Name: stab wound injury model of the adult optic tectum using zebrafish and medaka for the comparative analysis of regenerative capacity
Uploaded: 2022-02-10T14:30:00
Description: Watch this Scientific Journal Video about Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity at JoVE.com

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 Materi...

<ol>
	<li>Becker, T., Wullimann, M. F., Becker, C. G., Bernhardt, R. R., Schachner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+regrowth+after+spinal+cord+transection+in+adult+zebrafish.">Axonal regrowth after spinal cord transection in adult zebrafish.</a> Journal of Comparative Neurology. 377 (4), 577-595 (1997).</li><li>Raymond, P. A., Barthel, L. K., Bernardos, R. L., Perkowski, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+retinal+stem+cells+and+their+niches+in+adult+zebrafish.">Molecular characterization of retinal stem cells and their niches in adult zebrafish.</a> BMC Developmental Biology. 6, 36 (2006).</li><li>M&#228;rz, M., Schmidt, R., Rastegar, S., Str&#228;hle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+response+following+stab+injury+in+the+adult+zebrafish+telencephalon.">Regenerative response following stab injury in the adult zebrafish telencephalon.</a> Developmental Dynamics. 240 (9), 2221-2231 (2011).</li><li>Kang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+tissue+repair+by+regeneration+enhancer+elements.">Modulation of tissue repair by regeneration enhancer elements.</a> Nature. 532 (7598), 201-206 (2016).</li><li>Sim&#245;es, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+directly+contribute+collagen+to+scar+formation+during+zebrafish+heart+regeneration+and+mouse+heart+repair.">Macrophages directly contribute collagen to scar formation during zebrafish heart regeneration and mouse heart repair.</a> Nature Communications. 11 (1), 600 (2020).</li><li>Hoang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatory+networks+controlling+vertebrate+retinal+regeneration.">Gene regulatory networks controlling vertebrate retinal regeneration.</a> Science. 370 (6519), (2020).</li><li>Alunni, A., Bally-Cuif, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+view+of+regenerative+neurogenesis+in+vertebrates.">A comparative view of regenerative neurogenesis in vertebrates.</a> Development. 143 (5), 741-753 (2016).</li><li>Diotel, N., L&#252;bke, L., Str&#228;hle, U., Rastegar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+and+distinct+features+of+adult+neurogenesis+and+regeneration+in+the+telencephalon+of+zebrafish+and+mammals.">Common and distinct features of adult neurogenesis and regeneration in the telencephalon of zebrafish and mammals.</a> Frontiers in Neuroscience. 14, 568930 (2020).</li><li>Labusch, M., Mancini, L., Morizet, D., Bally-Cuif, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+and+divergent+features+of+adult+neurogenesis+in+zebrafish.">Conserved and divergent features of adult neurogenesis in zebrafish.</a> Frontiers in Cell and Developmental Biology. 8, 525 (2020).</li><li>Ito, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+reparative+phenotypes+between+zebrafish+and+medaka+after+cardiac+injury.">Differential reparative phenotypes between zebrafish and medaka after cardiac injury.</a> Developmental Dynamics. 243 (9), 1106-1115 (2014).</li><li>Lai, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+analyses+in+zebrafish+and+medaka+reveal+that+harnessing+the+immune+response+promotes+cardiac+regeneration.">Reciprocal analyses in zebrafish and medaka reveal that harnessing the immune response promotes cardiac regeneration.</a> eLife. 6, 25605 (2017).