Traumatic brain injury (TBI) is a prominent risk factor for dementias including tauopathies like chronic traumatic encephalopathy (CTE). The mechanisms that promote prion-like spreading of Tau aggregates after TBI are not fully understood, in part due to lack of tractable animal models. Here, we test the putative role of seizures in promoting the spread of tauopathy. We introduce ‘tauopathy reporter’ zebrafish expressing a genetically encoded fluorescent Tau biosensor that reliably reports accumulation of human Tau species when seeded via intraventricular brain injections. Subjecting zebrafish larvae to a novel TBI paradigm produced various TBI features including cell death, post–traumatic seizures, and Tau inclusions. Bath application of dynamin inhibitors or anticonvulsant drugs rescued TBI-induced tauopathy and cell death. These data suggest a role for seizure activity in the prion-like seeding and spreading of tauopathy following TBI. Further work is warranted regarding anti-convulsants that dampen post-traumatic seizures as a route to moderating subsequent tauopathy. Traumatic brain injury can result from direct head concussions, rapid head movements, or a blast wave generated by an explosion. Traumatic brain injury often causes seizures in the short term and is a risk factor for certain dementias, including Alzheimer’s disease and chronic traumatic encephalopathy in the long term. A protein called Tau undergoes a series of chemical changes in these dementias that makes it accumulate, form toxic filaments and kill neurons.The toxic abnormal Tau proteins are initially found only in certain regions of the brain, but they spread as the disease progresses. Previous studies in Alzheimer’s disease and other diseases where Tau proteins are abnormal suggest that Tau can spread between neighboring neurons and this can be promoted by neuron activity. However, scientists do not know whether similar mechanisms are at work following traumatic brain injury. Given that seizures are very common following traumatic brain injury, could they be partly responsible for promoting dementia?To investigate this, researchers need animal models in which they can measure neural activity associated with traumatic brain injury and observe the spread of abnormal Tau proteins. Alyenbaawi et al. engineered zebrafish so that their Tau proteins would be fluorescent, making it possible to track the accumulation of aggregated Tau protein in the brain. Next, they invented a simple way to perform traumatic brain injury on zebrafish larvae by using a syringe to produce a pressure wave. After this procedure, many of the fish exhibited features consistent with progression towards dementia, and seizure-like behaviors.The results showed that post-traumatic seizures are linked to the spread of aggregates of abnormal Tau following traumatic brain injury. Alyenbaawi et al. also found that anticonvulsant drugs can lower the levels of abnormal Tau proteins in neurons, preventing cell death, and could potentially ameliorate dementias associated with traumatic brain injury. These drugs are already being used to prevent post-traumatic epilepsy, but more research is needed to confirm whether they reduce the risk or severity of Tau-related neurodegeneration. Traumatic brain injury (TBI) is a leading cause of mortality and disability worldwide . It is aThe progressive deposition of hyperphosphorylated Tau protein in filamentous forms is a defining hallmark of tauopathies, which includes Alzheimer’s disease (AD), CTE, and several other dementias. Each of the tauopathies affects distinct brain regions and has a unique clinical presentation . Early iBeyond TBI, the self-propagation and prion-like spread of Tau aggregates is thought to play a key role in the progression of other tauopathies such as AD . MechaniIn this light, an intriguing aspect of TBI is the prominence of post-traumatic seizures that might be predicted to initiate the aggregation and/or exacerbate the spread of Tau pathology. Seizures are one of the key consequences of all types of TBI, and they have been more commonly reported in patients who suffered from blast injuries . AlthougThis knowledge gap is due in part to a lack of accessible in vivo models that can report the progression and spread of tauopathy, or that allow neural activity associated with TBI to be measured and manipulated. To address these issues, we engineered a tauopathy biosensor transgenic zebrafish that develops GFP+ puncta when Tau aggregates within the brain or spinal cord. Additionally, we introduce a simple medium-throughput method to induce TBI in zebrafish larvae. Combining these novel approaches, we found that post-traumatic seizures correlate strongly with spreading tau pathology following TBI. Manipulating this seizure activity mitigated Tau aggregation and revealed a critical role for endocytosis in the prion-like spread of Tau seeds in vivo following TBI. The results from our novel in vivo TBI model implicate seizures and dynamin-dependent endocytosis in the spread of Tau seeds, thereby offering potential therapeutic targets.neuronal enolase 2 , and assigned it allele number ua3171.Previous reports describe the assessment and quantification of Tau inclusions in living cells , via measuring aggregation of fluorescent proteins fused to Tau protein, providing sensitive detection of pathological Tau species and strain variants . Tau is Simultaneously, we expressed the same biosensor in vitro to validate the construct we deployed in vivo . Both inP301L), were compared to normal non-Tg mouse homogenates as a negative control. Congruent with findings obtained in past similar cell assays (P310L mice)  . The in 0L mice) . To veri0L mice) . Tau fib0L mice) . Transdu0L mice) , confirmTo test if the Tau4R-GFP biosensor can report the in vivo progression of tauopathy, we emulated intracerebral injection methods that induce (prion-like) Tau pathology in mice . We injeThe abundance of GFP+ spinal cord inclusions was progressive and significantly higher in larvae injected with pathogenic Tau brain homogenate compared with various controls . A totalLike other protein misfolding diseases, tauopathies reflect a proteostatic imbalance wherein the clearance of pathological Tau species is insufficient relative to accumulation . We reasIt was striking that the zebrafish Tau biosensor was robustly able to discriminate brain homogenates that were burdened with human Tau aggregates versus those that were not. However, we considered an alternative explanation for the data: the difference may not depend directly on human Tau in the brain homogenate but could instead reflect other bioactive components of the degenerating Tg mouse brain. To verify that the formation of GFP+ puncta in zebrafish can be seeded by a protein-only injection, we delivered synthetic human Tau protein (2N4R). After confirming the recombinant Tau proteins were appropriately fibrillized via EM , we deliadult zebrafish that are comparable to mammalian TBI methods (We next sought to deploy our Tau biosensor in a tauopathy model that enables higher throughput than can be achieved with intraventricular injection methods. We considered TBI) as an inducer of the tauopathy in CTE; further, we were encouraged that innovations in this realm could fill an unmet need for a