Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

Zebrafish (Danio rerio) have an increased ability to regenerate their central nervous system (CNS) compared to other mammals^1,2,3. Recently, to better understand the molecular mechanisms underlying this increased regenerative capacity, comparative analyses of tissue regeneration using next-generation sequencing technology have been performed^4,5,6. The brain structures in zebrafish and tetrapods are quite different^7,8,9. 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 regeneration^10,11,12,13 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 animals^{19,20,21,22,23}. This young adult zebrafish stab wound injury model induced regenerative neurogenesis from RGCs^19,24,25. This stab wound injury in the optic tectum is a robust and reproducible method^{13,19,20,21,22,23,24,25}. 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.

Protokół

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

1. Prepare a 0.4% (w/v) tricaine stock solution for anesthesia. For a 100 mL stock solution, dissolve 400 mg tricaine methanesulfonate (see Table of Materials) in 90 mL of distilled water and adjust the pH to 7.0 using 1 M Tris HCl buffer (pH 9.0). Once the pH is adjusted, add water up to 100 mL, then make appropriate aliquots and store them at -20 °C.
2. To anesthetize the adult zebrafish or medaka, prepare a 0.02 % (w/v) tricaine solution by diluting the 0.4 % tricaine stock solution with fish facility water.
3. Anesthetize the fish with 0.02% tricaine solution; they become anesthetized when they do not move or respond to touch (1-2 min).
4. Place the anesthetized fish on a Styrofoam tray (see Table of Materials) and fix the fish upright between two 30 G needles inserted vertically into the Styrofoam.
5. Hold the fish body to prevent the fish head from moving and vertically insert a 30 G needle through the skull into the medial region of one of the optic tectum hemispheres (Figure 1A,B). The insertion depth is ~0.75 mm, almost equal to half the length of the 30 G bevel. This insertion depth induces brain injury on zebrafish and medaka because of the similar body size of these fish.
   NOTE: Medaka has scales on the skull. Scratch and remove the scales using the 30 G needle to efficiently and accurately induce the stab wound injury (Figure 1C).
6. Transfer the injured fish into a fish tank with fresh fish facility water and move the tank to the fish breeding system once they have fully recovered from the anesthesia.
   NOTE: Fish will recover and start freely moving within a few minutes.
7. For lineage analysis after the injury, prepare fresh fish facility water containing 5 mM bromodeoxyuridine (BrdU) (see Table of Materials) and then incubate the fish with this 5 mM BrdU solution for a predetermined amount of time^13,19 (as determined by an experimental design). Then dissect these fish as per step 2 and evaluate BrdU incorporation and cell lineage identity as appropriate.
   ​NOTE: This step is an optional step for cell lineage analysis. To detect BrdU signals, perform antigen retrieval as shown in step 4.3.

2. Brain dissection

1. Prepare 0.02% tricaine solution, a Styrofoam tray for dissection, and a 10 mL syringe containing phosphate-buffered saline (1x PBS) with an extension tube and 30 G needles (see Table of Materials) for intracardial perfusion.
2. Anesthetize the injured fish at selected time points.
3. Place paper towels on the Styrofoam tray and place the anesthetized fish on the paper towels.
4. Hold the fish body in place by vertically inserting two 30 G needles on either side of the anal fin (Figure 2A).
5. Make a ventral incision from the anus to the heart (Figure 2B) and keep the heart visible by inserting another two 30 G needles on each side of this organ (Figure 2C).
   NOTE: The heart is covered in the silvery epithelial layer of the hypodermis in both zebrafish and medaka (Figure 2C).
6. Gently remove the silver epithelial layer with the tip of the forceps (Figure 2D).
7. Insert the 30 G needle into the ventricle, keeping the bevel up, and make an incision into the atrium using the forceps (Figure 2E). Carefully press down on the syringe to push the 1x PBS and confirm that the atrium flushed the blood.
   NOTE: To prevent the needle from penetrating the ventricle, carefully insert the needle to the depth of the half bevel and then adjust the insertion depth. If the perfusion is well done, gills turn white (Figure 2F,G).
8. Stop the perfusion after the blood is drained and the gills turn white.
9. Remove the needles fixing the fish body in place and fix the fish ventral side down (Figure 2H). Cut the spinal cord and remove the skull from the optic tectum and telencephalon (Figure 2I). Then, cut the optic nerves and dissect the brain carefully.
   NOTE: Watch out for the optic nerves during the dissection because the nerves are connected to the tectum. When the optic nerve is pulled, the optic tectum can be detached from the brain. If the blood removal is not complete, the brain looks light pink (the right brain in Figure 2J).
10. Transfer the brains into 1.5 mL tubes with 1 mL of 4 % paraformaldehyde in 1x PBS and fix them overnight at 4 °C.

