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....

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....

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