The authors would like to thank the Hyde lab members for their thoughtful discussions, the Freimann Life Sciences Center technicians for zebrafish care and husbandry, and the University of Notre Dame Optical Microscopy Core/NDIIF for the use of instruments and their services. This work was supported by the Center for Zebrafish Research at the University of Notre Dame, the Center for Stem Cells and Regenerative Medicine at the University of Notre Dame, and grants from National Eye Institute of NIH R01-EY018417 (DRH), the National Science Foundation Graduate Research Fellowship Program (JTH), LTC Neil Hyland Fellowship of Notre Dame (JTH), Sentinels of Freedom Fellowship (JTH), and the Pat Tillman Scholarship (JTH).

Zebrafish were raised and maintained in the Notre Dame Zebrafish facility in the Freimann Life Sciences Center. The methods described in this manuscript were approved by the University of Notre Dame Animal Care and Use Committee.
1. Traumatic brain injury paradigm
<ol>
	<li>Add 1 mL of 2-phenoxyethanol to 1 L of system water (60 mg of Instant Ocean in 1 L of deionized RO water).</li>
	<li>Prepare an aerated recovery tank containing 2 L of system water at room temperature.</li>
	<li>Select th...

<ol>
	<li>Centers for Disease Control and Prevention. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveillance+Report+of+Traumatic+Brain+Injury-related+Emergency+Department+Visits,+Hospitalizations,+and+Deaths-United+States,+2014.">Surveillance Report of Traumatic Brain Injury-related Emergency Department Visits, Hospitalizations, and Deaths-United States, 2014.</a> Centers for Disease Control and Prevention, U.S. Department of Health and Human Services. , (2019).</li><li>Galgano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+brain+injury:+current+treatment+strategies+and+future+endeavors.">Traumatic brain injury: current treatment strategies and future endeavors.</a> Cell transplantation. 26 (7), 1118-1130 (2017).</li><li>Santiago, L. A., Oh, B. C., Dash, P. K., Holcomb, J. B., Wade, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clinical+comparison+of+penetrating+and+blunt+traumatic+brain+injuries.">A clinical comparison of penetrating and blunt traumatic brain injuries.</a> Brain injury. 26 (2), 107-125 (2012).</li><li>Korley, F. K., Kelen, G. D., Jones, C. M., Diaz-Arrastia, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergency+department+evaluation+of+traumatic+brain+injury+in+the+United+States,+2009-2010.">Emergency department evaluation of traumatic brain injury in the United States, 2009-2010.</a> The Journal of Head Trauma Rehabilitation. 31 (6), 379-387 (2016).</li><li>Faul, M., Xu, L., Wald, M., Coronado, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Traumatic Brain Injury in the United States: Emergency Department Visits, Hospitalizations and Deaths. , (2010).</li><li>Campbell, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch3+and+DeltaB+maintain+M&#252;ller+glia+quiescence+and+act+as+negative+regulators+of+regeneration+in+the+light-damaged+zebrafish+retina.">Notch3 and DeltaB maintain M&#252;ller glia quiescence and act as negative regulators of regeneration in the light-damaged zebrafish retina.</a> Glia. 69 (3), 546-566 (2021).</li><li>Green, L. A., Nebiolo, J. C., Smith, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+exit+the+CNS+in+spinal+root+avulsion.">Microglia exit the CNS in spinal root avulsion.</a> PLoS Biology. 17 (2), 3000159 (2019).</li><li>Hentig, J., Byrd-Jacobs, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+zinc+sulfate+results+in+differential+effects+on+olfactory+sensory+neuron+subtypes+in+the+adult+zebrafish.">Exposure to zinc sulfate results in differential effects on olfactory sensory neuron subtypes in the adult zebrafish.</a> International Journal of Molecular Sciences. 17 (9), 1445 (2016).</li><li>Ito, Y., Tanaka, H., Okamoto, H., Oshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+neural+stem+cells+and+their+progeny+in+the+adult+zebrafish+optic+tectum.">Characterization of neural stem cells and their progeny in the adult zebrafish optic tectum.</a> Developmental Biology. 342 (1), 26-38 (2010).</li><li>Becker, C., Becker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+zebrafish+as+a+model+for+successful+central+nervous+system+regeneration.">Adult zebrafish as a model for successful central nervous system regeneration.</a> Restorative Neurology and Neuroscience. 26 (2-3), 71-80 (2008).</li><li>Alyenbawwi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seizures+are+a+druggable+mechanistic+link+between+TBI+and+subsequent+tauopathy.">Seizures are a druggable mechanistic link between TBI and subsequent tauopathy.</a> eLife. 10, 58744 (2021).</li><li>Kaslin, J., Kroehne, V., Ganz, J., Hans, S., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+of+neuroepithelia-like+and+radial+glia-like+progenitor+cells+in+cerebellar+regeneration.">Distinct roles of neuroepithelia-like and radial glia-like progenitor cells in cerebellar regeneration.</a> Development. 144 (8), 1462-1471 (2017).</li><li>McCutcheon, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+model+of+traumatic+brain+injury+in+adult+zebrafish+demonstrates+response+to+injury+and+treatment+comparable+with+mammalian+models.">A novel model of traumatic brain injury in adult zebrafish demonstrates response to injury and treatment comparable with mammalian models.</a> Journal of Neurotrauma. 34 (7), 1382-1393 (2017).</li><li>Skaggs, K., Goldman, D., Parent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitotoxic+brain+injury+in+adult+zebrafish+stimulates+neurogenesis+and+long-distance+neuronal+integration.">Excitotoxic brain injury in adult zebrafish stimulates neurogenesis and long-distance neuronal integration.</a> Glia. 62 (12), 2061-2079 (2014).</li><li>Kishimoto, N., Shimizu, K., Sawamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+regeneration+in+a+zebrafish+model+of+adult+brain+injury.">Neuronal regeneration in a zebrafish model of adult brain injury.</a> Disease Models &amp; Mechanisms. 5 (2), 200-209 (2012).</li><li>Kroehne, V., Freudenreich, D., Hans, S., Kaslin, J., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+the+adult+zebrafish+brain+from+neurogenic+radial+glia-type+progenitors.">Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors.</a> Development. 138 (22), 4831-4841 (2011).</li><li>Marmarou, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+diffuse+brain+injury+in+rats.+Part+I:+Pathophysiology+and+biomechanics.">A new model of diffuse brain injury in rats. Part I: Pathophysiology and biomechanics.</a> Journal of Neurosurgery. 80 (2), 291-300 (1994).</li><li>Mussulini, B. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seizures+induced+by+pentylenetetrazole+in+the+adult+zebrafish:+a+detailed+behavioral+characterization.">Seizures induced by pentylenetetrazole in the adult zebrafish: a detailed behavioral characterization.</a> PloS One. 8 (1), 54515 (2013).</li><li>Kalueff, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+comprehensive+catalog+of+zebrafish+behavior+1.0+and+beyond.">Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond.</a> Zebrafish. 10 (1), 70-86 (2013).</li><li>Xiong, Y., Mahmood, A., Chopp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+traumatic+brain+injuries.">Animal models of traumatic brain injuries.</a> Nature Reviews Neuroscience. 14, 128-142 (2013).</li><li>Amamoto, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+axolotls+can+regenerate+original+neuronal+diversity+in+response+to+brain+injury.">Adult axolotls can regenerate original neuronal diversity in response to brain injury.</a> eLife. 5, 13998 (2016).</li><li>Yamamoto, S., Levin, H., Prough, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild,+moderate+and+severe:+terminology+implications+for+clinical+and+experimental+traumatic+brain+injury.">Mild, moderate and severe: terminology implications for clinical and experimental traumatic brain injury.</a> Current Opinion in Neurology. 31 (6), 672-680 (2008).</li><li>Lund, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderate+traumatic+brain+injury,+acute+phase+course+and+deviations+in+physiological+variables:+an+observational+study.">Moderate traumatic brain injury, acute phase course and deviations in physiological variables: an observational study.</a> Scandinavian Journal of Trauma Resuscitation and Emergency Medicine. 24, 77 (2016).</li><li>Levin, H., Arrastia, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis,+prognosis,+and+clinical+management+of+mild+traumatic+brain+injury.">Diagnosis, prognosis, and clinical management of mild traumatic brain injury.</a> The Lancet Neurology. 14 (5), 506-517 (2015).</li><li>Ruff, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+diagnosing+a+mild+traumatic+brain+injury:+a+National+Academy+of+Neuropsychology+education+paper.">Recommendations for diagnosing a mild traumatic brain injury: a National Academy of Neuropsychology education paper.</a> Archives of Clinical Neuropsychology: The Official Journal of the National Academy of Neuropsychologists. 24 (1), 3-10 (2009).</li><li>Ganz, J., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+in+fish.">Adult neurogenesis in fish.</a> Cold Spring Harbor Perspectives in Biology. 8 (7), 019018 (2016).</li><li>Grandel, H., Kaslin, J., Ganz, J., Wenzel, I., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+and+neurogenesis+in+the+adult+zebrafish+brain:+origin,+proliferation+dynamics,+migration+and+cell+fate.">Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration and cell fate.</a> Developmental Biology. 295, 263-277 (2006).</li><li>Lahne, M., Nagashima, M., Hyde, D. R., Hitchcock, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+Muller+glia+to+regenerate+retinal+neurons.">Reprogramming Muller glia to regenerate retinal neurons.</a> Annual Review of Visual Science. 6, 171-193 (2020).</li></ol>

