Name: spinal cord transection in the larval zebrafish
Uploaded: 2014-05-21T14:45:00
Description: Watch this Scientific Journal Video about Spinal Cord Transection in the Larval Zebrafish at JoVE.com

We are indebted to the University of Utah zebrafish facility for animal husbandry. R.I.D. was supported by NIH R56NS053897, and L.K.B. was a predoctoral trainee supported by the HHMI Med-Into-Grad initiative.

Zebrafish were raised and bred according to standard procedures; experiments were approved by the University of Utah Institutional Animal Care and Use Committee.
1. Preparation of Surgery Plates
<ol>
	<li>Make surgery plates using 60 mm Petri dishes and Sylgard 184 Silicone Elastomer Kit, following manufacturer&#8217;s instructions. Fill dishes no more than half-full and allow to polymerize. Store covered at room temperature.</li>
</ol>
2. Preparation of Micropipettes</...

<ol>
	<li>Houweling, D. A., B&#228;r, P. R., Gispen, W. H., Joosten, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury:+bridging+the+lesion+and+the+role+of+neurotrophic+factors+in+repair.">Spinal cord injury: bridging the lesion and the role of neurotrophic factors in repair.</a> Progress in brain research. 117, 455-471 (1998).</li><li>Mikami, Y., 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=Implantation+of+dendritic+cells+in+injured+adult+spinal+cord+results+in+activation+of+endogenous+neural+stem/progenitor+cells+leading+to+de+novo+neurogenesis+and+functional+recovery.">Implantation of dendritic cells in injured adult spinal cord results in activation of endogenous neural stem/progenitor cells leading to de novo neurogenesis and functional recovery.</a> Journal of neuroscience research. 76 (4), 453-465 (2004).</li><li>Chernoff, E. A. G., Sato, K., Corn, A., Karcavich, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+regeneration:+intrinsic+properties+and+emerging+mechanisms.">Spinal cord regeneration: intrinsic properties and emerging mechanisms.</a> Seminars in Cell &amp; Developmental Biology. 13 (5), 361-368 (2002).</li><li>Kuscha, V., Barreiro-Iglesias, A., Becker, C. G., Becker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+tyrosine+hydroxylase+and+serotonergic+systems+in+the+regenerating+spinal+cord+of+adult+zebrafish.">Plasticity of tyrosine hydroxylase and serotonergic systems in the regenerating spinal cord of adult zebrafish.</a> The Journal of comparative neurology. 520 (5), 933-951 (2012).</li><li>Becker, C. G., Lieberoth, B. C., Morellini, F., Feldner, J., Becker, T., Schachner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L1.1+is+involved+in+spinal+cord+regeneration+in+adult+zebrafish.">L1.1 is involved in spinal cord regeneration in adult zebrafish.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 24 (36), 7837-7842 (2004).</li><li>Hui, S. P., Dutta, A., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+response+after+crush+injury+in+adult+zebrafish+spinal+cord.">Cellular response after crush injury in adult zebrafish spinal cord.</a> Developmental Dynamics: An Official Publication of the American Association of Anatomists. 239 (11), 2962-2979 (2010).</li><li>Goldshmit, Y., Sztal, T. E., Jusuf, P. R., Hall, T. E., Nguyen-Chi, M., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf-dependent+glial+cell+bridges+facilitate+spinal+cord+regeneration+in+zebrafish.">Fgf-dependent glial cell bridges facilitate spinal cord regeneration in zebrafish.</a> The Journal of neuroscience: the official journal of the Society for Neuroscience. 32 (22), 7477-7492 (2012).</li><li>Reimer, M. M., 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=Motor+neuron+regeneration+in+adult+zebrafish.">Motor neuron regeneration in adult zebrafish.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 28 (34), 8510-8516 (2008).</li><li>Hale, M. E., Ritter, D. A., Fetcho, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+confocal+study+of+spinal+interneurons+in+living+larval+zebrafish.">A confocal study of spinal interneurons in living larval zebrafish.</a> The Journal of comparative neurology. 437 (1), 1-16 (2001).</li><li>Bhatt, D. H., Otto, S. J., Depoister, B., Fetcho, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP-induced+repair+of+zebrafish+spinal+circuits.">Cyclic AMP-induced repair of zebrafish spinal circuits.</a> Science. 305 (5681), 254-258 (2004).</li><li>McClenahan, P., Troup, M., Scott, E. 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When initially learning this technique, we recommend attempting no more than 50-100 transections in a single session. After mastering this technique, we are able to transect up to 300 embryos per&#160;hr; however, this level of throughput requires a few months of weekly practice. We also recommend practicing with a reporter line and verifying complete transection until the incidence of incomplete spinal cord transection is reduced to less than 1%.
Spinal cord transection in the adult zebrafish...