high-throughput, genetically tractable in vivo model of these devastating concussive injuries. Although a few methods have been reported to induce TBI in  methods , no suchWe devised a traumatic injury paradigm by loading zebrafish larvae  into a syringe with a closed valve stopper, and applying a hit on the plunger to produce a pressure wave through the fish body akin to pressure or shock waves experienced during human blast injury . To chalWe established the TBI method via empirical testing of various parameters, restricting ourselves to materials and methods that can be adopted inexpensively, with a goal of consistently inducing a robust injury (see phenotypes below) vs. a tradeoff with maximizing survival of the larvae. Subsequent to this optimization, we were able to characterize the pressure induced within the syringe during each injury . The maxTg(gata1a:DsRed) larvae that have red fluorescence in their blood cells  expressed throughout the CNS. CaMPARI fluoresces green in baseline conditions, and permanently converts to red fluorescent emission if high intracellular calcium levels  occur coincident with application of ‘photoconverting’ intense 405 nm blue light. We subjected our allele of ua3144Tg(elavl3:CaMPARI) larvae . Following TBI, and regardless of injury intensity, no GFP+ aggregates were detected in these controls . SimilarThe GFP+ Tau aggregates formed in the brain region following TBI tend to form fused shapes  reminiscConsidering the clinical prominence of post-traumatic seizures following TBI, and the suggested role of cell stress and increased neural activity in promoting protein misfolding diseases , we specTo assess the hypothesis that seizure activity has a causal role in increasing the abundance of Tau aggregates in our TBI model, we employed convulsant and anti-convulsant drugs to modulate the seizure intensity and in vivo neural activity. We selected drugs that are well-established to behave similarly in zebrafish as in mammals, although it is perhaps notable that the multi-day drug application used here is longer than the acute applications typically considered in zebrafish . Our hypSimilarly, intensifying post-traumatic seizures via application of the convulsant kainate increased the abundance on tauopathy fourfold . Surprisingly, higher doses of 4-AP consistently abrogated the appearance of Tau aggregates. Treating TBI larvae with 200 or 800 μM of 4-AP for a prolonged period  significantly inhibited the abundance of Tau4R-GFP+ puncta in the TBI group , a KBI group . AnalysiBI group . It is wBI group . A similBI group .Next, we considered if this unexpected inhibition of tauopathy by high-dose 4-AP convulsant is a direct consequence of increased neural activity . We found that larvae receiving TBI and 4-AP continued to exhibit a lack of Tau aggregates when co-treated with anti-convulsant retigabine (p<0.0001) . This suTo resolve a mechanism whereby high doses of 4-AP reduced Tau pathology, contrary to our predictions above regarding neural hyperactivity, we considered previous in vitro work that demonstrated high concentrations of 4-AP cause reduced endocytosis of synaptic vesicles . To examLimitations below) but by addressing many of these challenges in an accessible and vibrantly active vertebrate brain, we offer an innovative approach for the study of prion-like events, tauopathy and/or TBI.The consequences of concussive blasts and TBI extend beyond the proximate injury – they are prominent risk factors for devastating dementias including AD, CTE and other tauopathies. Identifying the causal links that entwine TBI with subsequent tauopathies would inspire improved diagnostics and therapeutics. Investigating these mechanisms has been hampered by lack of tractable models, since cell culture platforms cannot faithfully represent the injury or the response-to-injury or the treatment thereof. Indeed, TBI and tauopathies are complex tissue and systems-level events with pathobiology progressing on a backdrop of dynamic vigorous neural function, prion-like vectoring of misfolded proteins via glymphatic and blood vasculature, immune and support cells, sleep physiology, homeostatic regulation and complex drug metabolism. Rats have been the favored animal model for TBI, and mice can complement this as insightful models of tauopathy, yet both are challenged by expense, ethical considerations, and CNS tissues that are relatively inaccessible to  visualization of cellular events in living individuals . Here, oduring TBI and (ii) effectively document prion-like tauopathy in individual subjects over multiple days. The accessibility of this platform to pharmacology allowed us to query cell biology events in vivo and support a role for endocytosis in prion-like progression and TBI-induced tauopathy. Further, anti-convulsant drugs were potent mitigators of the tauopathy and cell death that emerged subsequent to TBI  uniquely quantify neural activity on freely behaving animals It is well established that TBI induces tauopathy, and over the past decade, numerous in vitro and in vivo studies have supported that Tau proteins possess prion-like properties. Indeed seeding, templated misfolding (conversion) and spread to synaptically connected regions has been documented in tauopathies such as AD and FTD . VariousRegarding TBI, a large knowledge gap exists regarding how Tau seeds are released and/or internalized by adjacent (or far-flung) cells - indeed the prion-like properties of Tau species following TBI had not been assessed until very recently . MoreoveWe inspected the role of seizure activity and/or neuronal excitability, as well as the role of dynamin-dependent endocytosis, during the progression of tauopathy after TBI. We focussed our attention on seizure activity in part because seizures frequently occur in TBI patients following blast traumatic injury . We hypoadult zebrafish when TBI was delivered via focused ultrasound  we were able to confirm the presence of various markers associated with brain injury, such as cell death, abnormalities in blood flow, hemorrhage and the occurrence of post-traumatic seizures. Recently, post-traumatic seizures were also observed in trasound  offeringduring injury in an unencumbered freely swimming animal. Here, we revealed for the first time a snapshot of neurons becoming active at the moment of TBI. Our results demonstrated an increase in neuronal excitability upon TBI, which may contribute to the frequency of post-traumatic seizures observed in our model, other blast TBI models and TBI patients  in the abundance of Tau aggregates following various insults and treatments, typically in a dose-dependent manner and in harmony with expected trends. Considering this success, it remains perplexing and intriguing that a subset of larvae exhibit GFP+ Tau puncta despite receiving no known tauopathy-inducing insults. This suggests that the larvae express the transgene at a level near to a threshold for producing spontaneous aggregates. We performed selective breeding to minimize these occurrences and tentatively believe, after too few generations, that genetics of the fish is a factor – substantial genetic variation exists in zebrafish in-bred lines . HoweverRegarding our TBI methods, further refinements may yet be able to improve consistency of the injury and reduce the apparent variability between individuals. This variability is real, but somewhat offset by the large sample sizes attainable: our TBI methods offer the potent advantages of zebrafish larvae with respect to genetic and drug accessibility in high-throughput formats, while also retaining the critical in vivo complexity required to investigate disease etiology and treatments. Further, it remains to be established if the mechanisms we reveal are ubiquitous across the various forms of TBI: our model fills a gap by supplying a rare ‘closed head’ TBI model  later progression of CTE, AD or other tauopathies.Zebrafish were raised and maintained following protocol AUP00000077 approved by the Animal Care and Use Committee: Biosciences at the University of Alberta, operating under the guidelines of the Canadian Council of Animal Care. The fish were raised and maintained within the University of Alberta fish facility under a 14/10 light/dark cycle at 28°C as previously described .ua3171Tg(eno2:Hsa.MAPT_Q244-E372−EGFP) was assigned the allele number ua3171.To engineer the transgenic Tau4R-GFP reporter zebrafish, the human wild-type MAPT sequence of the four-microtubule binding repeat domain  with a seven-amino acid C-terminal linker (RSIAGPA) was ordered as a gene block from IDT. The gene block was subcloned into a middle entry cloning vector . This was recombined with the p5E-enolase2 and p3E-GFP components into destination vector pDestTol2CG2 of the Tol2kit . The desTg[eno2:SOD1-GFP] zebrafish line that was assigned allele number ua3181.An equivalent transgenic zebrafish biosensor was engineered to detect human SOD1 aggregation. Subcloning from existing vectors  producedTCTCGTTGGGGTCTTTGCTC) confirmed the proper orientation. Purification of the plasmid was conducted with the Qiagen purification kit. HEK293T cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All cells were maintained at 37°C in a humidified 5% CO2 incubator. For passaging cells, cells were washed with phosphate-buffered saline (PBS) before trypsinization with 0.05 Trypsin-EDTA .To move the Tau4R-GFP reporter above into a vector appropriate for cell culture, BamHI and Xhol nuclease restriction enzymes were employed to remove the Tau4R-GFP fragment from pDest tol2CG2.eno2.Tau4R-GFP.pA. The Tau4R-GFP fragment was subcloned into the pCDNA3.1 vector using a T4 DNA ligase enzyme. Sequencing of the cloned vector with the following reverse primer for GFP . The expression of the fluorescent reporter was confirmed the next day through microscopic analysis. A stable cell line was established by replating the transfected cells at a 1:10 dilution and selecting in DMEM media containing 1200 μg/ml geneticin . Expression of the fused fluorescent proteins in the stable cell lines was confirmed using fluorescent microscopy. Polyclonal cells and monoclonal cells were grown to confluency in 10 cm dishes, then stored in liquid nitrogen until use.HEK293T cells were plated at 1 × 10For cell lysate preparation, cells were washed with cold PBS, then collected and incubated with cold lysis buffer , 1 mM EDTA and 1% Nonidet P-40) supplemented with protease inhibitor  for 10 min on ice. Cells were lysed using a bio-vortexer homogenizer for 20 s for two rounds. The lysate was centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was collected, and the protein concentration was determined using the Qubit Protein Assay Kit (Invitrogen).2, 20% glycerol, 0.1% Triton-X) with protease inhibitor and phospSTOP (Sigma-Aldrich) in the case of pt406 Tg. Brains were lysed using a bio-vortexer homogenizer and sonicated for 3 s for one round. Samples were centrifuged as above and concentration of the samples was assessed in a Qubit fluorometer (Invitrogen).For zebrafish brain lysate preparation, the brains of adult zebrafish were dissected. Brains were homogenized in cell lysis buffer  and boiled for 10 min before loading in 11% SDS-PAGE. Electrophoresis was performed using the Bio-Rad Power PAC system in running buffer . The gel was transferred to a PVDF membrane using a wet transfer system. All membranes were blocked for one hour in protein-free blocking buffer PBS (ThermoFisher) or TBST with 5% milk and then incubated with primary antibody overnight at 4°C with gentle agitation. The primary antibodies used in this study include rabbit monoclonal GFP  at 1:3000 dilution, rabbit anti-β-actin  at 1:10,000. All membranes were washed three times with 1X TBST before incubation with secondary antibody (goat-anti-mouse) HRP or HRP-conjugated anti-rabbit at 1:5000 dilution (Jackson ImmunoResearch) for 1 hr at room temperature. The membranes were washed for the final time before visualization using Pierce ECL Western Blotting Substrate (ThermoFisher) on a ChemiDoc (Biorad). For stripping and re-probing, the membranes were stripped using mild stripping buffer  before blocking them and repeating the methods described before.4 with 5% sucrose three times before washing with 1% Tween in H2O (pH 7.4), and then −20°C acetone. Larvae were incubated in PBS3+ containing 10% normal goat serum for 1 hr and then incubated with primary antibody with 2% normal goat serum in PBS3+. The primary antibody used was polyclonal Anti-Active-Caspase-3  at 1:500 dilution. The secondary antibody applied was Alexafluor 647 anti-rabbit at 1:200 dilution (Invitrogen). Larvae were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (ThermoFisher) for 30 min.Larvae were fixed overnight in 4% paraformaldehyde, either 1 day after being subjected to TBI or following the subsequent application of drugs as indicated. Immunostaining of Activated-Caspase3 on whole-mount larvae was carried out as previously described . Larvae P301L mice and non-Tg littermate controls (129/SvEvTac genetic background) were provided by Dr. David Westaway and Dr. Nathalie Daude . The samples were then incubated overnight under the previous conditions. The next day, the samples were centrifuged at 16,000 g for 30 mins at room temperature. The supernatant was discarded, while the resulting pellet was resuspended in 2% (vol/vol) PTA in ddH2O (pH 7.0) and 2% sarkosyl in DPBS. The samples were next incubated for one hour before the second centrifugation. The supernatant was removed and the pellet was re-suspended in DPBS. An aliquot of 5 μl of PTA purified brain homogenate was employed for electron microscopy (EM) analysis to confirm the presence of fibrils in each sample.The phosphotungstate anion (PTA)-precipitated brain homogenate was prepared as described . Briefly2O at a concentration of 2 mg/ml. The recombinant protein was fibrillized as described previously (Synthetic human Tau protein (wildtype full-length monomers) was purchased as a lyophilized powder  and resuspended in ddHeviously . RecombiNegative staining for EM analysis of fibrils was conducted as described elsewhere . Briefly5 per well in 24-well plates. Cells were transduced the next day, using 40 μl of 10% clarified brain homogenate combined with Opti-MEM to a final volume of 50 μl. A further 48 μl of Opti-MEM and 2 μl of Lipofectamine-2000 (Invitrogen) was added to the previous Opti-MEM mixture to a total volume of 100 μl and incubated for 20 min. The liposome mixture was applied to the cells for 18 hr, and cells were then washed with PBS, trypsinized, and re-plated on coated coverslips (ThermoFisher) for imaging and analysis.Polyclonal Tau4R-GFP cells were plated at 2 × 10For PTA-precipitated brain homogenate, 1:10 dilution of precipitated fibrils was used for the transfection. 