Name: advanced diffusion imaging in the hippocampus of rats with mild traumatic brain injury
Uploaded: 2019-08-14T15:00:00
Description: Watch this Scientific Journal Video about Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury at JoVE.com

The authors would like to thank Research Foundation - Flanders (FWO) for supporting this work (Grant number: G027815N).

The protocol has been approved by the Animal Ethics Committee at the University of Ghent (ECD 15/44Aanv), and all experiments were conducted in accordance with the guidelines of the European Commission (Directive 2010/63/EU).
1. Animal preparation and helmet attachment
<ol>
	<li>Weigh a female Wistar H rat (&#177; 250 g or 12 weeks of age) and anesthetize in a small induction chamber filled with a mixture of isoflurane (5%) and O2 for at least 1 min.</li>
	<li>Inject the rat with ...

<ol>
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E., van den Brink, W., Campbell, J., Kita, H., Demetriadou, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+diffuse+brain+injury+in+rats.">A new model of diffuse brain injury in rats.</a> Journal of Neurosurgery. 80 (2), 291-300 (1994).</li><li>Heim, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Invisibility+of+Mild+Traumatic+Brain+Injury:+Impaired+Cognitive+Performance+as+a+Silent+Symptom.">The Invisibility of Mild Traumatic Brain Injury: Impaired Cognitive Performance as a Silent Symptom.</a> Journal of Neurotrauma. 34 (17), 2518-2528 (2017).</li><li>Zohar, O., Rubovitch, V., Milman, A., Schreiber, S., Pick, C. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMIDE:+A+Free+Software+Tool+for+Multimodality+Medical+Image+Analysis.">AMIDE: A Free Software Tool for Multimodality Medical Image Analysis.</a> Molecular Imaging. 2 (3), 131-137 (2003).</li><li>Veraart, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denoising+of+diffusion+MRI+using+random+matrix+theory.">Denoising of diffusion MRI using random matrix theory.</a> NeuroImage. 142, 394-406 (2016).</li><li>Veraart, J., Fieremans, E., Novikov, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+MRI+noise+mapping+using+random+matrix+theory.">Diffusion MRI noise mapping using random matrix theory.</a> Magnetic Resonance in Medicine. 76 (5), 1582-1593 (2016).</li><li>Braeckman, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+changes+in+hippocampal+diffusion+and+kurtosis+metrics+following+experimental+mTBI+correlate+with+glial+reactivity.">Dynamic changes in hippocampal diffusion and kurtosis metrics following experimental mTBI correlate with glial reactivity.</a> NeuroImage: Clinical. 21 (August 2018), 101669 (2019).</li><li>Jones, D. K., Kn&#246;sche, T. R., Turner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=White+matter+integrity,+fiber+count,+and+other+fallacies:+The+do's+and+don'ts+of+diffusion+MRI.">White matter integrity, fiber count, and other fallacies: The do's and don'ts of diffusion MRI.</a> NeuroImage. 73, 239-254 (2013).</li><li>. Matlab code DKI and WMTI model Available from: <a target="_blank" href="https://github.com/NYU-DiffusionMRI/Diffusion-Kurtosis-Imaging">https://github.com/NYU-DiffusionMRI/Diffusion-Kurtosis-Imaging</a> (2019)</li></ol>

Since mTBI often is the result of a diffuse and subtle injury that shows no abnormalities on CT and conventional MRI scans, the evaluation of microstructural damage after a mild trauma remains a challenge. Therefore, more advanced imaging techniques are needed to visualize the full extent of the trauma. The application of diffusion magnetic resonance imaging in TBI research has gained more interest during the last decade, where diffusion tensor imaging is most frequently used5. A limitation of the...

