We would like to thank Dr David Spiller and the University of Manchester Systems Microscopy Core Facility for use of the equipment, Prof. Richard Baines for the use of DanioVision and Dr. Jack Rivers-Auty for statistical consultation. The bbh line was kindly shared by Nicole Munsie from Dr. Sarah Child&#39;s lab at the University of Calgary. We also thank Prof. Stephen Renshaw, Dr. Adam Hurlstone, Dr. Andrew Badrock and Dr. Helen Young for fish lines and equipment.
This study was supported by the NC3Rs (NC/N002598/1), Stroke Association (TSA LECT 2017/02), ERA-NET NEURON (MR/M501803/1) and The British Heart Foundation (FS/15/67/32038). We are also particularly thankful to The Natalie Kate Moss Trust and the University of Manchester Faculty of Biology, Medicine and Health for their continued financial support.

Zebrafish were raised and maintained at The University of Manchester Biological Services Unit under standard conditions as previously described21. Adult zebrafish husbandry was approved by the University of Manchester Animal Welfare and Ethical Review Board. All experiments were performed in accordance with U.K. Home Office regulations (PPL:P132EB6D7).
NOTE: Transgenic lines used in this study include macrophage-specific lineage mpeg1:mCherry (constructed in-house as previously described22), neutrophil-specific mpo:GFP23,erythroid-specific gata1:dsRed24 and ubiq:secAnnexinV-mVenus, a reporter for cell death (re-derived in house25)on wild-type, nacre (mitfaw2/w2) and mutant (bbhm292) backgrounds. Figure 1 shows the experimental timeline.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59716/59716fig1.jpg" style="opacity: 0.9; font-size: 14px;" /> 
Figure 1:&#160;Graphic of experimental timeline to characterize brain injury, locomotor and neuroinflammatory outcomes.&#160;ICH, intracerebral hemorrhage; bbh, bubblehead. Figure has been reproduced from Crilly et al.20&#160;with permission under a Creative Commons license.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59716/59716fig1large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>
1. Day 0: Egg production and collection
<ol>
	<li>Collect fertilized embryos from natural spawning in breeding boxes produced from 1 male and 1-2 female adult zebrafish. 
	NOTE: For atorvastatin protocol any wildtype/transgenic animals can be used however hemorrhage rates differ slightly between strains.</li>
	<li>Incubate 100 embryos at 28 &#176;C in standard E3 embryo medium per Petri dish and stage according to standard guidelines26.</li>
	<li>At ~6 hours post fertilization (hpf) remove dead and unfertilized embryos from the dish using a Pasteur pipette.</li>
</ol>
2. Day 1: Atorvastatin treatment at 24 hpf
<ol>
	<li>Dechorionate embryos for atorvastatin treatment using sharp ultra thin dissection forceps27. Numbers required for experimentation can be adjusted accordingly.</li>
	<li>Add 30 mL of E3 embryo medium to two clean Petri dishes. Use one dish for 100 embryos. 
	NOTE: If plates are designed for cell culture, dechorionated zebrafish at this early stage often stick to the bottom. To avoid this, rinse the plates thoroughly in clean water before use.</li>
	<li>Remove 60 &#181;L of embryo water from the treatment plate and add 60 &#181;L of 0.5 mM atorvastatin (ATV). At a 0.5 mM stock concentration, the above dilution will result in a final concentration of 1 &#181;M which will result in ~20% of larvae non-hemorrhaged (ICH-) and ~80% of larvae hemorrhaged (ICH+). Use the other plate for untreated controls 
	NOTE: Atorvastatin is solubilized in distilled water (3 mg into 10 mL) to make a 0.5 mM stock solution. Incubate overnight at room temperature in the dark with agitation as solubilization takes some time. Complete solubilization can take up to 1 week. Do not use DMSO. Solution is aliquoted and stored at -20&#176;C. Do not freeze thaw.</li>
	<li>Using a Pasteur pipette, transfer 100 embryos in as little water as possible to the treatment plates.</li>
	<li>Incubate the plates at 28 &#176;C. 
	NOTE: ICH will occur between 33 and 48 hpf. Atorvastatin does not need to be removed as incubation longer than 24 h does not cause any further developmental issues.</li>
</ol>
3. Day 2: Separating ICH- and ICH+ populations at 50 hpf
<ol>
	<li>Separate ICH+ fish from ICH- populations and transfer to new dishes for ease.
	<ol>
		<li>If using the ATV model at a concentration of 1 &#181;M, 75-100% of larvae will exhibit hemorrhage (ICH+) at this time point. 
		NOTE: The response of the larvae differs between strains, if larvae are not hemorrhaging at the desired frequencies, use a fresh batch of atorvastatin or a higher concentration. If larvae have not exhibited hemorrhage by 48 hpf then consider them ICH-.</li>