3. Preparation of frozen sections

1. Wash the fixed brains three times in 1x PBS for 5 min each.
2. To cryoprotect the brains, transfer them into 1.5 mL tubes with 1 mL of 30 % (w/v) sucrose in 1x PBS and incubate them overnight at 4 °C. Prepare the embedding compound (2:1 mixture of OCT compound and 30% sucrose, see Table of Materials) by combining 30 mL of OCT and 15 mL of 30 % sucrose. Store this at 4 °C.
   NOTE: To remove the bubbles from the mixed compound, centrifuge the 50 mL tubes at 10,000 x g for 2 min at room temperature or place the tubes upright overnight.
3. Incubate an aluminum block overnight at -80 °C in order to cool the cryomold (see Table of Materials) immediately after the dissected brain is embedded in the cryomold with the embedding compound.
   NOTE: Liquid nitrogen can also be used to cool the aluminum block. In that case, the aluminum block should be placed in a Styrofoam box filled with enough liquid nitrogen to cover the aluminum block just before embedding. After confirming the liquid nitrogen sublimation, place the cryomold on the precooled aluminum block. It is better to confirm how many cryomolds can be placed on the aluminum block at once. It is advisable to use liquid nitrogen or an additional precooled block to ensure enough space to cool all samples quickly.
4. Put the precooled aluminum block in a Styrofoam box. Use a Styrofoam box with a lid to prevent the aluminum block from warming.
5. For coronal sections, embed the brain in a cryomold and adjust the orientation using forceps under the microscope (Figure 3A).
6. Place the cryomold on the precooled aluminum block in the Styrofoam box and allow the OCT compound to freeze (Figure 3B). When the OCT compound starts to freeze, it becomes white.
7. Apply a small circle of OCT compound on a specimen disc, and mount the cryoblock to attach the cryoblock to the specimen disc.
8. Place the specimen disc in the cryostat and freeze the OCT compound completely.
9. Place the specimen disc on the specimen head in the cryostat.
10. Set the orientation of the cryoblock and trim the OCT to remove any extra regions.
    NOTE: Adjust the orientation of the cutting plane while cutting the telencephalon. The blade is equipped with a cryostat. It is possible to adjust the orientation of the cutting plane by changing the angle of the specimen head.
11. Cut 14 µm thick serial sections through the whole optic tectum using a cryostat. Store the slides at -25 °C.
    ​NOTE: Long-term storage should be at -80 °C.

4. Fluorescent immunostaining

1. Dry glass slides in a slide rack for 30 min at room temperature (RT). Wash slides in 1x PBS for 30 min at RT. If immunostaining with anti-PCNA or BrdU antibodies is to be performed, proceed to step 4.2 or step 4.3.
2. Prepare 500 mL of 10 mM sodium citrate in a 500 mL beaker and warm the citrate buffer to 85 °C on a hot plate magnetic stirrer, then place the slides in a slide staining rack and incubate the rack in the citrate buffer at 85 °C for 30 min. To avoid boiling, keep the temperature around 85 °C. After antigen retrieval, wash the slides three times in 0.1 % Triton in 1x PBS (PBSTr), with each wash taking 5 min at RT.
   NOTE: This step is an optional step for proliferating cell nuclear antigen (PCNA) retrieval.
3. Prepare a 2 N HCl solution by diluting 12 N HCl in distilled water. Using a liquid blocker (see Table of Materials), draw a line as a hydrophobic barrier to keep the 2 N HCl solution around the sections.
   1. Place the slides on immunostaining trays and apply 200-300 µL of the 2N HCl solution. Incubate the trays at 37 °C for 30 min. After antigen retrieval, wash three times in 0.1 % PBSTr with each wash taking 5 min at RT.
      NOTE: This step is an optional step for BrdU antigen retrieval.
4. Absorb and remove the remaining solution using paper towels. Then, use a liquid blocker to draw a hydrophobic barrier to keep the antibody solution around the sections as necessary.
5. Place the slides on trays and apply 200-300 µL of blocking solution, 3% (v/v) horse serum in PBSTr) to every slide, and incubate the slides for 1 h at RT.
6. Wash the sections with PBSTr for 5 min at RT and repeat three times.
7. Prepare the primary antibodies (see Table of Materials) using the blocking solution (200-300 µL of antibody solution for each slide). Remove the remaining PBSTr with paper towels and place the slides on the immunostaining trays. Apply the primary antibody solution to every slide and incubate trays overnight at 4 °C.
8. Wash the sections with PBSTr for 5 min at RT and repeat three times.
9. Prepare fluorescent secondary antibody solution (see Table of Materials) using the blocking buffer (1:500). Remove the remaining PBSTr with paper towels and place the slides on the immunostaining trays. Apply the secondary antibody solution to every slide and shade trays with aluminum foil. Incubate for 1-2 h at RT.
10. Wash the sections with PBSTr for 5 min at RT three times without exposing them to light.
11. Prepare Hoechst solution (1:500 dilution in 1x PBS, see Table of Materials) for nuclear staining. Remove the remaining PBSTr with paper towels and place the slides on the immunostaining trays. Apply the Hoechst solution to every slide and cover the trays with aluminum foil. Incubate for 30 min at RT.
12. Wash the sections with 1x PBS for 10 min at RT without exposing them to light.
13. Absorb and remove the remaining 1x PBS using paper towels and mount the sections on slides using a water-soluble mounting medium (see Table of Materials) for fluorescent immunostaining. Slowly set a 24 x 45 mm coverslip onto the sections. Store at 4 °C, without exposing the slides to light.
14. Observe and image the sections under fluorescent microscopy or confocal microscopy.

Wyniki

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 secti...

Dyskusje

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 developed^3,19,

Materiały

Name	Company	Catalog Number	Comments
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