Name: controlled semi-automated laser-induced injuries for studying spinal cord regeneration in zebrafish larvae
Uploaded: 2021-11-22T15:00:00
Description: Watch this Scientific Journal Video about Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae at JoVE.com

This study was supported by the BBSRC (BB/S0001778/1). CR is funded by the Princess Royal TENOVUS Scotland Medical Research Scholarship Programme. We thank David Greenald (CRH, University of Edinburgh) and Katy Reid (CDBS, University of Edinburgh) for the kind gift of transgenic fish (See Supplementary File). We thank Daniel Soong (CRH, University of Edinburgh) for the kind access to the 3i spinning-disk confocal.

All animal studies were carried out with approval from the UK Home Office and according to its regulations, under project license PP8160052. The project was approved by the University of Edinburgh Institutional Animal Care and Use Committee. For experimental analyses, zebrafish larvae up to 5-day-old of either sex were used of the following available transgenic lines: Tg(Xla.Tubb:DsRed;mpeg1:GFP), Tg(Xla.Tubb:DsRed), Tg(betaactin:utrophin-mCherry), Tg(Xla.Tubb:GCaMP6s), and Tg(mnx1:gfp) (see Supplementary File 1<...

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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=Adult+zebrafish+as+a+model+for+successful+central+nervous+system+regeneration.">Adult zebrafish as a model for successful central nervous system regeneration.</a> Restorative Neurology and Neuroscience. 26 (2-3), 71-80 (2008).</li><li>Ohnmacht, J., 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=Spinal+motor+neurons+are+regenerated+after+mechanical+lesion+and+genetic+ablation+in+larval+zebrafish.">Spinal motor neurons are regenerated after mechanical lesion and genetic ablation in larval zebrafish.</a> Development. 143 (9), 1464-1474 (2016).</li><li>Anguita-Salinas, C., 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=Cellular+dynamics+during+spinal+cord+regeneration+in+larval+zebrafish.">Cellular dynamics during spinal cord regeneration in larval zebrafish.</a> Developmental Neuroscience. 41 (1-2), 112-122 (2019).</li><li>Chapela, D., 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=A+zebrafish+drug+screening+platform+boosts+the+discovery+of+novel+therapeutics+for+spinal+cord+injury+in+mammals.">A zebrafish drug screening platform boosts the discovery of novel therapeutics for spinal cord injury in mammals.</a> Scientific Reports. 9, 1-12 (2019).</li><li>Schl&#252;&#223;ler, R., 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=Mechanical+mapping+of+spinal+cord+growth+and+repair+in+living+zebrafish+larvae+by+brillouin+imaging.">Mechanical mapping of spinal cord growth and repair in living zebrafish larvae by brillouin imaging.</a> Biophysical Journal. 115 (5), 911-923 (2018).</li><li>Cavone, L., 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=A+unique+macrophage+subpopulation+signals+directly+to+progenitor+cells+to+promote+regenerative+neurogenesis+in+the+zebrafish+spinal+cord.">A unique macrophage subpopulation signals directly to progenitor cells to promote regenerative neurogenesis in the zebrafish spinal cord.</a> Developmental Cell. 56 (11), 1617-1630 (2021).</li><li>Keatinge, 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=CRISPR+gRNA+phenotypic+screening+in+zebrafish+reveals+pro-regenerative+genes+in+spinal+cord+injury.">CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury.</a> PLoS Genetics. 17 (4), 1-21 (2021).</li><li>. 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There is an urgent need for a deeper understanding of the processes at play during regeneration in zebrafish. This animal model offers many benefits for biomedical research, in particular for spinal cord injuries1. Most of the studies involve manual lesions that require a well-trained operator and induce multi-tissue damage. A laser lesion protocol is presented here, allowing control over the lesion characteristics and reduced damage to the surrounding tissues. Furthermore, this technique is easy ...

In contrast to mammals, zebrafish (Danio rerio) can repair their central nervous system (CNS) after injury1. The use of zebrafish larvae as a model for spinal cord regeneration is relatively recent. It has proven valuable to investigate the cellular and molecular mechanisms underlying repair2. This is due to the ease of manipulation, the short experimental cycle (new larvae every week), the tissues&#39; optical transparency, and the larvae&#39;s small size, ideally suited for in vivo fluorescence microscopy.
In the case of spinal cord regeneration, two additional advantages of using larvae are the speed of recovery, a few days compared to a few weeks for adults, and the ease of inducing injuries using manual techniques. This has been successfully used in many studies3,4,5, including recent investigations6,7. Overall, this leads to increased meaningful data production, high adaptability of experimental protocols, and decreased experimental costs. The use of larvae younger than 5 days post-fertilization also reduces the use of animals following the 3R principles in animal research8.
After a spinal cord injury in zebrafish larvae, many biological processes occur, including inflammatory response, cell proliferation, neurogenesis, migration of surviving or newly generated cells, reformation of functional axons, and a global remodeling of neural processes circuits and spine tissues6,7,9,10. To be successfully orchestrated, these processes involve a finely regulated interaction between a range of cell types, extracellular matrix components, and biochemical signals11,12. Unraveling the details of this significant reorganization of a complex tissue such as the spinal cord requires the use and development of precise and controlled experimental approaches.
The primary experimental paradigm used to study spinal cord regeneration in zebrafish is to use surgical means to induce tissue damage by resection, stabbing, or cryoinjury3,13. These approaches have the disadvantage of requiring specific training in microsurgery skills, which is time-consuming for any new operator and may prevent their use in short-term projects. Furthermore, they usually induce damage to the surrounding tissues, which may influence regeneration.
Another approach is to induce cell damage chemically14 or by genetic manipulations15. The latter allows for highly targeted damage. However, such a technique requires long preparatory work to generate new transgenic fish before doing any experiment, renewed each time a unique cell type is targeted.
There is, thus, the need for a method allowing targeted but versatile lesions suitable to a variety of studies in regeneration. A solution is to use a laser to induce localized damage in the tissue of interest16,17,18,19,20. Indeed, the use of laser-induced tissue damage presents a robust approach for generating spinal cord lesions with many advantages. The microscopes equipped with such laser manipulation modules allow specifying a customized shaped area where cell ablation will occur, with the extra benefit of temporal control. The size and position of the lesion can be thus adapted to address any questions.
The missing feature of most laser lesion systems is the possibility to induce injuries in a highly reproducible way for a series of larvae. Here an original protocol is described using a UV laser to induce semi-automated precise and controlled lesions in zebrafish larvae based on a microfluidic platform designed for automated larvae handling21. Moreover, in the system presented here, larvae are inserted in a glass capillary, which permits free rotation of the animal around its rostrocaudal axis. The user can choose which side of the larva to present to the laser while allowing fluorescence imaging to precisely target the laser beam and assess the damage after the lesion.
The protocol described here is used with a semi-automated zebrafish larvae imaging system combined with a spinning disk equipped with a UV laser (designated hereafter as the VAST system). However, the main points of the protocol and most of the claims of the technique are valid for any system equipped with a laser capable of cell ablation, including two-photon laser scanning microscopes, spinning-disk microscopes provided with a UV laser (FRAP module), or video-microscopes with a laser module for photo manipulation. One of the main differences between the VAST system and conventional sample handling will be that for the latter, mounting larvae in low-melting-point agarose on glass coverslips/glass-bottom Petri dishes in place of loading them in a 96-well plate will be required.
The benefits offered by this method open opportunities for innovative research on the cellular and molecular mechanisms during the regeneration process. Moreover, the high data quality allows for quantitative investigations in a multidisciplinary context.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope software Zen Blue 2.0</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ/FIJI</td>
			<td>Open-Source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Visual Studio Code</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope and accessories</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ApoTome microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C-Plan-Apochromat 10X (0.5NA) dipping lens</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dual AxioCam 506 m CCD cameras</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscope LSM880</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning-disk module CSU-X1</td>
			<td>Yokogawa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Upright microscopeAxio Examiner D1</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UV laser</td>
			<td>Micropoint</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VAST BioImager</td>
			<td>Union Biometrica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Labware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>90 mm Petri dish</td>
			<td>Thermo-Fisher</td>
			<td>101R20</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning</td>
			<td>3841</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Click-It EdU Imaging Kit</td>
			<td>Invitrogen</td>
			<td>C10637</td>
			<td></td>
		</tr>
		<tr>
			<td>aminobenzoic-acid-ethyl methyl-ester (MS222)</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>phenylthiourea (PTU)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-chicken Alexa Fluor 488</td>
			<td>Jackson</td>
			<td>703-545-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-mouse Cy3</td>
			<td>Jackson</td>
			<td>715-165-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-GFP</td>
			<td>Abcam</td>
			<td>AB13970</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-tubulin acetylated antibody</td>
			<td>Sigma</td>
			<td>T6793</td>
			<td></td>
		</tr>
		<tr>
			<td>Transgenic zebrafish lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tg(beta-actin:utrophin-mCherry)</td>
			<td></td>
			<td>N/A</td>
			<td>Established by David Greenhald, University of Edinburgh</td>
		</tr>
		<tr>
			<td>Tg(mnx1:gfp)</td>
			<td></td>
			<td>N/A</td>
			<td>First described in [Flanagan-Steet et al. 2005]</td>
		</tr>
		<tr>
			<td>Tg(Xla.Tubb:DsRed)</td>
			<td></td>
			<td>N/A</td>
			<td>First described in [Peri and Nusslein-Volhard 2008]</td>
		</tr>
		<tr>
			<td>Tg(Xla.Tubb:DsRed;mpeg1:GFP)</td>
			<td></td>
			<td>N/A</td>
			<td>Established by Katy Reid, University of Edinburgh</td>
		</tr>
		<tr>
			<td>Tg(Xla.Tubb:GCaMP6s)</td>
			<td></td>
			<td>N/A</td>
			<td>Established by David Greenhald, University of Edinburgh</td>
		</tr>
	</tbody>
</table>

controlled semi-automated laser-induced injuries for studying spinal cord regeneration in zebrafish larvae

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes this animal model very useful for studying spinal cord injury and regeneration. The standard protocol for inducing such lesions is to transect the dorsal part of the trunk manually. However, this technique requires extensive training and damages additional tissues. A protocol was developed for laser-induced lesions to circumvent these limitations, allowing for high reproducibility and completeness of spinal cord transection over many animals and between different sessions, even for an untrained operator. Furthermore, tissue damage is mainly limited to the spinal cord itself, reducing confounding effects from injuring different tissues, e.g., skin, muscle, and CNS. Moreover, hemi-lesions of the spinal cord are possible. Improved preservation of tissue integrity after laser injury facilitates further dissections needed for additional analyses, such as electrophysiology. Hence, this method offers precise control of the injury extent that is unachievable manually. This allows for new experimental paradigms in this powerful model in the future.

Validation of spinal cord transection 
Structural and functional investigations were performed to assess whether the protocol allows a complete spinal cord transection.
First, to verify that the loss in fluorescence at the lesion site was due to neuronal tissue damage and not fluorescence photobleaching from the laser illumination, immunostaining using an antibody against acetylated tubulin (see Table of Materials and Supplementary File 1...

Watch this Scientific Journal Video about Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae at JoVE.com

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes ...

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Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...