Mechanical spinal cord transection in larval zebrafish and subsequent whole-mount histological processing

Abstract

Zebrafish regenerate their spinal cord after injury, both at larval and adult stages. Larval zebrafish have emerged as a powerful model system to study spinal cord injury and regeneration due to their high optical transparency for in vivo imaging, amenability to high-throughput analysis, and rapid regeneration time. Here, we describe a protocol for the mechanical transection of the larval zebrafish spinal cord, followed by whole-mount tissue processing for in situ hybridization and immunohistochemistry to elucidate principles of regeneration. For complete details on the use and execution of this protocol, please refer to Wehner et al. (2017) and Tsata et al. (2021).

Keywords: Developmental biology; Microscopy; Model Organisms; Neuroscience.

Graphical abstract

Figure 1

Essential equipment for transecting the larval zebrafish spinal cord (A) Surgery plate for perforation lesion: 10 cm petri dish coated with 1.5% agarose in E3 medium. (B) Surgery plate for incision lesion: 10 cm petri dish sealed with parafilm. Note the roughened surface (compact grid) for enhanced wettability. (C) Micro scalpel: 30 G × ½” hypodermic needle, 1 mL syringe with clipped barrel flange, handle of cell scraper (∼20 cm long; full length not shown) to extend the syringe length. Scale bar: 1 cm. (D) Stereo microscope for examination of needles and monitoring of larvae during and after surgery. (E) Finely drawn glass pipette with fitted rubber bulb for aspiration of excess E3 medium prior to surgery. Scale bar: 1 cm.

Figure 2

Mechanical spinal cord transection in larval zebrafish (A) Lateral view of a Tg(her4.3:EGFP) transgenic zebrafish larva at 3 dpf, labeling ependymoradial glia cells (astroglia-like cells) in the spinal cord with GFP protein (green). Anatomical structures relevant for the described protocol are indicated. Abbreviations: de, dorsal edge; nc, notochord; sc, spinal cord; urp, urogenital pore. (B) Larvae placed in lateral position on the surgery plate in preparation for perforation lesion. (C) Perforation lesion procedure to transect the larval zebrafish spinal cord. Images shown are single frames of the Methods video S1. Lateral view; dorsal is left. Abbreviations: D, dorsal; V, ventral. (D) Representative image of a perforation lesion immediately after injury. Note that the dorsal edge of the trunk remains intact. Lateral view; rostral is left, dorsal is up. (E) Larvae placed in lateral position on the surgery plate in preparation for incision lesion. (F) Incision lesion procedure to transect the larval zebrafish spinal cord. Images shown are single frames of the Methods video S2. Lateral view; dorsal is right. Abbreviations: D, dorsal; V, ventral. (G) Representative image of an incision lesion immediately after injury. Note that the notochord is left intact. Lateral view; rostral is left, dorsal is up. (H) Extensive injury to the notochord following an incision lesion leads to the formation of a bulgy tissue structure (arrowhead). Lateral view; rostral is left. (A–H) Scale bars: 500 μm (B, E), 200 μm (A, C, F), 100 μm (D, G, H)

Figure 3

Representative examples of whole-mount ISH in spinal cord lesioned larval zebrafish (A and B) ISH reveals local upregulation of col12a1a expression in the lesion site at 1 dpl (perforation lesion) as compared to adjacent unlesioned trunk tissue. col12a1a mRNA expression was visualized using either NBT/BCIP substrate (A; blue) or Fast Red substrate (B; red). Note that PTU was used to suppress developmental and lesion-induced pigmentation in (A) while the larva in (B) was untreated. Images shown are brightfield recordings (A) or a maximum intensity projection of a confocal image (B) of the lesion site (lateral view; rostral is left, dorsal is up). Abbreviation: BF, brightfield. Scale bars: 50 μm.

Figure 4

Representative examples of whole-mount IHC in larval zebrafish (A) Co-labeling of neurites (anti-acetylated tubulin; red) and astroglia-like processes (anti-GFAP; green) in the spinal cord of unlesioned larvae (top panel), immediately after incision lesion (middle panel) and at 2 dpl (bottom panel). Continuity of axonal labeling between rostral and caudal spinal cord stumps is restored within 2 dpl (bottom panel). (B) Co-labeling of neurites (anti-acetylated tubulin; red) and fibronectin matrix (anti-Fibronectin; green) in the lesion site at 1 dpl (perforation lesion). (C) Labeling of lesion-induced collagen type XII matrix deposition at 2 dpl (perforation lesion). (D) Labeling of neurites with anti-acetylated tubulin immunohistochemistry (red) preserves the GFP signal of the GFP reporter in Tg(pdgfrb:Gal4ff;UAS:EGFP) transgenic animals. GFP⁺ cells accumulate in the lesion site at 1 dpl (perforation lesion). (A–D) Images shown are maximum intensity projections of the unlesioned trunk or the lesion site at indicated timepoints after lesion (lateral view; rostral is left, dorsal is up). Scale bars: 50 μm (C) and 25 μm (A, B, D).