Traumatic brain injury (TBI) has gained more attention in recent years, as it has become clear that these brain injuries can result in lifelong cognitive, physical, emotional, and social consequences1. Despite this increasing awareness, mild TBI (mTBI, or concussion) is still often underreported and undiagnosed. MTBI has been referred to as a silent epidemic, and individuals with a history of mTBI show higher rates of substance abuse or psychiatric problems2. Several patients with mTBI go undiagnosed every year due to the diffuse and subtle nature of the injuries, which are often not visible on conventional computed tomography (CT) or magnetic resonance imaging (MRI) scans. This lack of radiological evidence of brain injury has led to the development of more advanced imaging techniques such as diffusion MRI, which are more sensitive to microstructural changes3.
Diffusion MRI allows in vivo mapping of the microstructure, and this MRI technique has been used extensively in TBI studies4,5,6. From the diffusion tensor, fractional anisotropy (FA) and mean diffusivity (MD) are computed to quantify alteration in the microstructural organization following injury. Recent reviews in mTBI patients report increases in FA and decreases in MD following injury, which can be indicative of axonal swelling7. Contrary, increases in MD and decreases in FA are also found and have been suggested to underlie disruptions in parenchymal structure following edema formation, axonal degeneration, or fiber misalignment/disruption8. These mixed findings can be partially explained by the significant clinical heterogeneity of mTBI caused by different types of impact and severity (e.g., rotation-acceleration, blunt force trauma, blast injury or combination of the former). However, currently there is no clear consensus about the underlying pathology and biological/cellular basis underpinning alterations in the microstructural organization.
Animal models provide a standardized and controlled setting to investigate biological mechanisms of injury and repair following TBI in greater detail. Several experimental models for TBI have been developed and represent different aspects of human TBI (e.g., focal vs. diffuse trauma or trauma caused by rotational forces)9,10. Commonly used animal models include the controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models11,12. Although the experimental parameters can be well-controlled, these models make use of a craniotomy to expose the brain. Craniotomies or skull fractures are not commonly seen in mTBI; therefore, these experimental models are not valid to mimic mTBI. The impact acceleration model developed by Marmarou et al.13 makes use of a weight that is dropped from a certain height onto the rat&#39;s head, which is protected by a helmet. This animal model induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma. Therefore, this Marmarou weight drop model is appropriate to investigate imaging biomarkers for diffuse mTBI14,15.
This report demonstrates the application of advanced diffusion MRI in an mTBI rat model using the Marmarou weight drop model. First shown is how to induce a mild and diffuse trauma, and analysis using diffusion tensor imaging (DTI) model is then provided. Specific biological information is obtained with the use of more advanced diffusion models [i.e., diffusion kurtosis imaging (DKI) and white matter tract integrity (WMTI) model]. Specifically, mild trauma is inflicted and microstructural changes are then evaluated in the hippocampus using conventional T2-weighted MRI and an advanced diffusion imaging protocol.