		<li>If using the bbh model, all homozygous mutants will exhibit hemorrhage by 48 hpf. If using a heterozygous incross, the ICH- heterozygous and wildtype siblings can be used as control animals for experiments.</li>
	</ol>
	</li>
	<li>If necessary, anaesthetize the larvae by adding 0.02% MS222 to the E3 media. Using a Pasteur pipette, sort the larvae for presence of blood in the head into fresh E3 media. See Figure 2. 
	NOTE: Blood in the head may appear in the fore, mid or hindbrain or in combination, and bleed volume can vary between animals. In the bbh mutants, ICH is often associated with severe edema recognizable by larger heads, wider space between the eyes and a more diffuse bleed. However not all ICH+ bbh larvae exhibit edema.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59716/59716fig2.jpg" style="opacity: 0.9; font-size: 14px;" /> 
Figure 2:&#160;ICH+ brain hemorrhage phenotypes.&#160;Examples of larval ICH phenotypes maintained on a transgenic gata1:DsRed reporter nacre background observed with a brightfield stereomicroscope (top panels) and fluorescence (bottom panel) at ~48 h post-fertilization. No hemorrhages were observed in ICH-larvae (left panels). A distinct accumulation of red blood cells in the forebrain and hindbrain (arrows) were observed in ICH+ larvae (right panels). Scale bars represent 250 &#181;m. Figure has been reproduced from Crilly et al.20&#160;with permission under a Creative Commons license.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59716/59716fig2large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>
4. Day 3: Cell death and leukocyte analysis at 72 hpf
<ol>
	<li>Screen the larvae using a fluorescence microscope to ensure the expression of fluorescent protein. 
	NOTE: In this study, transgenic ubiq:secAnnexinV-mVenus larvae were used to report brain cell death and double transgenic mpo:GFP;mpeg1:mCherry or ubiq:secAnnexinV-mVenus;mpeg1:mCherry used for leukocyte analysis.</li>
	<li>Fill the lightsheet mounting chamber with E3 media containing 0.02% MS222.</li>
	<li>Anaesthetize the larvae using 0.02% MS222. Transfer larvae for mounting (n = 1-6) to a dry Petri dish surface in a single droplet. Remove as much liquid as possible. 
	NOTE: 1.5% low melt agarose&#160;is prepared using 0.15 g of low melt agarose dissolved in 10 mL of E3 medium without methylene blue in a microwave and kept at 45 &#176;C until use.</li>
	<li>Add a drop of 1.5% low-melt agarose (maintained as liquid in a 45 &#176;C heat block) to the larvae and using a 800 &#181;m mounting capillary, and draw the larvae up head first. If positioning is not accurate the larvae can be expelled from the agarose and mounted again. Leave the capillary to cool before inserting into the lightsheet chamber. 
	NOTE: Alternatively, a confocal microscope could be used for this procedure. For this, larvae should be mounted laterally in agarose on a glass bottom dish.</li>
	<li>Acquire z-stack images of the head between the eye lenses (~300 &#181;m) and process to maximum intensity projection image.
	<ol>
		<li>Analyze brain region from images collected for total number of fluorescent cells and total intensity fluorescence20.</li>
	</ol>
	</li>
	<li>Create a time lapse video of multiple projection composites over 18-24 h to track leukocyte mobility and interaction with dying cells. 
	NOTE: If long term live imaging is performed, only one larva is mounted in the capillary.</li>
	<li>When imaging is completed expel the larvae from the mounting capillary into a lethal overdose of 4% MS222 to euthanize.</li>
</ol>
5. Day 3: Selecting larvae for motility assay at 72 hpf
<ol>
	<li>Anaesthetize larvae in the petri dishes with 0.02% MS222.</li>
	<li>Randomly select n = 24 larvae for motility assay and transfer into fresh E3 media and allow animals to recover from anesthetic. 
	NOTE: Anesthetic at this point removes selection bias for slow swimmers that are easy to catch.</li>
</ol>
6. Day 3-5: Assaying locomotion at 72, 96 and 120 hpf
<ol>
	<li>Transfer larvae selected at 72 hpf into E3 medium without methylene blue. 
	NOTE: For assaying at 3 dpf allow the larvae ample time to recover from anesthetic (&#62;1 h).</li>
	<li>Plate one larva in 1 mL per well of a 24-well plate using a pipette. 
	NOTE: Cut the end of the pipette tip to avoid damaging the larvae. Plate size can be changed according to experimental design.</li>
	<li>Load plates into the camera chamber and assay motion for 10 min using a white light startle routine to increase spontaneous swimming. 
	NOTE: Swimming behavior was tracked using a camera chamber and tracking software (see the Table of Materials).</li>
	<li>Repeat experiment with the same larvae at 96 and 120 hpf. Move larvae from individual housing in assay plate to a Petri dish and incubate at 28 &#176;C in between assays.</li>
	<li>At assay completion, euthanize larvae in a lethal overdose of 4% MS222.</li>
</ol>