5 μl of PTA-purified fibrils was diluted in 45 μl Opti-MEM to a final volume of 50 μl. The previous Opti-MEM mixture was added to 47 μl of Opti-MEM and 3 μl of Lipofectamine-2000 and incubated in room temperature for 2 hr as described in Prior to imaging, transfected cells were fixed 2% PFA in PBS for 15 mins. Samples were then washed twice with PBS then stained with DAPI (1:3000 from 1 mg/ml stock) for six mins. Cells were imaged using a Zeiss LSM 700 scanning confocal microscope featuring Zen 2010 software . Due to increased brightness of the GFP+ puncta formed after introduction of brain homogenate, GFP exposure was minimized for those cells only. To quantify the GFP+ puncta, a total of nine images were collected and analyzed for each condition, each with ~100 cells. DAPI-positive nuclei were utilized to determine the number of cells per image. The number of cells with inclusions (multiple nuclear inclusions or one cytoplasmic puncta) were counted and the percentage was calculated.P301L mice or wildtype littermate control), or synthetic tau, were mixed with 20% dextran Texas Red fluorescent dye (Invitrogen) and injected into the ventricles. For all the brain injection experiments, an uninjected control group and control group injected only with 20% red dextran fluorescent dye in PBS were included. After the injections, embryos were screened using a Leica M165 FC dissecting microscope and appropriately injected larvae were gathered for further analysis. The injections were considered appropriate if they had sharp edges and non-diffuse dye in the ventricle  were performed as described previously with few modifications . Embryosentricle . Larvae For the microscopic analysis of GFP-positive inclusions, larvae that were either injected or treated with traumatic injury, along with the control groups, were anesthetized via tricaine at the indicated time point  or post-traumatic injury (dpti) depending on the experiment). Images for GFP-positive puncta on the brain area or lateral line above the spinal cord were taken using a Leica M165 FC dissecting microscope and the number of GFP-positive puncta were manually counted.To induce TBI, 10–12 unanesthetized larvae (3 dpf) were loaded into a 10-ml syringe with 1 ml of E3 media. The syringe was blocked using a stopper valve to ensure no larvae or media left the syringe upon compression of the plunger. The syringe was held vertical using a metal tube holder at the bottom end of a 48’ tube apparatus. A defined weight (between 30 and 300 g) was dropped manually from the top of the tube. The tube diameter was matched to (slightly greater than) the weight’s diameter to enhance repeatability. This was either done once or repeated three times, with either 65 or 300 g weights. Once larvae were subjected to the TBI, they were moved back to a petri dish with fresh media and maintained for further analysis.To characterize the dynamic changes in pressure that occurred within the syringe during the TBI events, the stopper valve attached to the syringe (described immediately above) was replaced with a piezoresistive pressure transducer . Events were monitored via a PowerLab 2/26 data acquisition device and LabChart 7 software (AD Instruments). The pressure transducer was zeroed to report gauge pressure (pressure changes relative to atmospheric pressure) and was calibrated against a manometer . After each weight drop, the syringe apparatus was reset to remove any air bubbles and the pressure transducer was zeroed. Time courses of induced pressure were reported over a 350 msec time frame with 50 msec of base line recording, while mean and maximum pressure values were calculated from the initial 300 msec following the impact of the weight.Abnormalities of blood flow and circulation resulted from TBI was detected 5 to 10 mins after larvae were subjected to TBI. The blood flow in the tail area of zebrafish larvae, either those subjected to TBI or uninjured controls, was recorded using Leica DM2500 LED optical microscope.The seizure-like behavior and activity of zebrafish larvae post-traumatic injury experiment was quantified via behavioral tracking software as described in our recent publications . Brieflyua3144Tg[elavl3:CaMPARI (W391F+V398L)]. Due to a federal moratorium on importing zebrafish into Canada  using a laser point-scanning confocal microscope , and visualized as maximum intensity projections. The hindbrain area was analyzed, as it was the brain region most responsive to TBI. To specifically isolate the brain regions and obtain data points, a 3D area was isolated by creating a surface with Imaris 7.6  and the mean fluorescence intensities of the green and red channel intensities were calculated. Data points were presented as a red/green ratio for each individual larva and interpreted as relative neural activity, which is defined as red photoconverted CaMPARI in ratio to green CaMPARI .Tau biosensor larvae were treated with 20 μM of the proteasome inhibitor MG-132 at 2dpf, following injections with brain homogenate from Tg human Tau mice. The treatment was left for 48 hr before changing the media and evaluating the percentage of larvae developing GFP+ puncta in the brain region.For Kainic acid or kainate treatment (KA), the doses  were selected based on previous use of KA in zebrafish larvae , and addeno2:hsa.MAPT-ires-egfp)Pt406 throughout the zebrafish CNS (Pyrimidyn-7 (P7), the dynamin inhibitor, was purchased at a 50 mM concentration supplied in DMSO (Abcam). Larvae that were subjected to TBI were treated within six hours following the injury with 3 μM of P7. The dose was chosen based on the previous use of the P7 drug on zebrafish larvae . The larfish CNS .All statistical analyses were performed using GraphPad Prism Software . Sample sizes appropriate for our conclusions were estimated iteratively as the variance in each of our new methods became apparent; dose-response curves and significant differences amongst these dose were used to judge that any detected impacts of subsequent interventions would be valid. All experiments were independently replicated at least twice, individual larvae were the sampling unit , and no outliers or other data were excluded. The experimenters were blinded to the treatments prior to quantifying outcomes. Paired t-tests were used to compare between two groups, except for when sample sizes were too small to assess normality wherein Mann-Whitney tests were used. For comparison between three or more groups at various time points or the same time point, two-way and ordinary one-way ANOVA were used followed by post-hoc Mann-Whitney U tests and Kruskal-Wallace multiple comparison tests, respectively. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Your manuscript provides a new model for this form of brain injury. The accessibility of the work in larval zebrafish will enable a powerful combination of environmental, genetic and imaging tools to learn more about this pathogenic process – and to hopefully find new ways to treat any of us that are unfortunate to suffer from this kind of trauma.