Major trauma to the human spinal cord often results in permanent paralysis and loss of sensation below the level of injury, due to the inability to regrow axons or reinitiate neurogenesis1,2. In contrast to mammals, however,&#160;anamniotes including salamanders and zebrafish (Danio rerio) show robust recovery even after complete spinal cord transection3,4.
The adult zebrafish is a well-established model for studying the recovery process following spinal cord injury5-7. Following complete spinal cord transection, reestablishment of sensory and locomotive function is observed in the adult zebrafish by 6&#160;weeks post-injury8. In order to examine the regenerative process in vivo, we turned to the transparent larval zebrafish9.
Here we present a method to transect the spinal cord of a 5 days post-fertilization (dpf) larval zebrafish using a beveled microinjection pipette as a scalpel, modified from Bhatt, et al.10 This method supports high throughput, low mortality, and reproducibility. With practice, 300 larvae/hr can be transected, and over 6&#160;months of transections, including over 3,600 animals, 98.75% &#177; 0.72% survived until 7 days post-injury (dpi). Our data shows rapid recovery of sensory and locomotion as well: at 1 dpi, all movement by the injured fish is driven by pectoral fin locomotion only. However, larvae begin to respond to tungsten needle touch caudal to transection by 2 dpi, reestablish C-bend movement by 3 dpi, and display predatory swimming by 5 dpi11. Using antibody staining against acetylated tubulin, we have confirmed that axons are absent from the injury site at 1 dpi, but have crossed the injury site by 5 dpi. We believe this protocol will provide a valuable technique for the study of axonal regrowth and neurogenesis in the spinal cord following injury.

<table border="0" cellpadding="0" cellspacing="0" style="width:733px;" width="733">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Name of Material/ Equipment</td>
			<td style="width:179px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">60mm petri dish</td>
			<td style="width:179px;">VWR</td>
			<td style="width:119px;">82050-544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">100mm petri dish</td>
			<td style="width:179px;">VWR</td>
			<td style="width:119px;">89038-968</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">NC9644388</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">borosilicate capillary tubing: OD 1.00mm ID 0.78mm</td>
			<td style="width:179px;">Warner Instruments Inc.</td>
			<td style="width:119px;">64-0778</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">forceps</td>
			<td style="width:179px;">Fine Scientific Tools Inc.</td>
			<td style="width:119px;">11252-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">disssection microscope</td>
			<td style="width:179px;">Nikon</td>
			<td style="width:119px;">SMZ6454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">microgrinder</td>
			<td style="width:179px;">Narishige</td>
			<td style="width:119px;">EG-44</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Gentamycin Sulfate</td>
			<td style="width:179px;">Amresco Inc.</td>
			<td style="width:119px;">0304-5G</td>
			<td>dissolve in water 10mg/ml, store at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Tricaine</td>
			<td style="width:179px;">Acros Organics</td>
			<td align="right" style="width:119px;">118000100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">cotton tipped applicator, wood, 6-inch</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">23-400-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">1ml syringe</td>
			<td style="width:179px;">BD</td>
			<td align="right" style="width:119px;">309625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">27 ga. needle</td>
			<td style="width:179px;">BD</td>
			<td align="right" style="width:119px;">305109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fry food</td>
			<td style="width:179px;">Argent Labs</td>
			<td style="width:119px;">F-ARGE-PTL-CN</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">micropipette puller</td>
			<td style="width:179px;">Sutter Instrument Co.</td>
			<td style="width:119px;">Model P-97</td>
			<td>Box Filament FB330B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">20x E2 (1L)</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">17.5g NaCl</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">S671-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">0.75g KCl</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">P217-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:269px;">2.90g CaCl2&#183;2H2O</td>
			<td style="width:179px;">Sigma&#160;&#160;&#160;</td>
			<td style="width:119px;">C7902-500G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:269px;">4.90g MgSO4&#183;7H2O</td>
			<td style="width:179px;">Merck</td>
			<td style="width:119px;">MX0070-1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:269px;">0.41g KH2PO4</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">P285-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:269px;">0.12g Na2HPO4</td>
			<td style="width:179px;">Sigma&#160;&#160;&#160;</td>
			<td style="width:119px;">S0876-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:269px;">500x NaCO3 (10ml)</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td>make fresh, discard extra</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:269px;">0.35g NaCO3</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">1x E2 (1L)</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">50ml 20x E2</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:269px;">2ml fresh 500x NaCO3</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

spinal cord transection in the larval zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

To reduce severity of tissue damage surrounding the injury site, proper beveling of the micropipette is critical. Figure 1A shows a correctly beveled tip. Using a tip that is too wide (Figure 1B) tends to result in higher fatalities due to the increased likelihood of nicking the dorsal aorta, while a tip that is too narrow (Figure 1C) tends to glance off the skin rather than cutting tissue.
To practice this technique, it is advantageous to use...

Watch this Scientific Journal Video about Spinal Cord Transection in the Larval Zebrafish at JoVE.com

Spinal Cord Transection in the Larval Zebrafish

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

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