<ol>
	<li>An, S. J., Kim, T. J., Epidemiology Yoon, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology,+risk+factors,+and+clinical+features+of+intracerebral+hemorrhage:+an+update.">Epidemiology, risk factors, and clinical features of intracerebral hemorrhage: an update.</a> Journal of stroke. 19 (1), 3 (2017).</li><li>WHO. <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 top 10 causes of death. , (2017).</li><li>Casals, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+animal+models+for+stroke+research:+a+review.">The use of animal models for stroke research: a review.</a> Comparative medicine. 61 (4), 305-313 (2011).</li><li>Kellner, C. P., Connolly, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+Strategies+for+Intracerebral+Hemorrhage+Trials+and+Translation.">Neuroprotective Strategies for Intracerebral Hemorrhage Trials and Translation.</a> Stroke. 41 (10 Suppl 1), S99-S102 (2010).</li><li>Kirkman, M. A., Allan, S. M., Parry-Jones, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+intracerebral+hemorrhage:+avoiding+pitfalls+in+translational+research.">Experimental intracerebral hemorrhage: avoiding pitfalls in translational research.</a> Journal of Cerebral Blood Flow and Metabolism. 31 (11), 2135-2151 (2011).</li><li>ASPA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 1986 amendments. , (2012).</li><li>Butler, M. G., Gore, A. V., Weinstein, B. 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> Methods in cell biology. 105, 137-161 (2011).</li><li>Walcott, B. P., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+of+cerebrovascular+disease.">Zebrafish models of cerebrovascular disease.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 34 (4), 571-577 (2014).</li><li>Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+mediate+the+repair+of+brain+vascular+rupture+through+direct+physical+adhesion+and+mechanical+traction.">Macrophages mediate the repair of brain vascular rupture through direct physical adhesion and mechanical traction.</a> Immunity. 44 (5), 1162-1176 (2016).</li><li>Kasher, P. R. <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+samhd1+morphant+zebrafish+recapitulates+features+of+the+human+type+I+interferonopathy+Aicardi-Goutieres+syndrome.">Characterization of samhd1 morphant zebrafish recapitulates features of the human type I interferonopathy Aicardi-Goutieres syndrome.</a> The Journal of Immunology. 194 (6), 2819-2825 (2015).</li><li>Wen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+rnf213a+by+TALEN+causes+abnormal+angiogenesis+and+circulation+defects+in+zebrafish.">Mutation of rnf213a by TALEN causes abnormal angiogenesis and circulation defects in zebrafish.</a> Brain research. 1644, 70-78 (2016).</li><li>Eisa-Beygi, S., Rezaei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Etiology+of+intracerebral+hemorrhage+(ICH):+novel+insights+from+Zebrafish+embryos.">Etiology of intracerebral hemorrhage (ICH): novel insights from Zebrafish embryos.</a> International Journal of Developmental Biology. 60 (4-5-6), 119-126 (2016).</li><li>Mracsko, E., Veltkamp, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation+after+intracerebral+hemorrhage.">Neuroinflammation after intracerebral hemorrhage.</a> Frontiers in cellular neuroscience. 8, 388 (2014).</li><li>Eisa-Beygi, S., Hatch, G., Noble, S., Ekker, M., Moon, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+3-hydroxy-3-methylglutaryl-CoA+reductase+(HMGCR)+pathway+regulates+developmental+cerebral-vascular+stability+via+prenylation-dependent+signalling+pathway.">The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway regulates developmental cerebral-vascular stability via prenylation-dependent signalling pathway.</a> Developmental biology. 373 (2), 258-266 (2013).</li><li>Shen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+Rho-kinase+inhibitors+by+docking-based+virtual+screening.">Discovery of Rho-kinase inhibitors by docking-based virtual screening.