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

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.