</li><li>Lust, K., Wittbrodt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activating+the+regenerative+potential+of+M&#252;ller+glia+cells+in+a+regeneration-deficient+retina.">Activating the regenerative potential of M&#252;ller glia cells in a regeneration-deficient retina.</a> eLife. 7, 32319 (2018).</li><li>Shimizu, Y., Kawasaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regenerative+capacity+of+the+optic+tectum+of+adult+medaka+and+zebrafish.">Differential regenerative capacity of the optic tectum of adult medaka and zebrafish.</a> Frontiers in Cell and Developmental Biology. 9, 686755 (2021).</li><li>Adolf, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+and+acquired+features+of+adult+neurogenesis+in+the+zebrafish+telencephalon.">Conserved and acquired features of adult neurogenesis in the zebrafish telencephalon.</a> Developmental Biology. 295 (1), 278-293 (2006).</li><li>Grandel, H., Kaslin, J., Ganz, J., Wenzel, I., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+and+neurogenesis+in+the+adult+zebrafish+brain:+origin,+proliferation+dynamics,+migration+and+cell+fate.">Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration and cell fate.</a> Developmental Biology. 295 (1), 263-277 (2006).</li><li>Alunni, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+neural+stem+cells+in+the+medaka+optic+tectum+proliferation+zones.">Evidence for neural stem cells in the medaka optic tectum proliferation zones.</a> Developmental Neurobiology. 70 (10), 693-713 (2010).</li><li>Kuroyanagi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+zones+in+adult+medaka+(Oryzias+latipes)+brain.">Proliferation zones in adult medaka (Oryzias latipes) brain.</a> Brain Research. 1323, 33-40 (2010).</li><li>Ito, Y., Tanaka, H., Okamoto, H., Ohshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+neural+stem+cells+and+their+progeny+in+the+adult+zebrafish+optic+tectum.">Characterization of neural stem cells and their progeny in the adult zebrafish optic tectum.</a> Developmental Biology. 342 (1), 26-38 (2010).</li><li>Shimizu, Y., Ueda, Y., Ohshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+regulates+proliferation+and+differentiation+of+radial+glia+in+regenerative+processes+after+stab+injury+in+the+optic+tectum+of+adult+zebrafish.">Wnt signaling regulates proliferation and differentiation of radial glia in regenerative processes after stab injury in the optic tectum of adult zebrafish.</a> Glia. 66 (7), 1382-1394 (2018).</li><li>Ueda, Y., Shimizu, Y., Shimizu, N., Ishitani, T., Ohshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+sonic+hedgehog+and+notch+signaling+in+regenerative+neurogenesis+in+adult+zebrafish+optic+tectum+after+stab+injury.">Involvement of sonic hedgehog and notch signaling in regenerative neurogenesis in adult zebrafish optic tectum after stab injury.</a> Journal of Comparative Neurology. 526 (15), 2360-2372 (2018).</li><li>Kiyooka, M., Shimizu, Y., Ohshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+inhibition+promotes+regenerative+neurogenesis+after+stab+wound+injury+in+the+adult+zebrafish+optic+tectum.">Histone deacetylase inhibition promotes regenerative neurogenesis after stab wound injury in the adult zebrafish optic tectum.</a> Biochemical and Biophysical Research Communications. 529 (2), 366-371 (2020).</li><li>Shimizu, Y., Kawasaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetyltransferase+EP300+regulates+the+proliferation+and+differentiation+of+neural+stem+cells+during+adult+neurogenesis+and+regenerative+neurogenesis+in+the+zebrafish+optic+tectum.">Histone acetyltransferase EP300 regulates the proliferation and differentiation of neural stem cells during adult neurogenesis and regenerative neurogenesis in the zebrafish optic tectum.