</a> Molecular BioSystems. 9 (6), 1511-1521 (2013).</li><li>Huang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tanshinone+I+prevents+atorvastatin-induced+cerebral+hemorrhage+in+zebrafish+and+stabilizes+endothelial+cell-cell+adhesion+by+inhibiting+VE-cadherin+internalization+and+actin-myosin+contractility.">Tanshinone I prevents atorvastatin-induced cerebral hemorrhage in zebrafish and stabilizes endothelial cell-cell adhesion by inhibiting VE-cadherin internalization and actin-myosin contractility.</a> Pharmacological research. , (2017).</li><li>Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+a+ROCK+inhibitor,+FPND,+which+prevents+cerebral+hemorrhage+through+maintaining+vascular+integrity+by+interference+with+VE-cadherin.">Discovery of a ROCK inhibitor, FPND, which prevents cerebral hemorrhage through maintaining vascular integrity by interference with VE-cadherin.</a> Cell death discovery. 3, 17051 (2017).</li><li>Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+&#946;Pix-Pak2a+signaling+pathway+regulates+cerebral+vascular+stability+in+zebrafish.">A &#946;Pix-Pak2a signaling pathway regulates cerebral vascular stability in zebrafish.</a> Proceedings of the National Academy of Sciences. 104 (35), 13990-13995 (2007).</li><li>ten Klooster, P. J., Jaffer, Z. M., Chernoff, J., Hordijk, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+and+activation+of+Rac1+are+mediated+by+the+exchange+factor+&#946;-Pix.">Targeting and activation of Rac1 are mediated by the exchange factor &#946;-Pix.</a> The Journal of cell biology. 172 (5), 759-769 (2006).</li><li>Crilly, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+zebrafish+larval+models+to+study+brain+injury,+locomotor+and+neuroinflammatory+outcomes+following+intracerebral+haemorrhage.">Using zebrafish larval models to study brain injury, locomotor and neuroinflammatory outcomes following intracerebral haemorrhage.</a> F1000Research. 7, (2018).</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. , (2000).</li><li>Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A., Lieschke, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mpeg1+promoter+transgenes+direct+macrophage-lineage+expression+in+zebrafish.">mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish.</a> Blood. 117 (4), e49-e56 (2011).</li><li>Renshaw, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+zebrafish+model+of+neutrophilic+inflammation.">A transgenic zebrafish model of neutrophilic inflammation.</a> Blood. 108 (13), 3976-3978 (2006).</li><li>Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nature immunology. 4 (12), 1238 (2003).</li><li>Morsch, M. <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+characterization+of+microglial+engulfment+of+dying+neurons+in+the+zebrafish+spinal+cord.">In vivo characterization of microglial engulfment of dying neurons in the zebrafish spinal cord.</a> Frontiers in cellular neuroscience. 9, (2015).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental dynamics. 203 (3), 253-310 (1995).</li><li>Liang, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish in the Classroom. , (2011).</li><li>Yang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miconazole+protects+blood+vessels+from+MMP9-dependent+rupture+and+hemorrhage.">Miconazole protects blood vessels from MMP9-dependent rupture and hemorrhage.</a> Disease Models &amp; Mechanisms. 10 (3), 337-348 (2017).</li><li>Andaluz, N., Zuccarello, M., Wagner, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+animal+models+of+intracerebral+hemorrhage.">Experimental animal models of intracerebral hemorrhage.</a> Neurosurgery Clinics of North America. 13 (3), 385-393 (2002).</li><li>Rosenberg, G. A., Mun-Bryce, S., Wesley, M., Kornfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagenase-induced+intracerebral+hemorrhage+in+rats.">Collagenase-induced intracerebral hemorrhage in rats.</a> Stroke. 21 (5), 801-807 (1990).</li></ol>

