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>Fabricate micropipettes by heating and pulling thin-wall borosilicate capillary tubing in a micropipette puller using the same settings for making microinjection needles.</li>
	<li>Under a dissection microscope, snap off tip of micropipette to approximately 200 &#181;m in diameter with forceps.</li>
	<li>Bevel broken edge with a microgrinder initially to 35&#176;, followed by a second beveling at 25&#176;. Ensure tip is sharp and smooth. Store finished beveled micropipette in a Petri dish on a small amount of clay.</li>
</ol>
3. Preparation of Zebrafish Larvae
<ol>
	<li>7 days prior to surgery, set up mating tanks of male and female zebrafish.</li>
	<li>Collect embryos the following morning, 3&#160;hr&#160;after the lights come on to ensure maximum yield. If using a transgenic reporter line such as Tg(elevl3:eGFP)knu3, sort fertilized embryos 100/100 mm plate in 25 ml of E3 at 28.5 &#176;C. If using wildtype, sort fertilized embryos 25/100 mm plate in 25 ml of E3 at 28.5 &#176;C.</li>
	<li>If using a reporter line, screen embryos for fluorescent expression at 48 hpf. Allow identified embryos to mature at a density of 25/100 mm plate in 25 ml of E3 at 28.5 &#176;C.</li>
	<li>When larvae are 5 dpf, prepare surgery plate by covering Sylgard with E2 + 10 mg/L Gentamycin Sulfate (GS) + Tricaine.
	<ol>
		<li>Prepare recovery dish by adding 25 ml E2 + GS to a 100 mm Petri dish.</li>
		<li>Prepare scalpel by taping together three swabs. This will form a triangular tool with three grooves.</li>
		<li>Mount a prepared micropipette on the swabs by taping it into one of the grooves.</li>
	</ol>
	</li>
	<li>If reusing micropipettes, flush until clear with E2 + GS using a 1ml syringe and a 27 G&#160;needle prior to mounting on swabs.</li>
</ol>
4. Surgery
<ol>
	<li>Anesthetize 1 plate of larvae at a time (25 fish) with Tricaine. Fish are sufficiently anesthetized when they no longer exhibit touch response. It is important that fish are completely anesthetized prior to surgery, otherwise they will twitch when the scalpel touches them. Surgery is performed under a dissection microscope.</li>
	<li>Transfer larvae to surgery plate.
	<ol>
		<li>Under maximum magnification, rotate one larva at a time so that it lies on its side with its back closest to the hand holding the scalpel.</li>
		<li>Position forceps so that they rest on the Sylgard, angled over the width of the larva.</li>
		<li>Bracing the glass scalpel against one of the arms of the forceps, cut into the dorsal lateral face of the larva at the level of the anal pore, being sure not to cut beyond the ventral edge of the notochord. Twist the scalpel to sever the spinal cord.</li>
		<li>Repeat with remaining larvae. 
		Note: if a larva bleeds, it will not recover from the surgery. Immediately remove the larva from the surgery plate and euthanize it via Tricaine overdose.</li>
	</ol>
	</li>
	<li>Once surgery on the batch of larvae is complete, transfer injured animals to the recovery plate. This is to support the clearing of anesthesia.
	<ol>
		<li>Caution: when collecting injured larvae for transfer, make sure they are collected head or tail first: do not stress the injury site by bending the larvae. 
		Note: All devices used for surgery can be reused, including the micropipettes.</li>
	</ol>
	</li>
</ol>
5. Recovery
<ol>
	<li>Transfer injured larvae from the recovery plate to 100 mm plates filled with 25 ml E2 + GS at a density of 25/plate. Allow to recover in a 28.5 &#176;C incubator.</li>
	<li>Check plates daily, removing sick and dead animals. Do not change the media until Coleps (freshwater protozoa) are visible in the media. When changing the media, do not transfer the fish to a new plate; instead, remove as much media as possible and flood the same plate with new media. Repeat as necessary to reduce Coleps population.</li>
	<li>Feed daily with a small amount of powdered fry food. 
	Note: Live food (e.g., paramecia or rotifers) cannot be fed to injured larvae until after they have recovered locomotion. Otherwise, the live food will colonize the injury site and kill the larvae.</li>
</ol>

<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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fin-tail+coordination+during+escape+and+predatory+behavior+in+larval+zebrafish.">Fin-tail coordination during escape and predatory behavior in larval zebrafish.</a> PloS one. 7 (2), (2012).</li><li>Kim, C. H., 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=Repressor+activity+of+Headless/Tcf3+is+essential+for+vertebrate+head+formation.">Repressor activity of Headless/Tcf3 is essential for vertebrate head formation.</a> Nature. 407 (6806), 913-916 (2000).</li></ol>

The authors have nothing to disclose.

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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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 ...

spinal-cord-transection-in-the-larval-zebrafish

Research

JoVE Journal

Neuroscience

11.7K Views.  University of Utah. 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 days post-injury, and C-bend movement by 3 days post-injury.