primitive+fibres.">Experiments on the section of the glossopharyngeal and hypoglossal nerves of the frog, and observations of the alterations pro- duced thereby in the structure of their primitive fibres.</a> Philos. Trans. R. Soc. Lond. , 423-429 (1849).</li></ol>

The authors have nothing to disclose.

The most critical steps of this experimental design are: 1) properly mounting larvae for injury and subsequent in vivo imaging and 2) calibrating the laser and selecting the correct power settings in order to create a clean nerve transection that results in minimal extra-tissue damage. To help ensure a successful axotomy for in vivo imaging and subsequent analysis, mount multiple larvae in either individual glass bottom dishes or in a glass bottom dish with dividers. After calibrating the laser, ...

Zebrafish have been used extensively to study development of the nervous system because of their optical transparence and ease of transgenesis, which when coupled, allow for spectacular imaging of dynamic cell behaviors in a living embryo. Additionally, because zebrafish and mammals share nearly all of the genes required for nervous system formation, cellular and molecular information collected in this model organism is directly relatable to other vertebrate species. Although incredibly powerful for neural developmental studies, the zebrafish and its unique attributes have the potential to also elucidate the mechanisms that maintain and rebuild the nervous system after injury. Zebrafish larvae maintain their translucence into late larval stages and pigmentation can be effectively blocked with either the use of pharmacological inhibitors of melanin production or genetic mutants that lack pigment cells. Thus, using this model organism to study injury and regeneration in older animals is possible and offers the unique opportunity to directly investigate the cellular and molecular mechanisms that rebuild the nervous system. In this manuscript, we describe how to efficiently and reproducibly injure nerves in the PNS of zebrafish larvae. This injury paradigm lends itself to studying not only degeneration, but also the responses of peripheral glia and immune cells as well as the interactions between these populations during regeneration.
The PNS is a complex network of motor and sensory nerves that is necessary to pass information between the central nervous system (CNS) and the skin, organs and muscle of the body, allowing an organism to interact with its environment and survive. Along these nerves, peripheral glia, including myelinating and non-myelinating Schwann cells and perineurial glia, as well as connective tissue, encase the axons and ultimately form the mature nerve. Injury of these nerves initiates a process known as Wallerian degeneration 10. This mechanism of axonal fragmentation, immune recruitment, debris clearance and regeneration is very stereotyped and genetically regulated 1. Previous studies in mammalian systems have described the roles of Schwann cells during nerve degeneration and regeneration 1, 2, 6, 8. In these studies of fixed tissue or cell culture, Schwann cells not only recruited macrophages to the injury site to aid in debris clearance, but also aided in myelin phagocytosis themselves. While these studies have been incredibly informative, we have never before visualized glial responses to peripheral axon injury in vivo in real time, and no other studies have investigated the relationship between the different classes of peripheral glia during these events.
Recently, several labs have investigated Wallerian degeneration using zebrafish and laser-mediated axon injury similar to what we describe here 4, 5, 7, 9. In some of these studies, superficial sensory axons were axotomized in young larvae using a custom built, two-photon confocal microscope 4, 5, 9. In another study, which is very similar to our own, deeper axons within the ventral motor nerve were transected in 5 day old larvae using a commercially available laser ablation system 7. In both of these experimental set-ups, the focus was on Wallerian degeneration and both axons and immune cells were imaged. To expand on these studies, we describe injuring motor axons in older larvae with more mature, myelinated nerves and assay the response of all nerve-associated peripheral glia during degeneration and regeneration.
To do this, we transect motor nerves in 6 and 7 day post fertilization (dpf) larvae and visualize the responses of individual glial populations as well as investigate the interactions between these populations along injured axons. Using double and triple transgenic lines that label peripheral glia, including Schwann cells and perineurial glia, as well as a marker for axons, we use a commercially available laser ablation system consisting of a nitrogen-pumped dye laser (wavelength 435 nm) attached to a spinning disc confocal system to create axon transections. This experimental set-up allows us to visualize live, larval zebrafish, injure specific peripheral motor axon tracts and time-lapse image the responses of distinct glial populations to axon injury and their relationship to one another. This protocol can be further adapted to create nerve injuries in zebrafish of different ages, with different transgenic lines or genetic mutants to address different scientific questions.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Phenylthiourea</td>
 <td>Sigma</td>
 <td>P7629-100G</td>
 </tr>
 <tr>
 <td>Finquel Tricaine Methanesulfonate MS-222</td>
 <td>Argent Chemical</td>
 <td>C-FINQ-UE-100G</td>
 </tr>
 <tr>
 <td>Low melting point agarose</td>
 <td>Sigma</td>
 <td>A9414-10G</td>
 </tr>
 <tr>
 <td>Quad CELLview Cell Culture Dishes, Glass Bottom, Sterile, Greiner Bio One</td>
 <td>VWR/Greiner</td>
 <td>89125-444</td>
 </tr>
 <tr>
 <td>Single well glass bottom Petri dishes 35 x 10 mm, 12 mm thick</td>
 <td>Willco Wells</td>
 <td><a href="http://www.willcowells.com/en/products/willco-dish-glass-bottom-dishes/gwst-3512/" target="_blank">GWSt-3512</a></td>
 </tr>
 <tr>
 <td>MicroPoint Laser System with all components</td>
 <td>Andor Technology - purchased through Quorum Technologies, Inc.</td>
 <td>2203-SYS</td>
 </tr>
 <tr>
 <td>MicroPoint Laser Courmarin dye (435 nm)</td>
 <td>Andor Technology </td>
 <td>MP-27-435-DYE</td>
 </tr>
 <tr> </tr>
 </tbody>
 </table>

motor nerve transection and time-lapse imaging of glial cell behaviors in live zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

The assay described here can be used to assess the response of glial cells and other nerve-associated cell populations to axonal injury in vivo. Movie 1 shows an example of a nerve injury created using this method and the response of surrounding glial cells. This experiment was performed in Tg(nkx2.2a:megfp);Tg(olig2:dsred) zebrafish, in which perineurial glia express a membrane targeted EGFP and motor neurons express cytosolic DSRed. The injury was made along the rostral projection of ...

Watch this Scientific Journal Video about Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish at JoVE.com

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

motor-nerve-transection-time-lapse-imaging-glial-cell-behaviors-live

Research

JoVE Journal

Neuroscience

11.8K Görüntüleme.  University of Virginia. Periferal sinir sisteminin (PNS) yaralanmadan sonra önemli tamir yeteneğine sahip olmasına rağmen, küçük bu olgu yöneten hücresel ve moleküler mekanizmaları bilinmektedir. Canlı, transgenik zebrafish ve tekrarlanabilir bir sinir kesisi testi kullanarak, sinir dejenerasyonu ve rejenerasyonu sırasında dinamik glial hücre davranışlarını eğitim görebilirsiniz.