A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.
While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.
Zebrafish (Danio rerio) have an increased ability to regenerate their central nervous system (CNS) compared to other mammals1,2,3. Recently, to better understand the molecular mechanisms underlying this increased regenerative capacity, comparative analyses of tissue regeneration using next-generation sequencing technology have been performed4,5,6. The brain structures in zebrafish and tetrapods are quite different7,8,9. This means that several brain injury models using small fish with similar brain structures and biological features have been developed to facilitate the investigation of the underlying molecular mechanisms contributing to this increased regenerative capacity.
In addition, medaka (Oryzias latipes) is a popular laboratory animal with a low capacity for heart and neuronal regeneration10,11,12,13 compared with zebrafish. Zebrafish and medaka have similar brain structures and niches for adult neural stem cells (NSCs)14,15,16,17. In zebrafish and medaka, the optic tectum includes two types of NSCs, neuroepithelial-like stem cells and radial glial cells (RGCs)15,18. A stab wound injury for the optic tectum of adult zebrafish was previously developed, and this model was used to investigate the molecular mechanisms regulating brain regeneration in these animals19,20,21,22,23. This young adult zebrafish stab wound injury model induced regenerative neurogenesis from RGCs19,24,25. This stab wound injury in the optic tectum is a robust and reproducible method13,19,20,21,22,23,24,25. When the same injury model was applied to adult medaka, the low neurogenic capacity of RGCs in medaka optic tectum was revealed via the comparative analysis of RGC proliferation and differentiation following injury13.
Stab wound injury models in the optic tectum have also been developed in mummichog models26, but details of the tectum injury have been not well documented when compared with telencephalic injury27. The stab wound injury in the optic tectum using zebrafish and medaka allows the investigation of the differential cellular responses and gene expression between species with differential regenerative capacity. This protocol describes how to perform a stab wound injury in the optic tectum using an injection needle. This method can be applied to small fish like zebrafish and medaka. The processes for sample preparation for histological analysis and cellular proliferation and differentiation analysis using fluorescent immunohistochemistry and cryosections are explained here.
All experimental protocols were approved by the Institutional Animal Care and Use Committee at the National Institute of Advanced Industrial Science and Technology. Zebrafish and medaka were maintained according to standard procedures28.
1. Stab wound injury in the adult optic tectum
2. Brain dissection
3. Preparation of frozen sections
4. Fluorescent immunostaining
Stab wound injury in the optic tectum using needle insertion into the right hemisphere (Figure 1, Figure 4A, and Figure 5A) induces various cellular responses, including radial glial cell (RGC) proliferation and the generation of newborn neurons. Similarly, aged populations of zebrafish and medaka were used to counteract any aging effects in the regenerative response. Then fluorescent immunostaining was performed on the frozen sections, and the RGC proliferation and differentiation were analyzed after the tectum injury in the zebrafish and medaka (Figure 4-5)13.
Antibodies against a proliferating cell marker were used, proliferating cell antigen (PCNA), and an RGC marker, brain lipid-binding protein (BLBP), available in zebrafish and medaka to evaluate the RGC proliferation in these tissues13,19. As previously described, most of the RGCs were quiescent (PCNA negative) in the contralateral uninjured hemisphere (Figure 4B)13. Still, RGC proliferation was induced at 2 days post-injury (dpi) in the medaka tectum (Figure 4C,D)13. Induction of RGC proliferation after the injury is a common feature of both the zebrafish and medaka regenerative responses13,19.
BrdU labeling is a simple method used to evaluate cell lineage and analyze RGC differentiation after brain injury (Figure 5A)13. Immunostaining with antibodies for a pan-neuronal marker, HuC, and BrdU was previously used to compare RGC differentiation in the injured tectum of zebrafish and medaka (Figure 5B,C)13. If these antibodies are available in the target species, comparative analyses can be performed.
If the injury is appropriately induced, the injury site is located in the central-dorsal region in the optic tectum (Figure 4C). Nuclear staining and hematoxylin and eosin staining can then be used to confirm the injury site13,19,20,21,22,23,24,25. After the injury, a disturbed periventricular gray zone with nuclear staining can be observed (Figure 4C). If the injury is located in the medial dorsal region, RGC proliferation is not significantly increased25.