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Stephen C Ekker as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Didier Stainier as the Senior Editor.Thank you for submitting your article "Seizures are a druggable mechanistic link between TBI and subsequent tauopathy" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:The authors present a well-designed study to evaluate the validity of a larval model of traumatic brain injury in zebrafish, a biosensor to report on the prevalence of Tau aggregation and subsequent studies aimed at delineating some of the mechanistic pathways linked to the prion-like progression of tauopathy and relationship to post-traumatic epilepsy. They conclude with a demonstration test of anticonvulsants as a potential example intervention. The authors should be commended on their simple model and extensive body of work that has gone into validating the biosensor and its application in a zebrafish model. Moreover, this body of work further validates the use of zebrafish as an important tool and bridge organism in the field of neurotrauma.The work is also well described with suitable experimental study design to minimize bias and provides data for full transparency.We note a number of unresolved questions based on the body of work in the existing manuscript. We note these as opportunities for improvement through textual clarification. Should you have additional data on-hand to add, that is also appropriate but not required.Essential revisions:1) Acknowledging the previous work published by McCutcheon et al., 2016 that reported an excitotoxic model of TBI with potential for high throughput drug screening in TBI.2) Please provide a description and comparison of their work with recent data in an adult zebrafish model of TBI by Cho et al., 2020.3) Although the Materials and methods section of the manuscript is quite detailed, the Results section sometimes lacks experimental information. Some examples are itemized below.Points for Improvement:1) Figure 1G-I. The differences between fish injected with the TG brain  and controls is not large. Moreover, the presence of aggregates in control is surprising. Furthermore, such aggregates are less prominent in the controls of the experiment with the human protein  compared to the Tg mouse extract . Finally, the spontaneous aggregates in the spinal cord of fish in the experiment shown in Figure 5 is another surprising observation. This is discussed in the subsection “ Limitations of our approach” but the matter remains somewhat problematic.2) Why the increase in neural activity after TBI are particularly prominent in the hindbrain is something that could be discussed.3) In the first section of their Discussion, the authors state that their "zebrafish models imperfectly replicate human TBI and tauopathy…". It would have been helpful to the reader if they had first summed up why they thin this replication is imperfect. This comment has to do with the general organization of the Discussion and it could have preferable for the authors to start with the third section of the Discussion and perhaps introduce some elements found in subsection “ Limitations of our approach”. I leave it to the author's judgment.4) Subsection “Bath Application of Drugs”: How were these doses determined to be appropriate? Reference?5) Figure 1 panels E and F: figure legend should indicate when these microphotographs were taken after application or injection, respectively.6) Subsection “Validation of in vivo Tau biosensor via intra-ventricular brain injections of Tau fibrils”: The shape of the puncta. Can the authors refer to a specific figure or provide a higher magnification of a figure panel.7) Subsection “Validation of in vivo Tau biosensor via intra-ventricular brain injections of Tau fibrils” end of paragraph three: The text is redundant and reference to Figure 2A is not really appropriate. Some minor rewriting is necessary.8) Many figures are lacking scale bars.9) Paragraph four of subsection “Introduction of the first traumatic brain injury model for larval zebrafish”: It should be Figure 3F and not 3B. Furthermore, the signal is hard to see in the right half of the Figure.10) The red color images in panels B' and B' of Figure 4 are not particularly convincing, at least on my PDF version of the manuscript.11) The mass that was used for the experiment shown in Figure 5A-D should be indicated.12) Discussion paragraph four: The authors specify what a "blast injury" is: "(e.g as experienced by military.….)". However, this is at least the third time the term is mentioned. Perhaps move the explanation to the first occurrence.13) Typos, grammar: Subsection “Recording blood flow following TBI” "were subjected"14) "data" is plural. Please correct all instances of "data is" to "data are", "this data" to "these data" throughout the manuscript.15) It is not entirely clear in the manuscript why the SOD-GFP transgenic was an appropriate control for comparison to the Tau-GFP biosensor in terms of aggregation after trauma. Please elaborate.16) The model is simple to apply but raises some concerns about the interpretation of data and specific relevance to TBI. For example, the data in Figure 3 examines hemorrhage and blood flow impairments after trauma, however, the method of injury application is likely to extend injury to the entire larvae and not only to the head. This is highlighted in the data in Figure 3 that demonstrates blood flow impairments in the tail and hemorrhage in areas outside of the brain region. ). The authors should acknowledge the limitations of this particular injury paradigm and the interpretation of the data.17) The authors should provide some explanation as to why tau aggregation in the brain appears limited to a distinct region in the midline and specific somite regions within the spinal cord. Essential revisions:1) Acknowledging the previous work published by McCutcheon et al., 2016 that reported an excitotoxic model of TBI with potential for high throughput drug screening in TBI.We now acknowledge this excitotoxicity model in our Results.2) Please provide a description and comparison of their work with recent data in an adult zebrafish model of TBI by Cho et al., 2020.Thank you for directing us to this relevant work. We have now briefly contrasted this work with our larval approach in the Discussion.3) Although the Materials and methods section of the manuscript is quite detailed, the Results section sometimes lacks experimental information. Some examples are itemized below.We address each point in turn below.Points for Improvement:1) Figure 1 G-I. The differences between fish injected with the TG brain  and controls is not large.The purpose of Figures 1 and 2 is to validate our new Tau-GFP biosensor line, showing that we can detect Tau aggregates after an intervention known to provoke Tau aggregation. The differences shown are > 3-fold and statistically significant. We do not doubt that further optimization of Tau fibril injections in this model could increase the effect size, but this is not the objective of the present work.Moreover, the presence of aggregates in control is surprising.The detection of occasional aggregates in control zebrafish might reflect real stochastic events that are not observable in other models, since this is the first in vivo system allowing observation of Tau aggregation. It is conceivable that these events reflect the proteostatic concepts of dynamic balance between accumulation and clearance of misfolded proteins, in a rapidly developing CNS where some cells may express higher levels of Tau or the reporter gene. However, even if the detection of occasional aggregates in control zebrafish is a transient technical artifact, it is not “somewhat problematic”. All data are reported as comparative differences between experimental groups and controls. Figures 1, 2 and 5 show unequivocally that the number of GFP puncta was increased relative to controls after injection of transgenic mouse brain homogenate, synthetic fibrils, exposure to proteasome inhibitors or fluid shock wave injury, but not by control mouse brain homogenate, vehicle, Tau monomer, or sham injury. The reasonable and parsimonious interpretation is that the reporter shows Tau aggregates, allowing us to use this a tool to interrogate the mechanisms underlying tauopathy in TBI.Furthermore, such aggregates are less prominent in the controls of the experiment with the human protein  compared to the Tg mouse extract . Finally, the spontaneous aggregates in the spinal cord of fish in the experiment shown in Figure 5 is another surprising observation. This is discussed in the subsection “ Limitations of our approach” but the matter remains somewhat problematic.Minor quantitative differences between controls in replicate experiments are routinely seen in all biological studies, regardless of discipline, and are unavoidable because of the large number of undefined variables in complex biological systems. This is why all of our experiments include controls from the same clutch of embryos that replicate genetics, epigenetics, and environmental conditions exactly, so that any differences in the number of aggregates are unequivocally attributable to the experimental intervention. This is also why all of the experiments were carried out in biological triplicate, an advantage of using zebrafish models whose throughput allows this degree of experimental rigor.2) Why the increase in neural activity after TBI are particularly prominent in the hindbrain is something that could be discussed.We added some speculation on this point to the Discussion section that considers the link of post-traumatic seizures to subsequent tauopathy.3) In the first section of their Discussion, the authors state that their "zebrafish models imperfectly replicate human TBI and tauopathy…". It would have been helpful to the reader if they had first summed up why they thin this replication is imperfect. This comment has to do with the general organization of the Discussion and it could have preferable for the authors to start with the 3rd section of the Discussion and perhaps introduce some elements found in subsection “ Limitations of our approach”. I leave it to the author's judgment.We broadly have left the order of the information as originally submitted, and tried to partially fix this issue by adjusting the writing. We now indicate for the reader that the limitations (the “imperfect” part) will be discussed in a subsequent section.4) Subsection “Bath Application of Drugs”: How were these doses determined to be appropriate? Reference?We removed the word “appropriate” from this sentence and added references to the literature that guided our dose selection, including two papers that previously reported seizure responses to Kainate in zebrafish larvae. Please also note we established a dose-response curve using this drug .5) Figure 1 panels E and F: figure legend should indicate when these microphotographs were taken after application or injection, respectively.We have added a sentence clarifying this at the end of the legend to Figure 1.6) Subsection “Validation of in vivo Tau biosensor via intra-ventricular brain injections of Tau fibrils”: The shape of the puncta. Can the authors refer to a specific figure or provide a higher magnification of a figure panel.We have made this description less definitive, reflecting our perception that the puncta have various characters, and refer the reader to Figure 1F”.7) Subsection “Validation of in vivo Tau biosensor via intra-ventricular brain injections of Tau fibrils” end of paragraph three: The text is redundant and reference to Figure 2A is not really appropriate. Some minor rewriting is necessary.We have adjusted this writing.8) Many figures are lacking scale bars.We have added scale bars to the remaining figure panels.9) Paragraph four of subsection “Introduction of the first traumatic brain injury model for larval zebrafish”: It should be Figure 3F and not 3B. Furthermore, the signal is hard to see in the right half of the Figure.We have corrected this to reference to panel 3F. We have now changed the pseudocolored panel from magenta to grayscale to increase the visibility of the caspase-3 labeled cells, and added arrows to assist. Our quantification of the signals was performed while viewing the intact larvae in a fluorescent dissection scope , rather than from these types of images. The observers were blinded to the treatments. Further data convincing the results are consistent and robust include the quantification of a few dozen larvae in a separate experiment performed by separate observers .10) The red color images in panels B' and B' of Figure 4 are not particularly convincing, at least on my PDF version of the manuscript.Thank-you. We have adjusted the single-channel images to grayscale, and please note the red is pseudocoloured to magenta in the merged panels. In our view, the abundance of red/magenta is convincingly greater in B’ compared B”. One should not expect the difference between this to be large , because here the time window of photo-conversion was intentionally kept very brief. Regardless, this is a robust and reliable signal, that includes both an increase in red fluorescence and concomitant decrease in green fluorescence as the CaMPARI is photoconverted. That alteration is best appreciated from its quantification of ratiometric red:green CaMPARI fluorescence – it is readily obvious when that ratio is reported in the heatmaps of panel C, or when quantified (from 3D volumetric renderings) to show it is quite consistent between trials in panel D.11) The mass that was used for the experiment shown in Figure 5A-D should be indicated.We have added this to the beginning of the figure legend.12) Discussion paragraph four: The authors specify what a "blast injury" is: "(e.g as experienced by military.….)". However, this is at least the third time the term is mentioned. Perhaps move the explanation to the first occurrence.Thank-you. We have added a phrase to our first use of “blast injury”. Based on our experiencefrom other presentations of this work we think it is helpful to remind the reader of this mode of TBI. For example some readers will be more accustomed to rodent TBI models that typically implement a piston to deliver a direct physical blow. We feel it is worthwhile to remind the reader that while our model is different than (and complements) these approaches, our model is nonetheless representative of a mode of injury that important in human TBI.13) Typos, grammar: Subsection “Recording blood flow following TBI” "were subjected"Corrected.14) "data" is plural. Please correct all instances of "data is" to "data are", "this data" to "these data" throughout the manuscript.We have corrected every instance of incorrect usage we found.15) It is not entirely clear in the manuscript why the SOD-GFP transgenic was an appropriate control for comparison to the Tau-GFP biosensor in terms of aggregation after trauma. Please elaborate.SOD1-GFP was selected because it is a similar GFP-based biosensor that is designed to report protein aggregation, and because SOD1 aggregation is not thought to be involved in the response to TBI. The absence of aggregates in SOD1-GFP zebrafish after TBI provides unequivocal proof that the effect seen for the Tau biosensor is specific to Tau. This additional control enhances the degree of scientific rigor in our study. We now mention this rationale more clearly at first occurrence in the Results.16) The model is simple to apply but raises some concerns about the interpretation of data and specific relevance to TBI. For example, the data in Figure 3 examines hemorrhage and blood flow impairments after trauma, however, the method of injury application is likely to extend injury to the entire larvae and not only to the head. This is highlighted in the data in Figure 3 that demonstrates blood flow impairments in the tail and hemorrhage in areas outside of the brain region. ). The authors should acknowledge the limitations of this particular injury paradigm and the interpretation of the data.We agree with this important point, and have now alluded to this at the beginning of the Discussion  The authors should provide some explanation as to why tau aggregation in the brain appears limited to a distinct region in the midline and specific somite regions within the spinal cord.We added some speculation on this point to the Discussion section that considers the link of post-traumatic seizures to subsequent tauopathy.


Object: Predicting whether intramedullary slitlike cavity (SC) will worsen over time or remain stable is an outstanding clinical challenge. The aim of this study was to identify early features of SC .Methods: We prospectively included all patients referred to our institution following the discovery of a SC and divided them in two groups: typical SC  or atypical SC . Clinical evolution and changes in MRI features were evaluated during follow-up. In some patients, diffusion tensor imaging was performed and cervical cord cross-sectional area was analyzed.Results: A total of 48 consecutive patients were included in the study. The mean follow-up was 58 months. Of the seven patients presenting with deficits at first consultation, two worsened and five remained stable. Of the 41 patients without deficits, seven worsened and 34 remained stable. None of the patients developed severe motor deficits or experienced enlargement of the cavity; 7% of patients who presented with typical SC worsened compared with 35% with atypical SC. The negative predictive value was 0.93 (P = 0.02).Conclusion: Most patients remained stable and a subset of patients developed minor motor deficits. For clinical management, we propose surveillance of patients with a typical SC and close follow-up of those with an atypical SC and/or presenting with deficits. Neurologists and neurosurgeons regularly encounter patients referred following the discovery of an intramedullary slitlike cavity (SC). These patients usually undergo magnetic resonance imaging (MRI) for neck or upper limb pain mimicking cervical radiculopathy; an abnormal image may also be discovered as an incidental finding. These cavities are sometimes referred to as hydromyelia , 2 or asWhile physical examination frequently reveals minor abnormalities, it is important to determine whether patients will remain stable or will experience neurological deterioration. There is currently a lack of consensus regarding the modalities used in follow-up and the information delivered to patients.The aim of this study was to prospectively analyze the natural history and imaging features of SCs. We hypothesized that patients at risk of neurological deterioration who should be closely followed up are identifiable by spinal cord MRI.This is a prospective study performed at one academic neurosurgical center which is a reference center for syringomyelia and related diseases. Patients were included between January 2015 and January 2017.We included all consecutive patients referred for a SC. All patients with evidence of Chiari malformation, demyelinating disease, tumor related syringomyelia and post traumatic syringomyelia were excluded. The study was approved by the Ethics Committee of the Pitié-Salpêtrière Hospital . We obtained written informed consent for all participants.American Spinal Injury Association (ASIA) score was determined and pain was evaluated using a numeric pain rating scale (NPRS) ranging from 0 (no pain) to 10 (worst pain possible). Motor function and the presence of urinary symptoms were assessed at baseline and during follow-up by a neurosurgeon MF,SM,NA).Based on MRI T2/DRIVE analysis we described two type of cavities: A typical SC was defined as one meeting all of the following criteria: located at the midline  at the junction between the anterior third and posterior two-thirds of the spinal cord ; craniocaudal extension of less than three metamers; and no enlargement of the spinal cord at the level of the cavity . An atypDiffusion tensor imaging (DTI) metrics and spinal cross-sectional area were analyzed.Patients were clinically evaluated by a neurosurgeon and a physiotherapist.MRI scans were acquired on an Achieva 3Tesla scanner  with a multiple-element spine array coil that allowed parallel imaging. The MRI protocol was the same for all patients. Cavity size was determined by whole-spine high-resolution T2-weighted imaging (DRIVE). We also performed a 32-direction diffusion sequence with 3 mm-thick slices (voxel size: 2 × 2 × 3 mm) in the 1-h examination.If the patient had a previous MRI performed at another center showing the SC, we considered this MRI as the baseline of the follow-up.DTI analysis without spinal cord segmentation was performed on a voxel-by-voxel basis using DPTools software All diffusion-weighted images were corrected for motion artifacts using the sct_dmri_moco function in SpinalCordToolbox packageSpine cross-sectional area was measured from the T2-weighted images at the level of the C2 vertebra. Five 3 mm-thick slices perpendicular to the spinal cord were selected, with the most inferior slice passing centrally through the C2/C3 disk . The corStatistical analysis was performed using R softwareForty-eight consecutive patients were included in the study . The mean age at diagnosis was 41 years. Detailed information on the population is presented in All but one patient were symptomatic upon diagnosis, with one or more of the following symptoms at first consultation: axial pain  in 29 patients (60%), radicular pain in 23 patients (48%), and mild motor deficit (ASIA > 95/100) in seven patients (15%). No patient had urinary symptoms.SCs were located in the spinal cervical region in 13 patients (27%), the spinal thoracic region in 22 patients (46%), and in both regions in 13 patients (27%). A typical SC was observed in 28 patients (58%) by MRI and in nine (19%), the cavity spanned more than three vertebrae. The spinal cord was slightly enlarged in 17 patients (35%). The cavity was located between the anterior third and posterior two-thirds of the spinal cord in all patients.In 27 patients of this series there was a disc herniation at the level of the slitlike cavity  but only in 3 patients were the symptoms clearly related to degenerative disc disease, two of these patients had been operated on for cervical disc herniation. In one of these two patients the intramedullary slit was visible on preoperative imaging.Among the 47 patients who presented with pain, correlation between symptoms and imaging was good for only 9 patients (19%). In 32 patients (68%) it was impossible to attribute all symptoms to the SC since there were symptoms above the cavity level and/or evidence of degenerative spine disease on imaging. In 6 patients (13%) the symptoms were clearly not related to the SC. This disparity was irrespective of the typical or atypical nature of the cavity: over 20 patients with atypical cavities, concordance was good for 5 patients, was intermediate for 12 patients, and was poor for 3 patients. For the 28 patients with typical cavities, concordance was good for 4 patients, was intermediate for 20 patients, and was poor for 4 patients.Tractographic reconstructions were available for 34 patients. Visual inspection revealed abnormalities in 33 patients, with rarefaction and/or disorganization of fibers at the location of the cavity. Based on the assumption that the water content of the cavity could cause artifacts, we did not perform analyses at the level of the cavity; instead, we chose to analyze DTI metrics in the upper cervical cord including FA and MD, which were available for 32 and 31 patients, respectively. Mean FA was 0.38 ; MD was 1.79 ; mean transverse diffusivity (TD) was 1.32 ; and mean axial diffusivity (AD) was 2.18 .Given that these metrics could be influenced by perimedullary cerebrospinal fluid (CSF), we performed another analysis of the same data after automatic segmentation of the spinal cord which were available for 18 patients. Mean FA was 0.54 ; MD was 1.41 ; mean TD was 1.04 ; and mean AD was 2.27 .2 .Spine cross-sectional area data were available for six patients. Mean cross-sectional area at the upper cervical level was 98 mmMean follow-up from the time of diagnosis of the cavity was 58 months .At first consultation, seven patients presented with mild motor deficit (> 4/5); among them, two experienced worsening of the deficit or developed urinary symptoms, whereas five remained stable. Of the 41 patients who did not present any deficit at first consultation, seven developed mild motor deficit or urinary symptoms and 34 remained stable . The ASIMRI control scans with T2-DRIVE sequence were available for 42 patients; the cavity was stable in all patients.P = 0.02; Fisher's exact test). The positive and negative predictive values were 0.35 and 0.93, respectively  and seven (35%) patients, respectively, experienced worsening of their condition, with the appearance of motor deficit or urinary symptoms. A statistically significant difference was observed between the two groups (ectively .t-test) or MD .Among the nine patients whose condition worsened (appearance of a new deficit or aggravation of an existing deficit), DTI metrics of the segmented cervical spinal cord were available for four. Mean FA was 0.575  and MD was 1.410 . Among the 39 patients who remained stable (including those who presented with a deficit at first consultation), DTI metrics of the segmented cervical cord were available for 14; mean FA was 0.531  and MD was 1.414 . Differences between the two groups were not statistically significant for FA . Among the 39 patients who remained stable, data were available for three; the mean area was 95.81 mm2 .Among the nine patients whose condition worsened, data regarding the cross-sectional area of the spinal cord at the C2 level were available for three. The mean area was 100.26 mmThis is the largest study to prospectively assess clinical status and imaging findings in patients presenting with SCs. Our results revealed a different clinical course between patients with a typical and those with an atypical SC. Among the 28 patients in the former group, two worsened, with appearance of a mild motor deficit or urinary symptoms; among the 20 latter patients, seven experienced worsening of their condition. A typical intramedullary SC had very good negative predictive value (0.93), which can reassure patients. However, we did not identify any DTI imaging parameter that could predict an unfavorable evolution at the individual level.Our results are in accordance with the literature , 11, 13 In our study, diffusion imaging was not predictive at the individual level. All patients presented with rarefaction of fibers at the location of the cavity, which was likely an artifact. Our results at the cervical level (decreased FA and increased MD) could be due either to axonal degeneration or to low signal-to-noise ratio . We alsoDiffusion imaging data for syringomyelia is scarce . In a stThere were limitations to our study. The length of the follow-up was different for each patient. Importantly, we did not precisely measure chronic pain using appropriate scales. In terms of imaging, DTI sequences were not performed for all patients, and we did not obtain follow-up data for DTI imaging parameters for any patient. It would be interesting to investigate whether the evolution of DTI parameters mirrors clinical course . Moreover, the DTI imaging was not of sufficiently high resolution to permit analysis of specific tracts, thereby precluding correlational analyses between imaging and neurological findings.Based on clinical and imaging parameters, it remains difficult to distinguish at the individual level between patients who will remain stable and those who will experience neurological worsening. However, we showed that most patients with a typical SC remain stable. Accordingly, these patients should be reassured at first consultation and managed by yearly clinical and MRI follow-up. On the other hand, symptomatic patients or those showing atypical SC should be managed by a multimodal approach , with closer follow-up. We believe that technical improvements in imaging and electrophysiology will help identify the few SC patients with poor neurological outcome.The datasets generated for this study will not be made publicly available. The dataset will be made available for any reasonable request, every request for dataset should be sent to corresponding author.The studies involving human participants were reviewed and approved by the study was approved by the Ethics Committee of the Pitié-Salpêtrière Hospital . Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.MF and NA substantially contributed to the conception and design of the study, to the acquisition of data, and to the analysis and interpretation of data, and were involved in drafting the manuscript or revising it critically for important intellectual content and in producing the approved final version for publication. DD performed acquisition of MRI data. MF, DD, ME-M, and SD performed analysis of imaging data and wrote methodological section regarding each imaging modality. FP, NA, and SM reviewed the final manuscript. All agreed to be accountable for all aspects of the work by ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.