The authors have nothing to disclose.

Investigations of neurotrauma and associated sequelae have long been centered on traditional non-regenerative rodent models20. Only recently have studies applied various forms of CNS damage to regenerative models9,11,13,14,21. Though insightful, these models are limited by either their use of an injury method uncommonly seen in the huma...

Traumatic brain injuries (TBIs) are a global health crisis and a leading cause of death and disability. In the United States, approximately 2.9 million people experience a TBI each year, and between 2006-2014 mortality due to TBI or TBI sequelae increased by over 50%1. However, TBIs vary in their etiology, pathology, and clinical presentation due largely in part to the mechanism of injury (MOI), which also influences treatment strategies and predicted prognosis2. Though TBIs can result from various MOI, they are predominately the result of either a penetrating or blunt-force trauma. Penetrating traumas represent a small percentage of TBIs and generate a severe and focal injury that is localized to the immediate and surrounding impaled brain regions3. In contrast, blunt-force TBIs are more common in the general population, span a range of severities (mild, moderate, and severe), and produce a diffuse, heterogeneous, and global injury affecting multiple brain regions1,4,5.
Zebrafish (Danio rerio) have been utilized to examine a wide range of neurological insults spanning the central nervous system (CNS)6,7,8,9. Zebrafish also possess, unlike mammals, an innate and robust regenerative response to repair CNS damage10. Current zebrafish trauma models use various injury methods, including penetration, excision, chemical insult, or pressure waves11,12,13,14,15,16. However, each of these methods utilizes an MOI that is rarely experienced by the human population, is not scalable across a range of injury severities, and does not address the heterogeneity or severity-dependent TBI sequela reported after blunt-force TBI. These factors limit the use of the zebrafish model to understand the underlying mechanisms of the pathologies associated with the most common form of TBI in the human population (mild blunt-force injuries).
We aimed to develop a rapid and scalable blunt-force TBI zebrafish model that provides avenues to investigate injury pathology, progression of TBI sequela, and the innate regenerative response. We modified the commonly used rodent Marmarou17 weight drop and applied it to adult zebrafish. This model yields a reproducible range of severities ranging from mild, moderate, to severe. This model also recapitulates multiple facets of human TBI pathology, in a severity-dependent manner, including seizures, edema, subdural and intracerebral hematomas, neuronal cell death, and cognitive deficits, such as learning and memory impairment. Days following injury, pathologies and deficits dissipate, returning to levels resembling undamaged controls. Additionally, this zebrafish model displays a robust proliferation and neuronal regeneration response across the neuroaxis concerning injury severity.
Here, we provide details toward the set up and induction of blunt-force trauma, scoring post-traumatic seizures, assessment of vascular injuries, instructions on preparing brain sections, approaches to quantifying edema, and insight into the proliferative response following injury.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-phenoxyethanol</td>
			<td>Sigma Alderich</td>
			<td>77699</td>
			<td></td>
		</tr>
		<tr>
			<td>#00 buckshot</td>
			<td>Remington</td>
			<td>RMS23770</td>
			<td>3.3g weight for sTBI</td>
		</tr>
		<tr>
			<td>#3 buckshot</td>
			<td>Remington</td>
			<td>RMS23776</td>
			<td>1.5g weight for miTBI/moTBI</td>
		</tr>
		<tr>
			<td>#5 Dumont forceps</td>
			<td>WPI</td>
			<td>14098</td>
			<td></td>
		</tr>
		<tr>
			<td>5-ethynyl-2&#8217;-deoxyuridine</td>
			<td>Life Technologies</td>
			<td>A10044</td>
			<td>EdU</td>
		</tr>
		<tr>
			<td>5ml glass vial</td>
			<td>VWR</td>
			<td>66011-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Click-iT EdU Cell Proliferation Kit</td>
			<td>Life Technologies</td>
			<td>C10340</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoOne 12-well plate</td>
			<td>USA Scientific</td>
			<td>CC7682-7512</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant Ocean</td>
			<td>Instant Ocean</td>
			<td>SS15-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Super frost postiviely charged slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>Super PAP Pen Liquid Blocker</td>
			<td>Ted Pella</td>
			<td>22309</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue freezing medium</td>
			<td>VWR</td>
			<td>15148-031</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Preparing the injury-induction rig allows for a rapid and simplistic means of delivering a scalable blunt-force TBI to adult zebrafish. The graded severity of the injury model provides several easily identifiable metrics of successful injury, though the vascular injury is one of the easiest and most prominent pathologies (Figure 3). The strain of fish used during the injury can make this indicator easier or harder to identify. When using wild-type AB fish(WTAB, <...