Figure 1: Stab wound injury in adult optic tectum using a needle. (A) Dorsal view of an adult zebrafish. Zebrafish is kept upright between two bent needles inserted vertically into a Styrofoam. (A') Magnified image of the boxed area in (A). 30 G needle is inserted into the medial region of the border between two skulls called os frontale and os parietale on the optic tectum. The yellow circle indicates the injury site, and white dashed lines indicate the two skulls on the optic tectum. (B) Dorsal view of adult medaka. (B') A magnified image of the boxed area in (B). 30 G needle is inserted into the border on the optic tectum. The yellow circle indicates the injury site, and white dashed lines indicate two skulls on the optic tectum. (C) Medaka has scales on the skull. Skull scales are to be removed before the stab wound injury. The dashed line indicates the scale on the optic tectum. Scale bar: 2 mm in A-B, 1 mm in A', B' and C. Telencephalon (Tel), optic tectum (OT), os frontale (F), and os parietale (P). Please click here to view a larger version of this figure.
Figure 2: Intracardiac perfusion in small adult fish. (A) Ventral view of a zebrafish fixed on Styrofoam using bent needles ready for intracardiac perfusion and brain dissection. (B) A ventral incision is made from the origin of the anal fins to the chest. (C) The heart is behind a silvery epithelial layer called the hypodermis in both zebrafish and medaka. Another fixation using a bent needle beside the silver epithelial layer allows for easier access. The solid white line indicates the ventricle (V), and the dotted line indicates the hypodermis. (D) The silver epithelial layer is removed before the intracardial perfusion of 1x PBS. (E-G) Canula is inserted into the ventricle for intracardiac perfusion. Gills before (F) and after (G) the intracardiac perfusion. If the blood removal is not complete, the gills remain red. (H-J) Brain dissection after PBS perfusion to remove the blood from the tissues. Remove skulls on the optic tectum and telencephalon as shown in (I). If the blood removal is not complete, the brain looks light pink (the right brain in (J)). Scale bar: 2 mm in A-C and H, 1 mm in D-G and I-J. The olfactory bulb (OB), telencephalon (Tel), optic tectum (OT), bulbus arteriosus (Ba), ventricle (V), and atrium (At). Please click here to view a larger version of this figure.
Figure 3: Brain embedding for frozen sections. (A) The brain is embedded in a cryomold with an embedding compound. Anterior is down. (B) Cryomolds are cooled on a precooled aluminum block. Telencephalon (Tel), optic tectum (OT). Please click here to view a larger version of this figure.
Figure 4: Representative results of fluorescent immunostaining against RGC proliferation after tectum injury in adult medaka. (A) Schematic view of the stab wound injury to the right hemisphere of the optic tectum and coronal section. (B-C) Representative results of proliferative RGCs (PCNA + BLBP + cells) in the contralateral uninjured (B) and injured (C) side at 2 days post-injury. White arrowhead in C' indicates a disturbed periventricular gray zone by the stab wound injury. (D) Magnified images of the boxed area in (C). White arrowheads in (D) indicate PCNA + BLBP + cells. Scale bar: 50 µm in B-D. Adapted with permission from Reference13. Please click here to view a larger version of this figure.
Figure 5: Representative results of the fluorescent immunostaining for the generation of newborn neurons after tectum injury. (A) Schematic view of bromodeoxyuridine (BrdU) treatment and the stab wound injury in the optic tectum and coronal section. (B-C) Representative results of newborn neurons (BrdU + HuC + cells) at 7 days post-injury in the injured zebrafish (B) and medaka (C). Scale bar: 50 µm in B-C. Adapted with permission from Reference13. Please click here to view a larger version of this figure.
Here a set of methods is described which can be used to induce stab wound injuries in the optic tectum utilizing a needle to facilitate the evaluation of RGC proliferation and differentiation after brain injury. Needle-mediated stab wounds are a simple, efficiently implemented method that can be applied to many experimental samples using a standard set of tools. Stab wound injury models for several regions of the zebrafish brain have been developed3,19,29. The optic tectum is one of the most largest parts of the brain and is easy to manipulate. Moreover, most RGCs in the optic tectum are quiescent under physiological conditions when compared to the telencephalon, making it easier to observe RGC proliferation and differentiation depending on the injury3,19.