The authors have nothing to disclose.

This study shows that ICH in zebrafish larvae induces a brain injury response that recapitulates key aspects of the human condition that can be systematically assayed and quantified. Zebrafish offer a consistent and reproducible model of spontaneous ICH which will assist with future drug intervention studies focused on targeting blood-induced brain injury, rather than preventing vessel rupture17,28. Indeed, given the rapid nature of disease onset akin to the clinical situation, such an approach offers exciting prospects for successful translation in the future.
Some limitations are associated with the use of zebrafish larvae, such as the use of a developing system and taxonomic rank, however the practical and scientific advantages of this model must be considered to offer new insights into ICH. No surgery is required to initiate a haemorrhage or to monitor cellular processes over extended periods of time after injury. High fecundity of zebrafish pairings generate easily accessible and large sample sizes, and due to the fast development of the larvae the experimental timeline is significantly reduced compared to rodent studies29,30. 
Currently these models are fit to use for elucidating the immediate pathological and immunological response to spontaneous ICH in the brain of live intact animals. Potentially, this model can be adapted for medium-high throughput drug screens for ICH therapies, whether preventative or recovery promoting. As such, the post-ICH pathologies presented in this study represent an alternative, complementary platform for pre-clinical ICH research.

Intracerebral hemorrhage (ICH) is the most severe sub-type of stroke associated with spontaneous cerebral vessel rupture and bleeding into the parenchyma leading to brain damage, physical disability and often death1. Despite the high mortality and morbidity rate associated with ICH2, understanding of the underpinning etiology and post-hemorrhage pathology is still lacking. As such, there are no specific treatments to prevent ICH or improve patient outcomes. Most of our understanding of disease biology has come from pre-clinical rodent models of ICH3, however studies to-date in these models have failed to translate any successful therapeutic to the clinic4,5. This failure may be due in part, to some limitations of these preclinical models, including the inability to easily recapitulate the spontaneous nature of human disease and the requirement for invasive surgery to generate the models in mammals6. Additionally, rodents pose practical problems with regards to observing the rapid onset of cellular responses to ICH in intact tissue. Given the lack of translation from rodent models, developing alternative models of spontaneous ICH is imperative if we are to overcome these practical problems and help identify novel drug targets.
The molecular mechanisms of vascular development are well conserved amongst vertebrates including zebrafish (Danio rerio)7. As such, the adoption of this model organism is becoming an ever more useful mechanistic strategy for studying cerebrovascular disease8. A number of zebrafish models have been generated which recapitulate phenotypes associated with stroke-related conditions9,10,11,12. The use of zebrafish larvae to investigate disease pathogenesis offers both practical and scientific advantages over mammalian models8. This includes high reproduction rates, rapid development and larval transparency that allows for intravital imaging without the invasive constraints associated with rodents. Coupling these advantages with the wide range of transgenic reporter lines available within the zebrafish research community amounts to a powerful in vivo approach for studying disease biology, not yet utilized for studying the pathological consequences of ICH.
The injury response to blood in the brain is biphasic13; the primary insult causes neuronal death and cell necrosis, which then initiates a secondary wave of damage that is induced by innate immune activation. The second phase of brain injury, in particular the neuroinflammatory component, is considered a realistic target for future drug treatment13. Spontaneous and cerebral-specific hemorrhages have been described in zebrafish larvae previously14,15,16,17,18,19. Two such models are the use of atorvastatin (ATV) at 24 h post-fertilization (hpf) to inhibit the HMGCR pathway and cholesterol biosynthesis14, and a bubblehead (bbh) mutant which express a hypomorphic mutation in the arhgef7 gene, &#946;pix, and subsequently inhibits actin remodeling for tight endovascular junctions18. These models exhibit spontaneous cerebral-specific blood vessel rupture at the onset of circulation (~33 hpf).&#160;Recently, we have characterized these models further to reveal that key aspects of the brain injury response is conserved between humans and zebrafish larvae20. This study demonstrates the methodology required to obtain and visualize spontaneous brain hemorrhages in zebrafish larvae and how to quantify brain injury, and locomotor and neuroinflammatory phenotypes that relate to the human condition. These data and techniques support the use of this model species as a valuable complementary system for pre-clinical ICH research.