Watch this Scientific Journal Video about A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish at JoVE.com

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

This protocol provides a simple, rapid, and reproducible method to induce a scalable blunt force traumatic brain injury in adult zebrafish. The adult zebrafish that undergo this blunt force traumatic brain injury exhibit many of the characteristics observed in humans that suffer from a blunt force TBI. Demonstrating the procedure will be Mr.James Hentig and Ms.Kaylee Cloghessy, two doctoral students from my laboratory.Begin by filling a Petri dish with modeling clay. Then use fingers or the back of a pair of forceps to create a raised platform with additional modeling clay. Divide the raised platform lengthwise into two approximately equal halves using a razor blade.Form the two halves into a channel, such that it accommodates the length of an adult fish. Use additional clay to build walls to secure 2/3 of the fish body with the head exposed. Mold a small support in the exposed head region perpendicular to the walls to avoid rotation or recoil of the head upon injury.Ensure that the channel's sides do not impede the dropped weight. Use a mini hole punch to create a 3-millimeter steel disc from a 22-gauge steel flashing. Place an anesthetized fish unresponsive to tail pinch on the clay mold within the channel with its dorsal side up so that its body is secured on the sides.Place the 3-millimeter 22-gauge steel disc on the head, centered over the desired impact point. Align the fish perpendicularly to avoid its head from tilting to one side, which could cause an uneven impact. Use a standard ring stand and arm clamp to secure the steel or plastic tubing, ensuring it is straight, so the bottom of the tubing is 1.5 centimeters above the head of the zebrafish.Look down the tubing to ensure it is aligned above the steel plate. Choose the appropriate ball bearing based on the desired severity of injury. Drop the ball bearing from a predetermined height down the tubing onto the steel plate.Then place the injured fish in the recovery tank to be monitored. Fill a Petri dish with modeling clay and create a small cavity to support the body during dissection. Place the fish in the clay mold with the dorsal side up.Place two dissection pins, one through the midline halfway down the body and the other around 5 millimeters behind the base of the head. Use a pair of 5 Dumont forceps to bluntly sever the optic nerve and remove the eyes. Place one end of the 5 forceps under the right parietal plate.Make a deliberate scissor action moving toward the rostral end and remove the right frontal plate. Then rotate the fish 90 degrees clockwise. Place one end of the 5 forceps under the left parietal plate and use the same scissor motion to remove the left parietal and frontal plates, exposing the entire dorsal aspect of the brain.Use the 5 forceps to bluntly transect the maxilla such that the olfactory bulbs are preserved and not damaged. Using the same forceps, remove the right opercle, preopercle, interopercle, and subopercle. Then bluntly resect the musculature at the caudal end of the calvarium to expose the spinal cord.Using the forceps, bluntly transect the spinal cord. Place the forceps carefully under the brain and gently remove the brain from the calvarium. Dissect the whole brain or region of interest using the instructions in the text manuscript, and place the brain immediately on a small weigh boat using fine forceps, taking care not to stab