<table>
	<tbody>
		<tr>
			<td>Induction of trauma</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% NaCl physiologic solution</td>
			<td>B Braun</td>
			<td>394496</td>
			<td></td>
		</tr>
		<tr>
			<td>brass weight 450g</td>
			<td>custom made</td>
			<td>custom made</td>
			<td>diamter 18mm and 210 mm height</td>
		</tr>
		<tr>
			<td>catheter</td>
			<td>Terumo</td>
			<td>Versatus-W</td>
			<td>26G</td>
		</tr>
		<tr>
			<td>ethilon II</td>
			<td>Ethicon</td>
			<td>EH7824</td>
			<td>FS-3, 4-0, 3/8, 16mm</td>
		</tr>
		<tr>
			<td>Matrass</td>
			<td>Foam to Size</td>
			<td>Type E</td>
			<td></td>
		</tr>
		<tr>
			<td>Plexiglas tube</td>
			<td>ISPA Plastics</td>
			<td>416564</td>
			<td>M1 PMMA XT GOO tube 25x19 mm (inner diamter 19 mm, minimal length of 1.50 m)</td>
		</tr>
		<tr>
			<td>Preclinical CT scanner</td>
			<td>Molecubes</td>
			<td>X-cube</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel helmet</td>
			<td>custom made</td>
			<td>custom made</td>
			<td>diameter 10 mm and 3 mm thickness</td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetergesic (buprenorphin)</td>
			<td>EcuPhar</td>
			<td>VETERG20</td>
			<td>0.05 mk/kg</td>
		</tr>
		<tr>
			<td>Xylocaine 2% gel</td>
			<td>AstraZeneca</td>
			<td>Xylocaine 2%</td>
			<td>gel</td>
		</tr>
		<tr>
			<td>Xylocaine (lidocain 2%)</td>
			<td>Aspen/AstraZeneca</td>
			<td>Xylocaine 2% gel</td>
			<td>100 &#956;l injection</td>
		</tr>
		<tr>
			<td>Diffusion MRI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Preclinical MRI acquisition software</td>
			<td>Bruker Biospin MRI GmbH</td>
			<td>Z400_PV51_CENTOS55</td>
			<td>ParaVision 5.1 MRI software</td>
		</tr>
		<tr>
			<td>Preclinical MRI scanner</td>
			<td>Bruker Biospin MRI GmbH</td>
			<td>PharmaScan 70/16</td>
			<td>7T MRI scanner</td>
		</tr>
		<tr>
			<td>Quadrature volume coil</td>
			<td>Bruker Biospin MRI GmbH</td>
			<td>RF RES 300 1H 075/040 QSN TR</td>
			<td>Model No: 1P T13161C3</td>
		</tr>
		<tr>
			<td>Small animal physiological monitoring unit</td>
			<td>Rapid Biomedical</td>
			<td>EKGHR02-0571-043C01</td>
			<td>Unit for respiratory monitoring</td>
		</tr>
		<tr>
			<td>Water-based heating unit</td>
			<td>Thermo Fisher Scientific</td>
			<td>Haake S 5P</td>
			<td>Model No: 1523051</td>
		</tr>
		<tr>
			<td>Anaesthesia</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anaesthesia movable unit</td>
			<td>Veterenary technics</td>
			<td></td>
			<td>BDO - Medipass, Ijmuiden</td>
		</tr>
		<tr>
			<td>isoflurane: Isoflo</td>
			<td>Zoetis</td>
			<td>B506</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen generator</td>
			<td>Veterenary technics</td>
			<td>7F-3</td>
			<td>BDO - Medipass, Ijmuiden</td>
		</tr>
		<tr>
			<td>Diffusion image processing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amide</td>
			<td>http://amide.sourceforge.net</td>
			<td>Version 1.0.5.</td>
			<td>Medical Imaging Data Examiner Toolbox (Loening AM, Gambhir SS, &#34; AMIDE: A Free Software Tool for Multimodality Medical Image Analysis&#34;, Molecular Imaging, 2(3):131-137, 2003)</td>
		</tr>
		<tr>
			<td>ExploreDTI</td>
			<td>http://www.exploredti.com</td>
			<td>Version 4.8.6</td>
			<td>Toolbox for (pre-)processing and analysis of diffusion weighted MR images (Leemans A, Jeurissen B, Sijbers J, and Jones DK. ExploreDTI: a graphical toolbox for processing, analyzing, and visualizing diffusion MR data. In: 17th Annual Meeting of Intl Soc Mag Reson Med, p. 3537, Hawaii, USA, 2009)</td>
		</tr>
		<tr>
			<td>MRtrix3</td>
			<td>http://www.mrtrix.org</td>
			<td>Version 3.0_RC3-86-g4b523b41</td>
			<td>Toolbox for (pre-)processing and analysis of diffusion weighted MR images</td>
		</tr>
	</tbody>
</table>

advanced diffusion imaging in the hippocampus of rats with mild traumatic brain injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

In the study, all TBI rats (n = 10) survived the impact and were able to recover from the impact and anesthesia within 15 min after detachment from anesthesia23. On the CT images, there was no evidence of skull fractures and the T2 images did not show any abnormalities such as bleeding, enlarged ventricles, or edema formation at the contusion site 1 day after trauma (Figure 5). Thus, based on these visual inspections of the anatomical images, large focal lesions were ...

Watch this Scientific Journal Video about Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury at JoVE.com

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

With this diffusion imaging protocol, it is possible to investigate microstructural changes in the hippocampus of a rat with a mild traumatic brain injury that are otherwise not visible on anatomical MRI. This technique can detect alterations in the brain following a mild and diffuse trauma which cannot be detected with CT or anatomical MRI. This technique makes it easier to monitor the recovery process after sustaining a mild traumatic brain injury in an objective and quantitative manner.This diffusion imaging and analysis technique can also be applied in other disorders affecting the brain, such as dementia and multiple sclerosis, not only in preclinical studies but also in humans. In this protocol, it's important that the quality of the diffusion scans and correction steps is high, therefore guidance from experienced technicians and analysts is suggested. Place the animal on a 37-degrees Celsius heating pad after confirming a lack of response to toe pinch in a 12-week old, female, Wistar H rat and insert a catheter into a lateral tail vein.Inject 100 microliters of 2%lidocaine locally into the shaved and disinfected scalp and make a midline incision to expose the skull. Use a small scissors to remove any excess membranes and rub a cotton bud across the skull until the periosteum is no longer present, then use a drop of tissue glue to attach a 10-millimeter diameter, three-millimeter thick, metallic disk approximately 1/3 in front of and 2/3 behind the bregma. For traumatic brain injury induction, place the rat on a custom made bed with a foam mattress of a specific spring constant and place the rat directly under a transparent plastic tube with a 450-gram brass weight with the helmet as horizontal as possible.Pull the weight up to one meter. With a second experimenter present, release the weight and have the second experimenter move the rat away from the tube immediately after the impact to prevent a second injury. Gently pull the helmet from the skull and use a gauze to stem any bleeding.Close the skin with a suture and apply local analgesia gel to the incision. Place the rat on the bed of a CT scanner and administer a general-purpose, low-dose CT scan to rule out skull fractures, then place the rat in a clean cage on a 37-degrees Celsius heating pad with monitoring until full recumbency before returning the animal to its cage. Before and one day following the trauma induction, confirm a lack of response to toe pinch in the experimental animal and place the animal on the MR scanner bed in a headfirst, prone position.Slide the quadrature volume coil over the head and advance the scanner bed into the scanner bore. To ensure a correct positioning, obtain a default 3-plane scout scan. When the scan is finished, load the scan in the image display and ensure that the head is lying straight and that the brain is positioned in the center of the magnet and coil.Acquire T2-weighted images using the default settings, except for the field, the view, and matrix size which should be adjusted to a higher in-plane resolution of 109 by 109 micrometers. Open the Geometry Editor and place the slice package in the correct position, including the bulbus of the brain and the cerebellum and load three new echo-planar, diffusion-weighted, spin-echo sequences from the B_diffusion folder into the scan control protocol. Acquire diffusion-weighted images using the default settings and open the Edit Scan tab.Set the slice orientation to axial and the number of slices to 25 to achieve a slice thickness of 500 micrometers and an inner slice distance of 600 micrometers and amend the readout direction into left-right. Under the Geometry tab, adjust the geometrical parameters and adjust the field of view and matrix size to 105 by 105 to ensure a resolution of 333 by 333 micrometers. Click the Diffusion tab within the Research tab for each of the three diffusion shells and adjust the number of diffusion directions to 32 for the first shell, 46 for the second shell, and 64 for the third shell.Change the number of B0 images to five for the first shell, five for the second shell, and seven for the third shell, and adjust the gradient directions with custom gradient direction files. Adjust the B value per direction to 800 seconds per millimeter squared for the first shell, 1, 500 seconds per millimeter squared for the second shell, and 2, 000 seconds per millimeter squared for the third shell, then open the Geometry Editor and place the field of view between the bulbus and cerebellum containing only the cerebrum