</a> Neuroscience Letters. 756, 135978 (2021).</li><li>Shimizu, Y., Kiyooka, M., Ohshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analyses+reveal+IL6/Stat3+signaling+involvement+in+radial+glia+proliferation+after+stab+wound+injury+in+the+adult+zebrafish+optic+tectum.">Transcriptome analyses reveal IL6/Stat3 signaling involvement in radial glia proliferation after stab wound injury in the adult zebrafish optic tectum.</a> Frontiers in Cell and Developmental Biology. 9, 668408 (2021).</li><li>Lindsey, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midbrain+tectal+stem+cells+display+diverse+regenerative+capacities+in+zebrafish.">Midbrain tectal stem cells display diverse regenerative capacities in zebrafish.</a> Scientific Reports. 9 (1), 4420 (2019).</li><li>Yu, S., He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+cell-cycle+entry+and+cell-state-dependent+fate+outputs+of+injury-reactivated+tectal+radial+glia+in+zebrafish.">Stochastic cell-cycle entry and cell-state-dependent fate outputs of injury-reactivated tectal radial glia in zebrafish.</a> eLife. 8, 48660 (2019).</li><li>Bisese, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+transcriptome+response+of+the+midbrain/diencephalon+to+injury+in+the+adult+mummichog+(Fundulus+heteroclitus).">The acute transcriptome response of the midbrain/diencephalon to injury in the adult mummichog (Fundulus heteroclitus).</a> Molecular Brain. 12 (1), 119 (2019).</li><li>Schmidt, R., Beil, T., Str&#228;hle, U., Rastegar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stab+wound+injury+of+the+zebrafish+adult+telencephalon:+a+method+to+investigate+vertebrate+brain+neurogenesis+and+regeneration.">Stab wound injury of the zebrafish adult telencephalon: a method to investigate vertebrate brain neurogenesis and regeneration.</a> Journal of Visualized Experiments. (4), e51753 (2014).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio) 5th ed. , (2007).</li><li>Kaslin, J., Kroehne, V., Ganz, J., Hans, S., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+of+neuroepithelial-like+and+radial+glia-like+progenitor+cells+in+cerebellar+regeneration.">Distinct roles of neuroepithelial-like and radial glia-like progenitor cells in cerebellar regeneration.</a> Development. 144 (8), 1462-1471 (2017).</li><li>Curado, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+targeted+cell+ablation+in+zebrafish:+a+new+tool+for+regeneration+studies.">Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies.</a> Developmental Dynamics. 236 (4), 1025-1035 (2007).</li><li>Shimizu, Y., Ito, Y., Tanaka, H., Ohshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radial+glial+cell-specific+ablation+in+the+adult+zebrafish+brain.">Radial glial cell-specific ablation in the adult zebrafish brain.</a> Genesis. 53 (7), 431-439 (2015).</li><li>Godoy, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+neurons+regenerate+following+chemogenetic+ablation+in+the+olfactory+bulb+of+adult+zebrafish+(Danio+rerio).">Dopaminergic neurons regenerate following chemogenetic ablation in the olfactory bulb of adult zebrafish (Danio rerio).</a> Scientific Reports. 10 (1), 12825 (2020).</li><li>Sawahata, M., Izumi, Y., Akaike, A., Kume, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+brain+ischemia-reperfusion+model+induced+by+hypoxia-reoxygenation+using+zebrafish+larvae.">In vivo brain ischemia-reperfusion model induced by hypoxia-reoxygenation using zebrafish larvae.</a> Brain Research Bulletin. 173, 45-52 (2021).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>Danio rerio 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>Oryzias latipes 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 ...