or scrape the brain.Transfer the brain to the tared drying weigh boat and record the wet weight of the brain. Orient the brains to lay them flat on the weigh boat with the dorsal side facing up. Then place the brain and the drying weigh boat in a hybridization oven set to 60 degrees Celsius for eight hours.To transfer the brain to a new tared small weigh boat once it is dry, pinch the fine forceps together and, starting at the ventral side of the brain, scoop in an upward motion. Make a partial incision on a wet sponge. Place one fish at a time in the opening with the ventral side up and use a 30-gauge needle to inject approximately 40 microliters of 10 millimolar EdU into the fish's body.Then return the fish to the holding tank filled with system water. Collect the brains as mentioned previously, and place them as a group in a 9-milliliter glass vial containing 2 milliliters of nine parts 100%ethanol to one part 37%formaldehyde. Fix the brains at 4 degrees Celsius on a rocker platform.Use a cryostat chuck to embed the brains in TFM in the desired orientation on dry ice. Vascular injury was found to be one of the easiest and most prominent pathologies to identify successful injury via this model. The ability to identify the indicator changed with the strain of fish used during injury.Identification of vascular injury in wild type AB was difficult to distinguish between either mild or moderate TBI and undamaged control fish due to the pigmentation. Following injury, the mild TBI fish displayed minimal surface abrasions, while the moderate TBI fish exhibited limited cerebral hemorrhaging. The extent of the injury was apparent in severe TBI fish.In contrast, vascular injury can be easily identified when using albino or Casper fish. Swelling of the cerebrum due to injury was assessed using edema. In contrast, both moderate TBI and severe TBI had significant edema, 1 dpi and 3 dpi, but fluid content of both moderate TBI and severe TBI returned to levels resembling undamaged controls by 5 dpi.This blunt force injury resulted in a robust cell proliferation response spanning the neuroaxis. Increased EdU labeling was observed in the ventricular and subventricular zones of the forebrain compared to undamaged controls. The injured brains displayed increased EdU labeling in the periventricular gray zone, the optical tectal lobes, and aspects of the anterior hypothalamus compared to the undamaged fish brain.Following severe TBI, neurogenic regions in the hindbrain exhibited increased cell proliferation as compared to the undamaged brain. For proper induction of the injury, ensure the fish are secured and stable in the mold, and that the tubing is straight to avoid off-center impact or alter the weight drop trajectory. This procedure provides a rapid and cost-effective blunt force injury induction method applied to a regenerative model that would be especially applicable for repeated head trauma or injury-induced regenerative studies.

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3.1K vistas.  University of Notre Dame. Este protocolo proporciona un método simple, rápido y reproducible para inducir una lesión cerebral traumática por fuerza contundente escalable en peces cebra adultos. Los peces cebra adultos que sufren esta lesión cerebral traumática de fuerza contundente exhiben muchas de las características observadas en humanos que sufren de una lesión cerebral traumática de fuerza contundente. Demostrando el procedimiento estarán el Sr. James Hentig y la Sra. Kaylee Cloghessy, dos estudiantes d...