This work was supported by JSPS KAKENHI Grant Number 18K14824 and 21K15195 and an internal grant of AIST, Japan.

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
<ol>
	<li>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 &#176;C.</li>
	<li>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.</li>
	<li>Anesthetize the fish with 0.02% tricaine solution; they become anesthetized when they do not move or respond to touch (1-2 min).</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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 time13,19 (as determined by an experimental design). Then dissect these fish as per step 2 and evaluate BrdU incorporation and cell lineage identity as appropriate. 
	&#8203;NOTE: This step is an optional step for cell lineage analysis. To detect BrdU signals, perform antigen retrieval as shown in step 4.3.</li>
</ol>
2. Brain dissection
<ol>
	<li>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.</li>
	<li>Anesthetize the injured fish at selected time points.</li>
	<li>Place paper towels on the Styrofoam tray and place the anesthetized fish on the paper towels.</li>
	<li>Hold the fish body in place by vertically inserting two 30 G needles on either side of the anal fin (Figure 2A).</li>
	<li>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).</li>
	<li>Gently remove the silver epithelial layer with the tip of the forceps (Figure 2D).</li>
	<li>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).</li>
	<li>Stop the perfusion after the blood is drained and the gills turn white.</li>
	<li>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).</li>
	<li>Transfer the brains into 1.5 mL tubes with 1 mL of 4 % paraformaldehyde in 1x PBS and fix them overnight at 4 &#176;C.</li>
</ol>
3. Preparation of frozen sections
<ol>
	<li>Wash the fixed brains three times in 1x PBS for 5 min each.</li>
	<li>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 &#176;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 &#176;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.</li>
	<li>Incubate an aluminum block overnight at -80 &#176;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.</li>
	<li>Put the precooled aluminum block in a Styrofoam box. Use a Styrofoam box with a lid to prevent the aluminum block from warming.</li>
	<li>For coronal sections, embed the brain in a cryomold and adjust the orientation using forceps under the microscope (Figure 3A).</li>
	<li>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.</li>
	<li>Apply a small circle of OCT compound on a specimen disc, and mount the cryoblock to attach the cryoblock to the specimen disc.</li>
	<li>Place the specimen disc in the cryostat and freeze the OCT compound completely.</li>
	<li>Place the specimen disc on the specimen head in the cryostat.</li>
	<li>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.</li>
	<li>Cut 14 &#181;m thick serial sections through the whole optic tectum using a cryostat. Store the slides at -25 &#176;C. 
	&#8203;NOTE: Long-term storage should be at -80 &#176;C.</li>
</ol>
4. Fluorescent immunostaining
<ol>
	<li>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.</li>
	<li>Prepare 500 mL of 10 mM sodium citrate in a 500 mL beaker and warm the citrate buffer to 85 &#176;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 &#176;C for 30 min. To avoid boiling, keep the temperature around 85 &#176;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.</li>
	<li>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.
	<ol>
		<li>Place the slides on immunostaining trays and apply 200-300 &#181;L of the 2N HCl solution. Incubate the trays at 37 &#176;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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Place the slides on trays and apply 200-300 &#181;L of blocking solution, 3% (v/v) horse serum in PBSTr) to every slide, and incubate the slides for 1 h at RT.</li>
	<li>Wash the sections with PBSTr for 5 min at RT and repeat three times.</li>
	<li>Prepare the primary antibodies (see Table of Materials) using the blocking solution (200-300 &#181;L 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 &#176;C.</li>
	<li>Wash the sections with PBSTr for 5 min at RT and repeat three times.</li>
	<li>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.</li>
	<li>Wash the sections with PBSTr for 5 min at RT three times without exposing them to light.</li>
	<li>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.</li>
	<li>Wash the sections with 1x PBS for 10 min at RT without exposing them to light.</li>
	<li>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 &#176;C, without exposing the slides to light.</li>
	<li>Observe and image the sections under fluorescent microscopy or confocal microscopy.</li>
</ol>

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</ol>

The authors have nothing to disclose.

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,</sup...

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.