Stab wound injury is a mechanical injury method that induces nonspecific cell ablation. This method facilitates the evaluation of RGC proliferation and differentiation after brain injury. Needle-mediated stab wound injury is a simple, efficiently-implemented method to induce brain injury.This method can be applied to many experimental samples using a standard set of tools. The regenerative capacity of zebrafish was illustrated using the stab wound injury model. New insights into molecular mechanism regulating neuronal regeneration contribute to regenerative therapy in the central nervous system.Begin by placing an anesthetized adult zebrafish or medaka on a styrofoam tray. Fix the fish upright between two 30-gauge needles inserted vertically into the styrofoam. Hold the fish body to prevent the fish head from moving, and then vertically insert a 30-gauge needle through the skull into the medial region of one of the optic tectum spheres.Next, transfer the injured fish into a fish tank with fresh fish facility water. Once the fish have fully recovered from the anesthesia, move the tank to the breeding system. Put paper towels on the styrofoam tray to place the anesthetized fish.Then hold the fish body in place by vertically inserting two 30-gauge needles on either side of the anal fin. Once the fish is placed, make a ventral incision from the anus to the heart. Keep the heart visible by inserting two 30-gauge needles on each side.Then use the tip of the forceps to remove the silver epithelial layer. Insert the 30-gauge needle into the ventricle, keeping the bevel up, and make an incision into the atrium using the forceps. Next, push PBS into the atrium by pressing down on the syringe to flush the blood from the atrium.When the blood is drained and the gills turn white, stop the PBS perfusion. Later, remove the needles fixing the fish body in place, and fix the fish ventral side down. After cutting the spinal cord and removing the skull from the optic tectum and telencephalon, cut the optic nerves and dissect the brain.Next, transfer the brains into 1.5-milliliter tubes with one milliliter of 4%paraformaldehyde in PBS, to fix overnight at four degrees Celsius. After washing the fixed brains three times in PBS for five minutes each, transfer the brains into 1.5-milliliter tubes with one milliliter of 30%weight by volume sucrose in PBS as a cryoprotectant, and incubate them overnight at four degrees Celsius. Prepare the embedding compound by combining 30%sucrose with 30 milliliters of optimal cutting temperature, or OCT, compound, and store the compound at four degrees Celsius.To cool the cryomold once, incubate an aluminum block overnight at minus 80 degrees Celsius, and put the pre-cooled block in a styrofoam box with a lid to prevent warming. For coronal sections, embed the dissected brain in a cryomold by adjusting the orientation using forceps under the microscope. Next, place the cryomold on the pre-cooled aluminum block in the styrofoam box, and allow the OCT compound to freeze and become white.As the compound freezes, apply a small circle of OCT compound on a specimen disc before mounting the cryoblock on the specimen disc. By placing the specimen disc in the cryostat, freeze the OCT compound. Then place the specimen disc on the specimen head in the cryostat.After setting the orientation of the cryoblock, trim the OCT to remove any extra regions. Cut 14-micrometer thick serial sections through the whole optic tectum using a cryostat, and store the slides at minus 25 degrees Celsius. The radial glial cell, or RGC, proliferation in the brain tissue of zebrafish was evaluated.Most RGCs were proliferating cell antigen negative in the contralateral uninjured hemisphere. At two days post-injury, RGC proliferation was induced on the injured side of the medaka tectum. After the brain injury, cell lineage and RGC differentiation were evaluated with bromodeoxyuridine, or BrdU, labeling.The representative analysis shows newborn neurons at seven days post-injury in the injured zebrafish and medaka. One of the critical steps in stab wound injuries is manual needle insertion. A consistent injury is necessary for creating reproducible results.Fluorescent dyes are effective as wound markers. The extent of fluorescent dye diffusion indicates the location and depth of the stab wound injury. This technique is potentially adapted to other small fish species, and adaptation to medaka allows for comparisons of regenerative capacity.

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Research

JoVE Journal

Developmental Biology

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 ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L&#8722;1 in different concentrations of ERM at pH 7 and 25 &#176;C in the dark.
We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

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 ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its permeability, thereby allowing different molecules to be introduced to the cell. In this paper, electroporation is used to introduce plasmids to ependymoglial cells, which line the ventricular zone of the adult zebrafish telencephalon. A fraction of these cells shows stem cell properties and generates new neurons in the zebrafish brain; therefore, studying their behavior is essential to determine their roles in neurogenesis and regeneration. The introduction of plasmids via electroporation enables long-term labeling and tracking of a single ependymoglial cell. Furthermore, plasmids such as Cre recombinase or Cas9 can be delivered to single ependymoglial cells, which enables gene recombination or gene editing and provides a unique opportunity to assess the cell&#8217;s autonomous gene function in a controlled, natural environment. Finally, this detailed, step-by-step electroporation protocol is used to obtain successful introduction of plasmids into a large number of single ependymoglial cells.

Presented here is an electroporation method for plasmid DNA delivery and ependymoglial cell labeling in the adult zebrafish telencephalon. This protocol is a quick and efficient method to visualize and trace individual ependymoglial cells and opens up new possibilities to apply electroporation to a broad range of genetic manipulations.

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its ...