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 human population (penetrating traumas, chemical ablation, blast) and/or the injury is not scalable and therefore does not fully address the heterogeneity of severity-dependent pathologies observed in the human population22,23,24,25. Here, we provide a damage paradigm that applies the most common form of clinically relevant head trauma (blunt-force)4 that produces many of the pathological metrics established in human diagnosis22,23,24,25. When applied to the regenerative zebrafish, the model provides avenues to investigate the progression and recovery of injury-induced pathologies across severities, such as edema or post-traumatic seizures, as well as elucidating the mechanisms behind the innate regenerative recovery.
There are two key features of our model in producing a blunt-force TBI in zebrafish. First, our model delivers an inexpensive and simple injury paradigm that is rapid, which permits the successive injury to a large number of individuals or repeated injury to an individual to investigate the cumulative effect of blunt-force TBI. Second, this model is easily scalable to examine the effects of different force impacts. By changing the length of the tube (the height from which the ball bearing is dropped) and the weight of the ball bearing, the energy delivered to the skull of the fish, and the impact force can be easily modified and computed. This scalability of the injury allows for multiple avenues of investigation in regard to severity-dependent TBI sequelae progression and regenerative mechanisms of CNS repair.
With multiple metrics to access successful injury application, careful consideration should still be given to the genetic background of fish to be utilized. The casper or albino mutant fish will be favorable for novice investigators to reliably place the fish under the drop shaft, the placement of the steel disk over the desired neuroanatomical impact point, and assessing vascular injury. Furthermore, careful removal of the brain is simplified by the visual accessibility of the bones and brain in the casper and albino mutant&#160;fish. However, pigmented wild-type fish can be used though identification of landmarks and successful dissection may come with notable practice. Furthermore, pigmented fish can be used when producing either a moTBI or sTBI insult, as the subsequent pathologies allow for proper characterization of injury.
One major reason to study the effects of blunt-force TBI in zebrafish is to examine the source of injury-induced cell proliferation and the mechanisms underlying neuronal regeneration. Developmental and basal levels of constitutive proliferation have been identified in neurogenic niches across the zebrafish neuroaxis26,27, and injury-induced regeneration has been observed localized or adjacent to the injury site in adult zebrafish8,12,15. However, our blunt-force TBI model demonstrates that the diffuse injury also results in a severity-dependent cell proliferation event within the neurogenic niches across the neuroaxis. Identification of the source and extent of cell proliferation following TBI will permit the application of single-cell RNA-Seq to identify the changes in gene expression in the proliferative niche and testing the role of different signaling pathways, through the application of specific agonists and antagonists, in regulating this regeneration response. This approach has proven useful in elucidating the mechanisms underlying neuronal regeneration in the damaged zebrafish retina28 and should be equally useful in the brain following TBI.
In conclusion, our model provides a rapid, simple, and cost-effective injury method to deliver a scalable blunt-force TBI. This model will be useful to further investigate the effects of severity-dependent or repeated blunt-force TBI, as well as elucidating therapeutic targets of genetic regulation improving neuronal protection or inducing neuronal regeneration for functional cognitive recovery in adult vertebrates.

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, Figure 3A-D), identification of vascular injury can be difficult to distinguish between either miTBI or moTBI and undamaged control fish due to the pigmentation (Figure 3A-C). Following injury, miTBI fish display minimal surface abrasions (Figure 3B), while moTBI exhibit limited cerebral hemorrhaging (Figure 3C). While sTBI can still be challenging, the extent of injury is often apparent (Figure 3D). In contrast, when using albino (Figure 3E-H) or casper fish (Figure 3I-L), vascular injury is easily identified. Additionally, impact seizures are often observed following injury and seizure rate among the group is another representative metric of injury (Table 1). Injured fish will display tonic-clonic seizures (ataxia, ZBC 1.9, bending, ZBC 1.16, circling, ZBC 1.32, and corkscrew swimming, ZBC 1.37)19 that are easily observed following injury, regardless of the background strain. Seizures will be observed with increasing prevalence in relation to severity. After injury, miTBI do not display seizure-like behaviors; however, moTBI will display seizure behaviors (10.66% &#177; 1.37%, p &#60; 0.0001, Table 1) and the incidence is further elevated in sTBI fish (19.93% &#177; 1.49%, p &#60; 0.0001, Table 1).
Successful removal of the brain is critical for a myriad of further investigations, such as edema and assessing cell proliferation. Perform dissections with the utmost care to avoid damaging brain regions (most often by unintentional puncture) and to conserve all regions (the olfactory bulbs can easily be lost). Following the brain dissection procedure and schematic (section 3, Figure 2A-F) allows for a complete brain removal (Figure 2G,H). Investigators should consider whether their analysis requires the entire brain or whether a collection of specific brain regions may suit their needs. Dependent on the severity of the injury and time of collection, the brains may exhibit attached subdural hemorrhages, however, these are often adhered to the underside of the skull and lost during dissection. Swelling of the cerebrum is at times apparent, but due to anatomical differences and variation in general size, edema is the best method to assess swelling. Following the protocol outlined (section 4), undamaged brains exhibit a fluid content of 73.11% &#177; 0.80%, and miTBI, though slightly elevated, do not display a significant increase in edema at 1, 3, or 5 dpi (1 dpi: 76.33% &#177; 1.32%, p = 0.36, 3 dpi: 75.33 &#177; 1.37%, p = 0.84, 5 dpi: 74.14 &#177; 1.50%, p &#62; 0.99, Figure 4). In contrast, both moTBI and sTBI had significant edema 1 dpi (moTBI: 80.55 &#177; 0.94%, p &#60; 0.0001, sTBI: 86% &#177; 1.05%, p &#60; 0.0001), and 3 dpi (moTBI: 78.11 &#177; 0.93%, p &#60; 0.018, sTBI: 77.77% &#177; 1.02%, p &#60; 0.036, Figure 4). However, fluid content of both moTBI and sTBI returned to levels resembling undamaged controls by 5 dpi (moTBI: 74.42 &#177; 1.25%, p &#62; 0.99, sTBI: 73.85% &#177; 1.01%, p &#62; 0.99, Figure 4).
Cell proliferation, following TBI in zebrafish, is a robust assessment of the extent of injury. While the cell proliferation response has been studied previously in zebrafish following other forms of brain injury9,12, in most instances, the investigation was limited to the injury site. This blunt-force TBI results in a robust proliferation response spanning the neuroaxis. In a severity-dependent manner (sTBI data shown), increased EdU labeling is observed in the ventricular and subventricular zones of the forebrain (telencephalon, Figure 5B) relative to undamaged controls (Figure 5A). As sections moved caudally into the midbrain (mesencephalon and diencephalon), injured brains displayed increased EdU labeling in the periventricular grey zone (PGZ) the optic tectal lobes (TeO), and aspects of the anterior hypothalamus compared to undamaged fish (Figure 5D and Figure 5C, respectively). In the hindbrain, neurogenic regions that are evident in the undamaged brain (Figure 5E,G) exhibit increased cell proliferation following the sTBI (Figure 5F,H).
To summarize, a modified Marmarou weight drop applied to adult zebrafish provides a reproducible and scalable mild, moderate, or severe blunt-force TBI. Zebrafish, in a severity-dependent manner, display various pathologies, including seizures and vascular injury (i.e., subdural and intracerebral hematomas). Additionally, injured fish display decreased recovery rate (analogous to loss of consciousness, cognitive deficits in the form of learning and memory issues, and neuronal cell death (data not shown). The pathologies observed, rapidly recover over the span of 4-7 days coinciding with robust proliferative events across the neuroaxis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62709/62709fig01.jpg" /> 
Figure 1: Setting up the scalable injury apparatus. Graphical representation of the setup, the model, and delivery of scalable TBIs to the zebrafish. Steps 1-4 provide instructional overview of the steps to form the support mold that immobilizes the fish and exposing the head during damage. Steps 5-7 provide instructions on delivering the injury with insight on aspects to consider when troubleshooting the model. The figure was created with BioRender.com. <a href="https://www.jove.com/files/ftp_upload/62709/62709fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62709/62709fig02.jpg" /> 
Figure 2: Skull removal for brain dissection. Schematic of a simplified zebrafish skull and the step-by-step removal of bone (blue sections) to expose the adult zebrafish brain. (A,B) The eyes are bluntly removed with #5 forceps severing the optic nerves. (C) Forceps are placed into the musculature directly caudal to the parietal plates (black arrow) to remove the right parietal bone and then the right frontal bone. (D,E) The left parietal bone and left frontal bone are removed. (F) The right opercle, preopercle, interopercle, and subopercle are removed, providing lateral and dorsal access to the brain. (G,H) Undamaged and sTBI brains were removed. Scale bar = 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62709/62709fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62709/62709fig03.jpg" /> 
Figure 3: Vascular injury in various backgrounds across injury-severities. Dorsal view of undamaged and TBI wild-type AB, albinob4, and casper adult zebrafish displaying vascular injury. (A-D) Adult wild-type AB fish are heavily pigmented and abrasions following miTBI (B) are difficult to visualize. Vascular injury was more apparent in moTBI (C) and sTBI (D) fish compared to undamaged controls (A). (E-H) albino fish were less pigmented and visualization of the brain was more distinct. Vascular injury following TBI was clearly observed and distinguished across severities. (I-L) casper fish provided the most adaptable background for novice investigators as the transparency allowed for easy identification of desired neuroanatomical regions and clear observation and delineation of vascular injury by TBI-severity. Scale bar = 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62709/62709fig03large.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/62709/62709fig04.jpg" /> 
Figure 4: Zebrafish experience injury-induced edema following TBI. Zebrafish were exposed to different severities of TBI (undamaged, miTBI, moTBI, and sTBI) and assessed at different days following damage for percent fluid content (edema). Statistical analyses were performed with a Browns-Forsythe and Welch ANOVA followed by a Dunnett&#39;s T3 multiple comparison post-hoc test. n = total number of individual fish. All statistical analyses were performed with the Prism (Graphpad 9.0) software package. <a href="https://www.jove.com/files/ftp_upload/62709/62709fig04large.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/62709/62709fig05.jpg" /> 
Figure 5: TBI-induced proliferation across the neuroaxis. (A-H) Confocal images of coronal and sagittal brain sections of undamaged and sTBI fish that were IP-injected with EdU 12 h prior to collection. Increased EdU incorporation was observed in multiple neurogenic niches following injury across the forebrain (A,B), midbrain (C,D), and hindbrain (E-H). Cerebellum, CCe, Granule Layer, GL, Medial Valvula Cerebelli, Vam, Molecular Layer, ML, Optic Tectum, TeO, Periventricular Grey Zone, PGZ, Telencephalon, and Tel. All scale bars are 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62709/62709fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Group</td>
			<td>N</td>
			<td>n</td>
			<td>Mean Seizures (%) &#177; SEM</td>
			<td>p</td>
		</tr>
		<tr>
			<td>Undam</td>
			<td>10</td>
			<td>74</td>
			<td>0%</td>
			<td></td>
		</tr>
		<tr>
			<td>miTBI</td>
			<td>10</td>
			<td>100</td>
			<td>0%</td>
			<td>&#62;0.99</td>
		</tr>
		<tr>
			<td>moTBI</td>
			<td>10</td>
			<td>184</td>
			<td>10.66% &#177; 1.37%</td>
			<td>&#60;0.0001</td>
		</tr>
		<tr>
			<td>sTBI</td>
			<td>10</td>
			<td>237</td>
			<td>19.93% &#177; 1.49%</td>
			<td>&#60;0.0001</td>
		</tr>
	</tbody>
</table>
Table 1. Zebrafish display severity-dependent impact seizures following TBI. Quantification of tonic-clonic seizures, which was recorded as the percent of an experimental injured group, that were observed within 1 h post-injury. Statistical analyses were performed with a one-way ANOVA followed by Tukey&#39;s post-hoc test. N = total number of experimental groups, n = total number of individual fish. Statistical analyses were performed using the Prism (Graphpad 9.0) software package.

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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University of Michigan: Ann Arbor

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JoVE Journal

Neuroscience

3.1K 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

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

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 ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

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 ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...

A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of human studies, animal models provide suitable alternatives to test treatment methods, and to study the mechanisms and related complications of the condition. Experimental rodent models have historically been the most widely used due to their accessibility, low cost, reproducibility, and validated approaches. A metric test, which tests the ability to recall the placement of two objects at various distances and angles from one another, is a technique to study impairment in spatial working memory (SWM) after TBI. The significant advantages of metric tasks include the possibility of dynamic observation, low cost, reproducibility, relative ease of implementation, and low stress environment. Here, we present a metric test protocol to measure impairment of SWM in adult rats after TBI. This test provides a feasible way to evaluate physiology and pathophysiology of brain function more effectively.

Traumatic brain injury (TBI) is commonly associated with memory impairment. Here, we present a protocol to assess spatial working memory after TBI via a metric task. A metric test is a useful tool to study spatial working memory impairment after TBI.

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of ...