<table><tbody><tr><td>10 mL syringe</td><td>TERUMO</td><td>SS-10ESZ</td><td></td></tr><tr><td>1M Tris-HCl (pH 9.0)</td><td>NIPPON GENE</td><td>314-90381</td><td></td></tr><tr><td>30 G needle</td><td>Dentronics</td><td>HS-2739A</td><td></td></tr><tr><td>4% Paraformaldehyde Phosphate Buffer Solution</td><td>Wako</td><td>163-20145</td><td></td></tr><tr><td>Aluminum block</td><td></td><td></td><td>115 x 80 x 37 mm (W x D x H) is enough size to freeze 6 cryomolds</td></tr><tr><td>Anti-BLBP</td><td>Millipore</td><td>ABN14</td><td>1:500</td></tr><tr><td>Anti-BrdU</td><td>Abcam</td><td>ab1893</td><td>1:500</td></tr><tr><td>Anti-HuC</td><td>Invitrogen</td><td>A21271</td><td>1:100</td></tr><tr><td>Anti-PCNA</td><td>Santa Cruz Biotechnology</td><td>sc-56</td><td>1:200</td></tr><tr><td>Brmodeoxyuridine</td><td>Wako</td><td>023-15563</td><td></td></tr><tr><td>Confocal microscope C1 plus</td><td>Nikon</td><td></td><td></td></tr><tr><td>Cryomold</td><td>Sakura Finetek Japan</td><td>4565</td><td>10 x 10 x 5 mm (W x D x H)</td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM1960</td><td></td></tr><tr><td>&lt;em&gt;Danio rerio&lt;/em&gt; WT strains RW</td><td></td><td></td><td></td></tr><tr><td>Extension tube</td><td>TERUMO</td><td>SF-ET3520</td><td></td></tr><tr><td>Fluoromount (TM) Aqueous Mounting Medium, for use with fluorescent dye-stained tissues</td><td>SIGMA-ALDRICH</td><td>F4680-25ML</td><td></td></tr><tr><td>Forceps</td><td>DUMONT</td><td>11252-20</td><td></td></tr><tr><td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488</td><td>Invitrogen</td><td>A32723</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546</td><td>Invitrogen</td><td>A11035</td><td></td></tr><tr><td>Hoechst 33342 solution</td><td>Dojindo</td><td>23491-52-3</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Wako</td><td>080-01066</td><td></td></tr><tr><td>Incubation Chamber for 10 slides Dark Orange</td><td>COSMO BIO CO., LTD.</td><td>10DO</td><td></td></tr><tr><td>MAS coat sliding glass</td><td>Matsunami glass</td><td>MAS-01</td><td></td></tr><tr><td>Micro cover glass</td><td>Matsunami glass</td><td>C024451</td><td></td></tr><tr><td>Microscopy</td><td>Nikon</td><td>SMZ745T</td><td></td></tr><tr><td>Normal horse serum blocking solution</td><td>VECTOR LABRATORIES</td><td>S-2000-20</td><td></td></tr><tr><td>O.C.T Compound</td><td>Sakura Finetek Japan</td><td>83-1824</td><td></td></tr><tr><td>&lt;em&gt;Oryzias latipes&lt;/em&gt; WT strains Cab</td><td></td><td></td><td></td></tr><tr><td>PAP Pen Super-Liquid Blocker</td><td>DAIDO SANGYO</td><td>PAP-S</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS) Tablets, pH 7.4</td><td>TaKaRa</td><td>T9181</td><td></td></tr><tr><td>Styrofoam tray</td><td></td><td></td><td>100 x 100 x 10 mm (W x D x H) styrofoam sheet is available as tray</td></tr><tr><td>Sucrose</td><td>Wako</td><td>196-00015</td><td>30 % (w/v) Sucrose in PBS</td></tr><tr><td>Tricaine (MS-222)</td><td>nacarai tesque</td><td>14805-24</td><td></td></tr><tr><td>Trisodium Citrate Dihydrate</td><td>Wako</td><td>191-01785</td><td></td></tr><tr><td>Triton X-100</td><td>Wako</td><td>04605-250</td><td></td></tr></tbody></table>

stab wound injury model of the adult optic tectum using zebrafish and medaka for the comparative analysis of regenerative capacity

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.

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...

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

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 ...

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Research

JoVE Journal

Developmental Biology

1.8K Views.  National Institute of Advanced Industrial Science and Technology. Steekwondletsel is een mechanische letselmethode die niet-specifieke celablatie induceert. Deze methode vergemakkelijkt de evaluatie van RGC-proliferatie en differentiatie na hersenletsel. Naaldgemedieerd steekwondletsel is een eenvoudige, efficiënt geïmplementeerde methode om hersenletsel op te wekken.Deze methode kan op veel experimentele monsters worden toegepast met behulp van een standaardset gereedschappen. Het regeneratieve vermogen van zebravissen werd geïllustreerd met behu...

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

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Neuroscience

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Biology

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and ...

Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. Indeed, the eye injuries most frequently encountered clinically are those to the cornea. While corneal wound healing has been extensively studied in small mammals (i.e., mice, rats, and rabbits), corneal physiology studies have neglected other species, including zebrafish, despite zebrafish being a classic research model.
This report describes a method of performing a corneal abrasion on zebrafish. The wound is performed in vivo on anesthetized fish using an ocular burr. This method allows for a reproducible epithelial wound, leaving the rest of the eye intact. After abrasion, wound closure is monitored over the course of 3 h, after which the wound is reepithelialized. By using scanning electron microscopy, followed by image processing, the epithelial cell shape, and apical protrusions can be investigated to study the various steps during corneal epithelial wound closure.
The characteristics of the zebrafish model permit study of the epithelial tissue physiology and the collective behavior of the epithelial cells when the tissue is challenged. Furthermore, the use of a model deprived of the influence of the tear film can produce&#160;new answers regarding corneal response to stress. Finally, this model also allows the delineation of the cellular and molecular events involved in any epithelial tissue subjected to a physical wound. This method can be applied to the evaluation of drug effectiveness in preclinical testing.

This protocol focuses on damaging the ocular surface of zebrafish through abrasion to assess the subsequent wound closure at the cellular level. This approach exploits an ocular burr to partly remove the corneal epithelium and uses scanning electron microscopy to track changes in cell morphology during wound closure.

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. ...

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

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