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

Summary

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.

Abstract

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.

Introduction

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' optical transparency, and the larvae's small size, ideally suited for in vivo fluorescence microscopy.

In the case of spinal cord regeneration, two additional advantages o....

Protocol

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 regarding the generation of the transgenic zebrafish lines). A schematic of the protocol using the automated zebrafish larvae handling platfo....

Results

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.......

Discussion

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 .......

Materials

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