<table><tbody><tr><td>24 well plates</td><td>Sigma-Aldrich</td><td>CLS3527</td><td></td></tr><tr><td>28 &amp;deg;C incubator</td><td>LMS</td><td>210</td><td></td></tr><tr><td>Atorvastatin</td><td>Sigma-Aldrich</td><td>PZ0001-5mg</td><td></td></tr><tr><td>Breeding boxes</td><td>Thoren Aquatics systems</td><td>10011</td><td></td></tr><tr><td>Daniovision observation chamber</td><td>Noldus</td><td>n/a</td><td></td></tr><tr><td>E3 medium 1x</td><td></td><td></td><td>4% Instant Ocean, 500 &amp;micro;L methylene blue in 1 L dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>EthoVision XT software</td><td>Noldus</td><td>version 11</td><td></td></tr><tr><td>Heat block</td><td>Grant-Bio</td><td>PHMT-PSC18</td><td></td></tr><tr><td>Instant ocean</td><td>Instant Ocean</td><td>SS15-10</td><td></td></tr><tr><td>Lightsheet microscope</td><td>Zeiss</td><td>Z.1</td><td></td></tr><tr><td>Lightsheet microscope mounting capillary</td><td>Zeiss</td><td>402100-9320-000</td><td></td></tr><tr><td>Low melt agarose</td><td>Promega</td><td>V2111</td><td></td></tr><tr><td>Methylene Blue</td><td>Sigma-Aldrich</td><td>319112-100ML</td><td></td></tr><tr><td>Microscope&amp;nbsp;</td><td>Leica</td><td>MZ95</td><td>dissection microscope</td></tr><tr><td>Microscope</td><td>Leica</td><td>M165FC</td><td>fluorescent microscope</td></tr><tr><td>MS222</td><td></td><td></td><td>4g tricaine powder, 500 mL of dH&lt;sub&gt;2&lt;/sub&gt;O, 10 mL of 1 M Tris (pH 9). Adjust pH to ~7</td></tr><tr><td>P1000 pipette</td><td>Gilson</td><td>F144059M</td><td></td></tr><tr><td>P1000 pipette tips</td><td>Starlab</td><td>S1122-1830</td><td></td></tr><tr><td>Pasteur pipettes</td><td>Starlab</td><td>E1414-0300</td><td></td></tr><tr><td>Petri dishes&amp;nbsp;</td><td>Corning</td><td>101VR20</td><td></td></tr><tr><td>Pipetboy</td><td>Integra Biosciences</td><td>PIPETBOY</td><td></td></tr><tr><td>Stripette 25ml</td><td>Corning</td><td>CLS3527</td><td></td></tr><tr><td>Tricaine powder</td><td>Sigma-Aldrich</td><td>A5040-25G</td><td></td></tr><tr><td>Tris Base</td><td>Fisher BioReagents</td><td>BP152-1</td><td></td></tr><tr><td>Ultra fine dissection forceps</td><td>Agar scientific</td><td>AGT502</td><td></td></tr><tr><td>Zen software</td><td>Zeiss</td><td>version 2.3</td><td></td></tr></tbody></table>

using zebrafish larvae to study the pathological consequences of hemorrhagic stroke