to reduce the artifact and scan time. At the completion of the scanning protocol, transfer the animal from the scanner bed to a clean cage with a 37-degrees Celsius heating pad with monitoring until full recumbency.For diffusion MRI image processing, load the images in MRtrix3 and perform noise correction and Gibbs ringing correction on the diffusion-weighted images in the software program. Convert the corrected, diffusion-weighted images in T2 image to the NIFTI format as indicated. To perform correction for echo-planar imaging, motion and eddy current distortions, in the Plug-ins menu of ExploreDTI select Correction for subject motion EC/EPI distortions and select the preprocessed diffusion data file.To calculate the diffusion tensor imaging metrics for each rat, click Plug-ins and Export stuff to NIFTI, then select the parametric maps of the diffusion tensor imaging model and export the parametric maps for the kurtosis and white matter tract integrity models. To create a mask file for the hippocampus of each rat, load the fractional anisotropy image of the rat in the MRtrix viewer and click the plus button to create a new region of interest. To extract the diffusion metrics of the hippocampus of the rat, import the created mask file in the AMIDE software and open the parametric maps and mask image of the rat.To add the region of interest of the mask file into AMIDE, select the mask file image, click Edit, Add Region Of Interest and 3D isocontour and give the region of interest a meaningful name. Click on the region of interest displayed in the mask image and confirm that this volume should only contain voxels with a value of one. To calculate the mean values of the diffusion metrics in the hippocampus, click Tools and Calculate Region Of Interest Statistics and indicate the images and the region of interest to be included.After clicking Execute, a pop-up window with the computed values that can be used for further statistical analysis will appear. In this representative experiment, there was no evidence of skull fracture as assessed by CT imaging and the T2 images did not show any abnormalities at the contusion site one day after the trauma. To examine the quality of the non-rigid co-registration step between the T2 image and the diffusion data set, an overlay of the T2 image was added to the color-encoded fractional anisotropy map.The parametric maps for fractional anisotropy, mean diffusivity, axial diffusivity, and radial diffusivity parametric maps could then be calculated. Within the region of interest, calculation of the mean values for the axial, mean, and radial kurtosis values as well as values for the axonal water fraction, axial and radial extra-axonal diffusivity, and tortuosity of the white matter tract integrity could also be performed. In this representative experiment, analysis of the diffusion tensor imaging metrics revealed a significant increase in the fractional anisotropy values and a decrease in the diffusivity values following impact in the mild traumatic brain injury group.Diffusion kurtosis metrics also showed a significant decrease in radial kurtosis following impact while no changes in the axial or mean kurtosis were observed. Using the white matter tract integrity model, the radial extra-axonal diffusivity displayed a significant decrease and the tortuosity demonstrated a significant increase in the mild traumatic brain injury group one day after the impact. During the image analysis, it's important to check whether the data format from MRtrix was properly converted and imported into ExploreDTI and that each correction step was performed correctly.Instead of ROI-based analysis, a voxel-by-voxel analysis can be applied to investigate whole-brain alterations. This technique is very valuable within the neuroimaging field and can be applied to other brain disorders as well, for example dementia and multiple sclerosis.

Research

JoVE Journal

Neuroscience

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

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 ...

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of ...

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 ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...

Assessing Changes in Synaptic Plasticity Using an Awake Closed-Head Injury Model of Mild Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of ...

Validating Injury Response and Behavioral Assessments in an mTBI Rat Model

Rat Model of Closed-Head Mild Traumatic Injury and its Validation

Author Spotlight: Advancing Traumatic Brain Injury Research - A Closed-Head Model for Accurate Replication and Rapid Assessment 

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful ...