A reproducible method to study traumatic injury-induced zebrafish brain regeneration

Abstract

Traumatic brain injury (TBI) can be caused by a sudden blow or jolt to the head, causing irreversible brain damage leading to cellular and functional loss. Mammals cannot repair such damage, which may increase the risk of progressive neurodegeneration. Unlike mammals, lower vertebrates such as zebrafish have the astounding capability to regenerate their brains. A model system would be of great value to study zebrafish brain regeneration. Here, we describe a physical method to induce traumatic injury in the zebrafish brain and outline a pipeline to utilize this model system to explore various aspects of brain regeneration. This will significantly advance the fields of regenerative biology and neuroscience. The method includes inducing TBI and validating this through histological assays, immunohistochemistry, and gene expression analysis. By using this model system, researchers will be able to gain valuable insights into the cellular and molecular mechanisms underlying brain regeneration. Understanding these mechanisms could lead to the identification of potential strategies to address neurodegenerative conditions in higher vertebrates.

Keywords: brain; histology; immunohistochemistry; regeneration; zebrafish.

Graphical Abstract

Figure 1.

Telencephalic stab-wound injury model of zebrafish brain regeneration. (A) Illustration of stab-wound injury. A2 and A3 show the injury in the right hemisphere (encircled in black). A4 shows toluidine blue O-stained zebrafish brain section in transverse orientation (injury encircled in black).

Figure 2.

An illustration showing the preparation of the paraffin block of zebrafish brain in the coronal orientation. The paraffin block is initially prepared with the brain in transverse orientation. Once the block is completely set in the fridge, it is cut as shown in step 1 to obtain only a small piece of the paraffin block containing the brain. The block is then placed in the mould as shown in step 2 and set in fridge to have the paraffin block of the brain in the desired orientation to obtain coronal sections.

Figure 3.

Histological assessment of the injury using HE staining at 7 dpl (A1), 14 dpl (A2) and 30 dpl (A3) (black arrow indicates the region of injury and accumulation of cells). Scale bar 100 μm.

Figure 4.

Immunostaining of regenerating brains showing increased proliferative response upon injury-induced regeneration. (A) Immunostaining of 7 dpl regenerating brain with anti-BrdU (A2) shows increased BrdU-positive cells (white arrows) in the injured/regenerating telencephalic hemisphere. Counterstaining is carried out with DAPI, and A3 shows merged image. (B) Immunostaining of 7 dpl and 14 dpl regenerating brain with anti-PCNA (B2 and B5) shows increased PCNA-positive cells (white arrows) in the injured/regenerating telencephalic hemisphere. Counterstaining is carried out with DAPI. B3 and B6 show the merged images. Scale bar 100 μm.

Figure 5.

Immunostaining of regenerating brains showing increased glial response upon injury-induced regeneration. A2 and B2 show immunostaining of 7 dpl and 14 dpl regenerating brains with anti-GFAP (white square), suggesting increased GFAP-positive cells. Counterstaining is carried out with DAPI. A3 and B3 show merged images. The area highlighted in white squares are shown in the panel as high magnification images (A4–A6 and B4–B6). Scale bar 100 μm (A1–A3 and B1–B3), Scale bar 50 μm (A4–A6 and B4–B6).

Figure 6.

RNA isolation and qRT-PCR of regeneration-specific markers. (A) 1% (w/v) agarose gel in 1xTBE shows two RNA-specific bands (28 s and 18 s), and control and 7 dpl brain samples are shown in the gel followed by 1 kb ladder. (B) 0.8% (w/v) agarose gel in 1xTBE gel shows primer-specific PCR products upon RT-PCR (Sox2-240bp, olig2-224bp, and gfap-158bp). The control and 7 dpl samples show the correct product size for their respective primers. As expected, NTC (no template control does not give a product). Note: the gel is cut to show only relevant lanes. (C) Analysis of qRT-PCR using delta-delta ct method (rpl13a was used as housekeeping). p-value < 0.05 for sox2 and olig2, p-value <0.01 for gfap.