Despite being the most severe subtype of stroke with high global mortality, there is no specific treatment for patients with intracerebral hemorrhage (ICH). Modelling ICH pre-clinically has proven difficult, and current rodent models poorly recapitulate the spontaneous nature of human ICH. Therefore, there is an urgent requirement for alternative pre-clinical methodologies for study of disease mechanisms in ICH and for potential drug discovery.
The use of zebrafish represents an increasingly popular approach for translational research, primarily due to a number of advantages they possess over mammalian models of disease, including prolific reproduction rates and larval transparency allowing for live imaging. Other groups have established that zebrafish larvae can exhibit spontaneous ICH following genetic or chemical disruption of cerebrovascular development. The aim of this methodology is to utilize such models to study the pathological consequences of brain hemorrhage, in the context of pre-clinical ICH research. By using live imaging and motility assays, brain damage, neuroinflammation and locomotor function following ICH can be assessed and quantified.
This study shows that key pathological consequences of brain hemorrhage in humans are conserved in zebrafish larvae highlighting the model organism as a valuable in vivo&#160;system for pre-clinical investigation of ICH. The aim of this methodology is to enable the pre-clinical stroke community to employ the zebrafish larval model as an alternative complementary model system to rodents.

Assessment of brain cell death using transgenic ubiq:secAnnexinV-mVenus results in clear definitive clusters of dying cells in ICH+ larvae in both ATV and bbh models that are absent in all ICH- larvae (Figure 3). Clusters recede before 96 hpf. Through image analysis, bleeding is associated with a significant two-fold increase in total intensity of fluorescence signal in the brain, indicating marked cell death.
A neuroinflammatory response is identified in ICH+ larvae by significantly increased numbers of mpeg1 positive macrophage cells in the brain. The number of total mpo positive neutrophil cells also increased however this did not reach statistical significance (Figure 4). The morphology of the mpeg1 positive macrophages can also be seen to change in ICH+ larvae as the cells adopt an active, rounded, amoeboid shape. These activated rounded cells can also be monitored over time to show an increased phagocytic response of the ubiq:secAnnexinV-mVenus expressing dying cells in ICH+ larvae (Figure 5). mpeg1 positive macrophages exhibiting ramified processes were categorized as inactive.
Brain hemorrhage is associated with a significant decrease in motility at 72 and 96 hpf in comparison to ICH- sibling controls in both bbh and ATV models (Figure 6). Motility at 120 hpf recovers to near baseline levels. There are often differences in baseline motility between egg clutches and strains and so comparison should be made to ICH- controls every time.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59716/59716fig3.jpg" /> 
Figure 3: Intracerebral hemorrhage (ICH) in zebrafish larvae results in a quantifiable brain injury. (A) Representative images of the brain injury phenotype in ICH+ larvae (right panels), in comparison to ICH- siblings (left panels), at 72 hpf. Brightfield images (bottom panels, scale bar = 250 &#181;m) demonstrate the presence of brain bleeds (arrows) in ICH+ larvae. Fluorescent microscopy was performed to visualize cell death in the ubiq:secAnnexinV-mVenus reporter line (top panels, scale bar = 100 &#181;m). Clusters of dying cells were observed in peri-hematomal regions. Images were cropped to brain-only regions and analyzed for total green fluorescence intensity in round particles larger than 30 pixels in diameter (white line) using the macro in Supplementary File 1. (B) Quantification of fluorescence signal in the brains of untreated, ICH- and ICH+ larvae obtained through the ATV model (n = 12 per group; 3 independent replicates) at 72 hpf. Significant differences were observed when comparing ICH+ with untreated (**p = 0.004) and with ICH- (*p = 0.03) siblings. (C) Quantification of fluorescence signal as a read out for annexinV binding in the brains of ICH- and ICH+ larvae obtained through the bubblehead (bbh) model (n = 12 per group; 2 independent replicates) at 72 hpf. Graphs show SD from the mean. A significant difference in mVenus fluorescence was observed between ICH+ and ICH- age-matched siblings (**p = 0.002). Figure has been reproduced from Crilly et al.20 with permission under a Creative Commons license. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59716/59716fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59716/59716fig4.jpg" /> 
Figure 4:&#160;Intracerebral hemorrhage (ICH) initiates an innate cellular immune response in the zebrafish larval brain. Numbers of leukocytes quantified within the brain regions previously described for mpo:GFP;mpeg1:dsRed double transgenic larvae (n = 8 per group; 2 independent replicates) at 72 hpf reveals a significant increase in macrophages (*p = 0.01), but not neutrophils (p = 0.5), in response to ICH. Figure has been reproduced from Crilly et al.20 with permission under a Creative Commons license. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59716/59716fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59716/59716fig5.jpg" /> 
Figure 5:&#160;Activated macrophage cells show a phagocytic response to the brain lesion. (A) Representative time-lapse stills20 showing a ramified patrolling macrophage migrating towards an annexinV positive cell (i - vi). Stills are obtained from a series of images taken of the whole brain using a 20x objective. Scale bar represents 50 &#181;m. The macrophage acquired an amoeboid morphology (v) before phagocytosing the annexinV-positive cell (vi, vii). After phagocytosis the macrophage resumes a ramified morphology and migrates away and the annexinV-positive cell can no longer be seen (viii). Ramified macrophage (#), annexinV positive cell (arrow), amoeboid macrophage (*) are indicated. (B) Representative images of mpeg1-positive cells in the ICH- and ICH+ larval brain exhibiting amoeboid and ramified morphologies. Scale bars represent 50 &#181;m. (C) An increased proportion of amoeboid (phagocytic) and decreased proportion of ramified (inactive) macrophages was observed in ICH+ brains in comparison to ICH- siblings. Figure has been reproduced from Crilly et al.20 with permission under a Creative Commons license. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59716/59716fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59716/59716fig6.jpg" /> 
Figure 6:&#160;ICH-induced brain injury results in a quantifiable locomotor deficit in zebrafish larvae. (A) Representative examples of the swimming tracks in ICH- and ICH+ bbh larvae at 72, 96 and 120 hpf. (B) ICH+ larvae exhibited a significant decrease in the cumulative time spent mobile during the 10 min recording period at both 72 and 96 hpf. Significance was lost at the 120 hpf time point potentially alluding to recovery from brain injury (n = 24 larvae per group; 3 independent replicates; ****p = 0.00006; **p = 0.003; ns: p = 0.08). (C) Quantification of cumulative time spent moving in untreated and ATV-treated ICH- and ICH+ larvae at 120 hpf. ICH+ larvae exhibited a significant decrease in the cumulative time spent mobile during the 10 min recording period. Three technical replicates (n = 24 larvae per group) were used to calculate SD from the mean (***p = 0.00004, **p = 0.0003). Figure has been reproduced from Crilly et al.20 with permission under a Creative Commons license. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59716/59716fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59716/Supplementary_File_1-Macro.docx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Using Zebrafish Larvae to Study the Pathological Consequences of Hemorrhagic Stroke at JoVE.com

