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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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>

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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 border="0" cellpadding="0" cellspacing="0" style="width:1159px;" width="1160">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">24 well plates</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:165px;">CLS3527</td>
			<td style="width:441px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">28 &#176;C incubator</td>
			<td style="width:227px;">LMS</td>
			<td align="right" style="width:165px;">210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Atorvastatin</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:165px;">PZ0001-5mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Breeding boxes</td>
			<td style="width:227px;">Thoren Aquatics systems</td>
			<td align="right" style="width:165px;">10011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Daniovision observation chamber</td>
			<td style="width:227px;">Noldus</td>
			<td style="width:165px;">n/a</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">E3 medium 1x</td>
			<td style="width:227px;"></td>
			<td style="width:165px;"></td>
			<td>4% Instant Ocean, 500 &#181;L methylene blue in 1 L dH2O</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:325px;">EthoVision XT software</td>
			<td style="width:227px;">Noldus</td>
			<td style="width:165px;">version 11</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Heat block</td>
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			<td>PHMT-PSC18</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Instant ocean</td>
			<td style="width:227px;">Instant Ocean</td>
			<td>SS15-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Lightsheet microscope</td>
			<td style="width:227px;">Zeiss</td>
			<td style="width:165px;">Z.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lightsheet microscope mounting capillary</td>
			<td style="width:227px;">Zeiss</td>
			<td>402100-9320-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Low melt agarose</td>
			<td style="width:227px;">Promega</td>
			<td style="width:165px;">V2111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Methylene Blue</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:165px;">319112-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Microscope&#160;</td>
			<td style="width:227px;">Leica</td>
			<td style="width:165px;">MZ95</td>
			<td>dissection microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Microscope</td>
			<td style="width:227px;">Leica</td>
			<td>M165FC</td>
			<td>fluorescent microscope</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;">MS222</td>
			<td style="width:227px;"></td>
			<td></td>
			<td>4g tricaine powder, 500 mL of dH2O, 10 mL of 1 M Tris (pH 9). Adjust pH to ~7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P1000 pipette</td>
			<td style="width:227px;">Gilson</td>
			<td>F144059M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">P1000 pipette tips</td>
			<td style="width:227px;">Starlab</td>
			<td style="width:165px;">S1122-1830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur pipettes</td>
			<td style="width:227px;">Starlab</td>
			<td style="width:165px;">E1414-0300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes&#160;</td>
			<td style="width:227px;">Corning</td>
			<td style="width:165px;">101VR20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipetboy</td>
			<td style="width:227px;">Integra Biosciences</td>
			<td style="width:165px;">PIPETBOY</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Stripette 25ml</td>
			<td style="width:227px;">Corning</td>
			<td>CLS3527</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Tricaine powder</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:165px;">A5040-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td style="width:227px;">Fisher BioReagents</td>
			<td style="width:165px;">BP152-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra fine dissection forceps</td>
			<td style="width:227px;">Agar scientific</td>
			<td style="width:165px;">AGT502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Zen software</td>
			<td style="width:227px;">Zeiss</td>
			<td style="width:165px;">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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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 de Cartagena UDEC

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

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

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 ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Using Zebrafish Larvae to Study the Pathological Consequences of Hemorrhagic Stroke

Summary

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Chapters in this video

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

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

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

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish