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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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 ...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63259">Controlled Semi-Automated Lased-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae</a>. The title was&#160;updated.
The title was updated from:
Controlled Semi-Automated Lased-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae
to:
Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63259">Controlled Semi-Automated Lased-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae</a>. The Title was&#160;updated.

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

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 ...

The technique we propose here allows us to overcome the limitations of manual lesions protocols by reducing damage to adjacent tissues and improving reproducibility, even for inexperienced operators. This technique allows us to choose precisely where to induce the lesion without damaging surrounding tissues thanks to rotating glass capillary combined with a targeted UV laser. Many steps are involved in operating the VAST equipment, so I would recommend ticking off every step as you go in order to ensure everything is done correctly.Begin by anesthetizing the larvae using the medium containing the anesthetic, and then transfer them to a 96-well plate containing 300 microliters of medium per well. Switch on all the system components including the laser for ablation. Then, launch the automated zebrafish imaging, or VAST software, choose Plate on the first window, and click on the Done button.A small window will pop up asking whether the capillary is empty and clean. Check the image of the capillary for any air bubbles inside. If there are no air bubbles inside, click on Yes on the popped-up window.Next in the LP Sampler window, go to the File menu and select the Open script option. Choose a file containing the script corresponding to the experiment to be performed. Then, in the main VAST software window, go to File and choose Open experiment.Choose the experiment file corresponding to the planned experiment. Launch ImageJ/Fiji software, go to the File menu, and choose New script to open the script window. Then, go to the File menu and choose Open to load the laser lesion script.Next, to launch the Python IDE, go to the File menu and choose Open file to load the script to manage the laser. Then, click on the Run menu and choose Run Without Debugging to run the script. Ensure that a sequence of messages in the terminal panel appears along with some noise while the laser attenuator initializes.In the main window of the VAST software, click on the arrow buttons to move the stage and center the capillary relative to the microscope objective. Look through the eyepieces and focus on the top of the capillary using the transmitted light of the microscope. Place a 96-well plate containing the larvae on the left plate holder of the LP sampler.Then, place another plate for collection on the right plate holder of the sampler. Ensure that the A1 well of the plate is in the front left corner of the holder. In the LP Sampler window of the VAST software, click on the Plate template button and select all the wells containing larvae.Click on the OK button to validate and close the window. Then, click on the Run plate button in the LP Sampler window to start loading the larva. Next, go to the microscope software and click on the Live button to image the larva.Turn the microscope focus knob on until the spinal cord central canal is visible. Take a snapshot in fluorescence and save the image to a folder. Open the image in ImageJ and adjust the contrast if required.Click on the region-of-interest line tool and draw a short line centered on the spinal cord. Switch the microscope to 100%reflective mirror position. Load the ImageJ script and set Repetition to 2, Sample to 1, Width to 40 micron, and Attenuation to 89.After setting all the parameters, click on the OK button. When the laser shot sequence is finished, switch to fluorescence imaging on the imaging software and adjust the focus. Take a new snapshot and save it.Open this new image in ImageJ and draw a new line larger than the spinal cord itself. Switch the microscope to 100%reflective mirror position. Go to the ImageJ script window and set Repetition to 2, Sample to 1, Width to 40 microns, and Attenuation to 89.After setting all the parameters, click on the OK button. When the laser shot sequence is finished, verify the transection quality by imaging fluorescence and focusing. Ensure that no cells or axons remain intact in the lesion site.Go to the main VAST software window and click on the Collect button to collect the lesioned larvae into the empty 96-well plate. Then, click on the checkbox tray light to switch back on the VAST system light. Take out the larva from the 96-well plate as soon as possible, and transfer them to a clean Petri dish with fresh fish water for the larva to recover post-lesion.Put the Petri dish in an incubator at 28 degrees Celsius. Acetylated tubulin immuno-staining and calcium imaging indicate that laser lesion entirely disrupts the continuity of spinal tissue. An intact spinal cord is shown in this image.A complete disruption of the axons between the caudal and rostral sides of the lesion confirms the complete transection of the spinal cord. An example of incomplete transection is shown here. The transected spinal cord on an NBTG cAMP 6-S larva is shown in this image.The rectangles show the ROIs used to quantify the fluorescence intensity in the lesions'rostral and caudal sides. The graphical image represents the fluorescence intensity change over time in the rostral and caudal analysis ROIs. The maximum intensity projection fluorescence images of an NBT:dsRed larva before the laser lesion, after three hours, 24 hours, and 48 hours of the laser lesion are shown here.After 24 hours post-injury, the wounds started to close, leading to a partial restoration of the initial structure of the spinal cord after 48 hours. A partial functional reconnection was confirmed after 48 hours post-injury using calcium imaging. The ratio between the amplitude of the spikes in the caudal area and the rostral area showed an increase between 3, 24, and 48 hours post-injury.Macrophage recruitment was observed after laser lesions using NBT:dsRed mpeg1 GFP larvae laser lesions. No difference was observed in the number of labeled cells between manual and laser lesions. Unlesioned fish displayed fewer double-labeled cells than lesioned fish in both lesion conditions.Laser lesion induces less muscle and skin damage than manual lesion. It is critical to verify whether the lesion is complete or not. No intact cell or structure should be visible in the lesion area, only a dim background.To study spinal cord regeneration, we use many methods, including immuno-histochemistry and calcium imaging. Importantly, we can also do electrophysiology, as the tissue can withstand dissection, contrary to manually-lesioned animals. This new technique allows us to quantitatively study the structural and functional tissue organization in the spinal cord after injury by allowing reproducible and controlled lesions.

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Medicine

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