One of the critical steps and limitations in stab wound injuries is manual needle insertion; a consistent injury is necessary for creating reproducible results and facilitating comparative analysis. The precise location and depth of insertion are crucial and help create reproducible injuries in experiments. This paper provides clear guidelines for making similar injuries each time. Moreover, the proliferation of RGC after the injury is essential for the neurogenesis of the injured tectum. In injured zebrafish and medaka, RGC proliferation increases at 1 dpi and returns to basal levels, the same as in the contralateral uninjured hemisphere, at 7 dpi13,19.
Stab wound injury is one of the mechanical injury methods that induce non-specific cell ablation. In contrast, transgenic approaches to cell-specific ablation such as nitroreductase/metronidazole system have also been developed30,31,32. These ablation models should be selected based on the experimental purpose. Non-specific ablation is suitable for brain injuries such as stab wounding and ischemic stroke. In contrast, cell-specific ablation might be more appropriate for evaluating the degeneration of specific cells associated with neurodegenerative diseases such as Parkinson's disease.
Recently, ischemic injury models using zebrafish have been developed33, but these models need transgenic lines and fluorescent microscopy to monitor blood flow. Therefore, these models are challenging to apply to species with poor genetic approaches, and their throughput is lower than the stab wound injury model.
As mentioned above, stab wound injury in the optic tectum is simple and easily applied in other small fish models such as African killifish and mummichog with common tools26. Furthermore, comparative analysis between species has been well investigated using sequencing technology. Therefore, this simple method remains essential when studying the regenerative capacity of NSCs in zebrafish using comparative analysis of cellular responses and gene expression.
The authors have nothing to disclose.
This work was supported by JSPS KAKENHI Grant Number 18K14824 and 21K15195 and an internal grant of AIST, Japan.
10 mL syringe | TERUMO | SS-10ESZ | |
1M Tris-HCl (pH 9.0) | NIPPON GENE | 314-90381 | |
30 G needle | Dentronics | HS-2739A | |
4% Paraformaldehyde Phosphate Buffer Solution | Wako | 163-20145 | |
Aluminum block | 115 x 80 x 37 mm (W x D x H) is enough size to freeze 6 cryomolds | ||
Anti-BLBP | Millipore | ABN14 | 1:500 |
Anti-BrdU | Abcam | ab1893 | 1:500 |
Anti-HuC | Invitrogen | A21271 | 1:100 |
Anti-PCNA | Santa Cruz Biotechnology | sc-56 | 1:200 |
Brmodeoxyuridine | Wako | 023-15563 | |
Confocal microscope C1 plus | Nikon | ||
Cryomold | Sakura Finetek Japan | 4565 | 10 x 10 x 5 mm (W x D x H) |
Cryostat | Leica | CM1960 | |
Danio rerio WT strains RW | |||
Extension tube | TERUMO | SF-ET3520 | |
Fluoromount (TM) Aqueous Mounting Medium, for use with fluorescent dye-stained tissues | SIGMA-ALDRICH | F4680-25ML | |
Forceps | DUMONT | 11252-20 | |
Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 | Invitrogen | A32723 | |
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 | Invitrogen | A11035 | |
Hoechst 33342 solution | Dojindo | 23491-52-3 | |
Hydrochloric Acid | Wako | 080-01066 | |
Incubation Chamber for 10 slides Dark Orange | COSMO BIO CO., LTD. | 10DO | |
MAS coat sliding glass | Matsunami glass | MAS-01 | |
Micro cover glass | Matsunami glass | C024451 | |
Microscopy | Nikon | SMZ745T | |
Normal horse serum blocking solution | VECTOR LABRATORIES | S-2000-20 | |
O.C.T Compound | Sakura Finetek Japan | 83-1824 | |
Oryzias latipes WT strains Cab | |||
PAP Pen Super-Liquid Blocker | DAIDO SANGYO | PAP-S | |
Phosphate Buffered Saline (PBS) Tablets, pH 7.4 | TaKaRa | T9181 | |
Styrofoam tray | 100 x 100 x 10 mm (W x D x H) styrofoam sheet is available as tray | ||
Sucrose | Wako | 196-00015 | 30 % (w/v) Sucrose in PBS |
Tricaine (MS-222) | nacarai tesque | 14805-24 | |
Trisodium Citrate Dihydrate | Wako | 191-01785 | |
Triton X-100 | Wako | 04605-250 |