Using Zebrafish Larvae to Study the Pathological Consequences of Hemorrhagic Stroke

Here we present a protocol to quantify brain injury, locomotor deficits and neuroinflammation following bleeding in the brain in zebrafish larvae, in the context of human intracerebral hemorrhage (ICH).

Despite being the most severe subtype of stroke with high global mortality, there is no specific treatment for patients with intracerebral hemorrhage ...

using-zebrafish-larvae-to-study-pathological-consequences-hemorrhagic

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

8.2K Views.  University of Manchester. Here we present a protocol to quantify brain injury, locomotor deficits and neuroinflammation following bleeding in the brain in zebrafish larvae, in the context of human intracerebral hemorrhage (ICH). 

Video: Using Zebrafish Larvae to Study the Pathological Consequences of Hemorrhagic Stroke

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Developmental Biology

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain&#39;s macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy Nanotomy of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution EM. Here we describe a universal method for nanotomy applied to investigate the zebrafish larval brain in health and upon non-invasive brain injury.

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously ...

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 ...

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 ...

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.

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 ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Biology

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...

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 ...

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes this animal model very useful for studying spinal cord injury and regeneration. The standard protocol for inducing such lesions is to transect the dorsal part of the trunk manually. However, this technique requires extensive training and damages additional tissues. A protocol was developed for laser-induced lesions to circumvent these limitations, allowing for high reproducibility and completeness of spinal cord transection over many animals and between different sessions, even for an untrained operator. Furthermore, tissue damage is mainly limited to the spinal cord itself, reducing confounding effects from injuring different tissues, e.g., skin, muscle, and CNS. Moreover, hemi-lesions of the spinal cord are possible. Improved preservation of tissue integrity after laser injury facilitates further dissections needed for additional analyses, such as electrophysiology. Hence, this method offers precise control of the injury extent that is unachievable manually. This allows for new experimental paradigms in this powerful model in the future.

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes ...