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 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.
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.
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
2. Scoring seizures post-TBI in the adult zebrafish
3. Brain dissection
4. Edema studies in the zebrafish brain
5. Labeling cellular proliferation across the neuroaxis and preparing fixed tissue.
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% ± 1.37%, p < 0.0001, Table 1) and the incidence is further elevated in sTBI fish (19.93% ± 1.49%, p < 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% ± 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% ± 1.32%, p = 0.36, 3 dpi: 75.33 ± 1.37%, p = 0.84, 5 dpi: 74.14 ± 1.50%, p > 0.99, Figure 4). In contrast, both moTBI and sTBI had significant edema 1 dpi (moTBI: 80.55 ± 0.94%, p < 0.0001, sTBI: 86% ± 1.05%, p < 0.0001), and 3 dpi (moTBI: 78.11 ± 0.93%, p < 0.018, sTBI: 77.77% ± 1.02%, p < 0.036, Figure 4). However, fluid content of both moTBI and sTBI returned to levels resembling undamaged controls by 5 dpi (moTBI: 74.42 ± 1.25%, p > 0.99, sTBI: 73.85% ± 1.01%, p > 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.
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. Please click here to view a larger version of this figure.
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 µm. Please click here to view a larger version of this figure.
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 µm. Please click here to view a larger version of this figure.
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'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. Please click here to view a larger version of this figure.
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 µm. Please click here to view a larger version of this figure.
Group | N | n | Mean Seizures (%) ± SEM | p |
Undam | 10 | 74 | 0% | |
miTBI | 10 | 100 | 0% | >0.99 |
moTBI | 10 | 184 | 10.66% ± 1.37% | <0.0001 |
sTBI | 10 | 237 | 19.93% ± 1.49% | <0.0001 |
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'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.
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 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.
The authors have nothing to disclose.
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).
2-phenoxyethanol | Sigma Alderich | 77699 | |
#00 buckshot | Remington | RMS23770 | 3.3g weight for sTBI |
#3 buckshot | Remington | RMS23776 | 1.5g weight for miTBI/moTBI |
#5 Dumont forceps | WPI | 14098 | |
5-ethynyl-2’-deoxyuridine | Life Technologies | A10044 | EdU |
5ml glass vial | VWR | 66011-063 | |
Click-iT EdU Cell Proliferation Kit | Life Technologies | C10340 | |
CytoOne 12-well plate | USA Scientific | CC7682-7512 | |
Instant Ocean | Instant Ocean | SS15-10 | |
Super frost postiviely charged slides | VWR | 48311-703 | |
Super PAP Pen Liquid Blocker | Ted Pella | 22309 | |
Tissue freezing medium | VWR | 15148-031 |