Name: a scalable model to study the effects of blunt-force injury in adult zebrafish
Uploaded: 2021-05-31T15:00:00.000+00:00
Duration: 8 min 13 s
Description: Watch this Scientific Journal Video about A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish at JoVE.com

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 the desired weight of ball bearing and a desired length and diameter of steel/plastic tubing and determine the energy and impact force. 
	NOTE: Tubing should have an inner diameter that allows the ball bearing to pass through without changing its path or speed of movement.
	<ol>
		<li>Determine the kinetic energy upon impact: 
		<img alt="Equation 1" src="/files/ftp_upload/62709/62709eq01.jpg" /> 
		Where, KE = kinetic energy, m = mass (in kg), g = gravitational force, h = height (in m) from drop point to fish. 
		NOTE: This provides kinetic energy in J. Multiply the value by 1,000 to determine mJ. KE is based on an accelerating object, which occurs when the ball bearing is dropped from a stationary position.</li>
		<li>Generate a mild TBI (miTBI) using 7.62 cm long steel/plastic tubing that ends 1.5 cm above the plate on the zebrafish skull (total distance 9.1 cm) and a 1.5 g (6.4 mm diameter) ball bearing. These produce a kinetic energy of 1.33 mJ. This damage was empirically decided to be equivalent to a miTBI based on key pathophysiological TBI markers, such as vascular injury, subdural/intracerebral hematoma formation, neuronal cell death, and cognitive impairments that largely recapitulated what was reported in the human population in a severity-dependent manner.</li>
		<li>Calculate kinetic energy miTBI = <img alt="Equation 2" src="/files/ftp_upload/62709/62709eq02.jpg" /></li>
		<li>Generate a moderate TBI (moTBI) using 12.7 cm long steel/plastic tubing that ends 1.5 cm above the plate on the zebrafish skull (total distance 14.2 cm) and a 1.5 g (6.4 mm diameter) ball bearing that produces kinetic energy of 2.08 mJ.</li>
		<li>Calculate kinetic energy moTBI = <img alt="Equation 3" src="/files/ftp_upload/62709/62709eq03.jpg" /></li>
		<li>Generate a severe TBI (sTBI) using 7.62 cm long steel/plastic tubing that ends 1.5 cm above the plate on the zebrafish skull (total distance 9.1 cm) and a 3.3 g (8.38 mm diameter) ball bearing that produces a kinetic energy of 2.94 mJ.</li>
		<li>Calculate kinetic energy sTBI =&#160; <img alt="Equation 4" src="/files/ftp_upload/62709/62709eq04.jpg" /></li>
	</ol>
	</li>
	<li>Fill a Petri dish with modeling clay (Figure 1, step 1) and use a blunt instrument (i.e., the back of a pair of forceps) to create a raised platform (5 cm x 1.5 cm) with additional modeling clay (Figure 1, step 2).
	<ol>
		<li>Use a razor blade to divide the raised platform lengthwise into two approximately equal halves (Figure 1, step 3, red dashed line). Form the two halves into a channel that accommodates the length of an adult fish (Figure 1, step 4). Use additional clay to build walls that will secure ~2/3 of the fish body, leaving the head exposed.</li>
		<li>Mold a small support in the exposed head region perpendicular to the walls to support the head to avoid rotation or recoil of the head upon injury (Figure 1, step 4). 
		NOTE: The channel should be deep enough to support the fish in a dorsal-up position but should still allow the head to rest above the surrounding clay. Additionally, the head support should follow the natural curvature of the fish, supporting the lower mandible and gills.</li>
		<li>Ensure that the dropped weight is not impeded by the channel&#39;s sides (Figure 1, step 4).</li>
	</ol>
	</li>
	<li>Create a 3 mm diameter steel disk using a mini hole-punch and 22 g steel flashing. Each disk may be used multiple times.</li>
	<li>Anesthetize one fish by placing it into a beaker with 50-100 mL of 1:1000 (1 mL/1 L, 0.1%) 2-phenoxyethanol until it is unresponsive to tail pinch.</li>
	<li>Place the fish, dorsal side up, on the clay mold within the channel so that the body is secured on the sides and place a 3 mm 22 g steel disc on the head, centered over the desired impact point (Figure 1, step 5).
	<ol>
		<li>Ensure that the fish is aligned as perpendicularly as possible to avoid its head from tilting to one side, which could cause an uneven impact.</li>
	</ol>
	</li>
	<li>Secure the steel/plastic tubing, using a standard ring stand and arm clamp, so the bottom of the tubing is 1.5 cm above the head of the zebrafish (Figure 1, step 6). Ensure the tubing is straight.
	<ol>
		<li>Look down the tubing and ensure that the tubing is aligned above the steel plate.</li>
	</ol>
	</li>
	<li>Drop the ball bearing (1.5 g for mild and moderate TBI, and 3.3 g for severe TBI) from a predetermined height (described in Steps 1.3.3-1.3.5) down the tubing onto the steel plate located over the desired neuroanatomical region of interest (e.g., cerebellum, Figure 1, step 5) to produce the blunt-force TBI for the desired severity of injury. Place the injured fish in a recovery tank to be monitored. 
	NOTE: Depending on the severity of the injury, mortality or tonic-clonic seizures may occur. If the fish are unresponsive for a prolonged period of time following the TBI, use a transfer pipette or forceps to administer a tail-pinch and evaluate for a pain response.</li>
</ol>
2. Scoring seizures post-TBI in the adult zebrafish
<ol>
	<li>Anesthetize and injure the fish according to the injury protocol outlined in section 1 and place the injured fish in an aerated recovery tank of 2 L of system water.</li>
	<li>Observe the fish for any signs of post-traumatic seizures beginning immediately after being placed in the recovery tank. Set an observation time (i.e., 1 h) and record all seizure activity, including the seizure score (described below), duration of each seizure, and the percentage of fish that experienced seizures (Figure 2A). 
	NOTE: Seizures can occur immediately upon injury, as well as hours or days post-TBI. Seizures can persist long term and a single fish can have multiple seizures. Setting an observation time of 1 h or greater will yield a good representation of the overall trend of seizure rates.</li>
	<li>Score fish using the Mussulini18 guidelines for adult zebrafish seizure phenotypes. 
	&#8203;NOTE: Without using the tracking software, it is difficult to assess seizure scores below 3 unbiasedly. Therefore, only seizure scores of 3 or greater should be recorded when the assessment is being performed without software.</li>
</ol>
3. Brain dissection
<ol>
	<li>Euthanize fish in a 1:500 (2 mL/1 L, 0.2%) solution of 2-phenoxyethanol, until gill movements cease, and they are unreactive to fin pinch, at the desired endpoint.</li>
	<li>Fill a Petri dish with modeling clay and create a small cavity to support the body during dissection.</li>
	<li>Place the fish, dorsal side up, in the clay mold. Place one dissection pin through the midline halfway down the body of the fish and a second pin ~5 mm behind the base of the head.</li>
	<li>Under a dissecting light microscope, bluntly sever the optic nerve with a pair of #5 Dumont forceps and remove the eyes (Figure 2A).</li>
	<li>Orientate the fish so that the rostral end is furthest when looking through the microscope (Figure 2B). 
	NOTE: The following steps are for right-handed individuals. Left-handed individuals may prefer to perform the following steps in a mirrored orientation.</li>
	<li>Use #5 forceps to slowly place one end of the forceps under the right parietal plate, making a deliberate scissor action moving toward the rostral end and remove the right parietal and frontal plates (Figure 2C).
	<ol>
		<li>Keep the forceps at an angle of 45&#176; or lower in order to avoid penetrating the brain during dissection.</li>
	</ol>
	</li>
	<li>Rotate the fish 90&#730; 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 (Figure 2D,E).</li>
	<li>Bluntly transect the maxilla with #5 forceps. Preserve the olfactory bulbs and do not damage them if this is the region of interest.</li>
	<li>Remove the right opercle, preopercle, interopercle, and subopercle with #5 forceps (Figure 2F).</li>
	<li>Bluntly resect the musculature at the caudal end of the calvarium opening using #5 forceps, exposing the spinal cord.</li>
	<li>Bluntly transect the spinal cord with #5 forceps. Carefully place forceps under the brain and gently remove the brain from the calvarium. 
	NOTE: Never pinch the brain. Use forceps to &#34;cradle&#34; the brain or resect caudally and utilize the exposed spinal cord as a pinch point to maneuver the brain.</li>
	<li>Fix the removed brains in 9 parts 100% ethanol to 1 part 37% formaldehyde overnight at 4&#176;C on a rocker platform.</li>
</ol>
4. Edema studies in the zebrafish brain
<ol>
	<li>Anesthetize and injure fish according to the injury protocol outlined in section 1 and allow the fish to recover in a recovery tank until they start swimming freely.</li>
	<li>Place fish back in the normal housing conditions post-injury for 1 day.</li>
	<li>Euthanize the fish in 1:500 2-phenoxyethanol, after time has elapsed.</li>
	<li>Dissect the whole brain or region of interest according to the protocol outlined in section 3 and place the brain immediately on a small weigh boat. 
	NOTE: Use caution when transferring the brain, using fine forceps to gently place it on the weigh boat without stabbing or scraping the brain, which could result in the loss of tissue.</li>
	<li>Label (with injury group and brain number) and tare an additional small drying weigh boat on a scale. Use a scale with the ability to measure a minimum of 0.001 g to get an accurate measurement.</li>
	<li>Transfer the brain to the tared drying weigh boat and record the wet weight of the brain. Orient the brains so that they lay flat on the weigh boat with the dorsal side facing up.</li>
	<li>Place the drying weigh boat and brain in a hybridization oven set to 60 &#176;C for 8 h.</li>
	<li>After drying, the brain may stick to the drying weigh boat and may be difficult to remove and transfer to a new tared small weigh boat. Avoid pinching the brain with forceps, as this might result in damage to the dry brain and loss of tissue. Instead, pinch the fine forceps together and, starting at the ventral side of the brain, scoop in an upward motion.</li>
	<li>Determine the water content of each brain using the formula (Figure 4): 
	<img alt="Equation 5" src="/files/ftp_upload/62709/62709eq05.jpg" /></li>
</ol>
5. Labeling cellular proliferation across the neuroaxis and preparing fixed tissue.
<ol>
	<li>Prepare 10 mM 5-ethynyl-2&#39;-deoxyuridine (EdU) in 2 mL of ddH2O.</li>
	<li>Anesthetize 3 to 4 fish at a time in 50-100 mL of 1:1000 (1 mL/1 L, 0.1%) 2-phenoxyethanol until the fish are unresponsive to tail pinch, at the desired time point following injury (using the protocol outlined in section 1).</li>
	<li>Make a partial incision on a wet sponge and place one fish at a time in the opening, ventral side up.</li>
	<li>Use a 30 G needle to inject ~40 &#181;L of 10 mM EdU into the fish&#39;s body. Return the fish to a holding tank filled with system water. 
	NOTE: Repeated injections may be performed at different time points to label a greater number of proliferating cells and may be needed if a chase period of greater than 1 week is desired.</li>
	<li>Collect brains as outlined in section 3 and place them as a group in a 5 mL glass vial containing 2 mL of 9 parts 100% ethanol to 1 part 37% formaldehyde. Fix the brains at 4 &#176;C on a rocker platform. 
	NOTE: A rocker or shaker platform prohibits the brains from resting at the bottom and the telencephalon of whole brains from curling.</li>
	<li>Rehydrate brains, in the same glass vial used to fix brains, in washes of descending ethanol series, 75%, 50%, and 25%, for 15 mins each, followed by a 1.5 h wash in 5% sucrose/PBS on a rocker at room temperature. Store the brains in a glass vial overnight in 30% sucrose/PBS at 4&#176; C on a rocker platform.</li>
	<li>Remove brains from 30% sucrose/PBS and transfer them with forceps into a 12-well plate (one treatment group per well) with wells filled with a 2:1 solution consisting of 2 parts tissue freezing medium and 1 part 30% sucrose/PBS. Incubate the brains overnight at 4 &#176;C on a rocker platform.</li>
	<li>Transfer brains to the next row of wells within the 12-well plate submerging brains in 100% TFM for 2-24 h at 4 &#176;C.</li>
	<li>Use a cryostat chuck to embed the brains in TFM in the desired orientation on dry ice.</li>
	<li>Perform cryosectioning (16 &#956;m thick sections) and collect the sections on positively charged slides. Dry slides on a slide warmer for 1 h and then store at -80 &#176;C or continue with immunohistochemistry.</li>
	<li>Prepare a hydrophobic barrier on the slide around the tissue sections and allow to dry on a slide warmer for 20 min.</li>
	<li>Briefly wash the slides in PBS for 5 min and then twice in PBS-Tween 20 (0.05%) for 10 min each.</li>
	<li>Perform EdU detection using the EdU Cell Proliferation Kit and the manufacturer&#39;s instructions.</li>
	<li>Analyze the slides and quantify the fluorescent EdU-labeled cells using either an epifluorescent microscope or a confocal microscope. A minimum of a 40x objective will be required to clearly distinguish individual cells.</li>
</ol>

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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><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&amp;rsquo;-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 ...

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Research

JoVE Journal

Neuroscience

3.2K Views.  University of Notre Dame. 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.

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

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

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

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

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

Developmental Biology

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 ...

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

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

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

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

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 ...

Removing Zebrafish Scales for Culturing and Bone Biology Studies

In Vitro Culturing Technique for Studying Cellular Dynamics in Zebrafish Scales

Zebrafish scales offer a variety of advantages for use in standard laboratories for teaching and research purposes. Scales are easily collected without the need for euthanasia, regenerate within a couple of weeks, and are translucent and small, allowing them to be viewed using a standard microscope. Zebrafish scales are especially useful in educational environments, as they provide a unique opportunity for students to engage in hands-on learning experiences, particularly in understanding cellular dynamics and in vitro culturing methods. The main objective of this protocol is to describe a method for collecting and maintaining zebrafish scales in culture for use in a variety of biological studies using basic laboratory equipment. Additionally, the protocol details their use in understanding bone homeostasis by examining the activity of bone cells involved in bone resorption and deposition. It also includes additional protocols for general techniques, such as the visualization of nuclei and apoptotic cells. The in vitro culturing protocol produces reliable results with minimal reagents and equipment. This article discusses the benefits of using in vitro cultures of zebrafish scales to foster scientific inquiry and outlines the resources needed to support their integration into educational settings.

Author Spotlight: Advancing Bone Cell Activity Research with Zebrafish Scales 

This protocol offers a method to study cellular dynamics using a simple in vitro culture technique. It provides an opportunity for zebrafish researchers and educators to study cellular processes, such as those related to bone homeostasis and basic cell biology, by visualizing fluorescent nuclei and apoptotic cells within the scales.

Zebrafish scales offer a variety of advantages for use in standard laboratories for teaching and research purposes. Scales are easily collected without ...

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.

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 ...

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 ...

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.

Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration.

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...