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 0.05 mg/kg buprenorphine subcutaneously in the neck, return it to the home cage, and allow pre-emptive analgesia for at least 30 min to take an effect. 
	NOTE: During the 30 min wait, the surgical site can be prepared.</li>
	<li>Place a heating pad kept at 37 &#176;C under the surgical field.&#160;Place the sterilized surgical instruments on the surgical field that was disinfected with 70% ethanol.</li>
	<li>Place the rat back in the induction chamber and anesthetize the rat until it is non-responsive to a paw or tail pinch.</li>
	<li>Place the rat on the surgical field and insert a catheter in the lateral tail vein.&#160;Next, shave the rat&#8217;s head, remove excess fur and disinfect the scalp and the rest of the surgical area with chlorohexidine.&#160;</li>
	<li>Inject 100 &#956;L of 2% lidocaine locally in the scalp.</li>
	<li>Make a midline incision using a scalpel size 11 to expose the skull, removing any excess membranes with small scissors. Retract the skin using an ocular speculum with a maximum spread of 1 cm.&#160;&#160;Additionally, remove the periosteum by gently rubbing a sterile cotton bud across the skull until the periosteum is no longer present.</li>
	<li>Put one drop of tissue glue on the skull and one on the sterilized metallic disc (diameter of 10 mm and 3 mm thickness), which acts as the helmet. Glue the disc approximately one-third before and two-thirds behind the bregma. Allow the glue to dry for 1 min.</li>
</ol>
2. Induction of traumatic brain injury (TBI)
<ol>
	<li>Place the rat on the custom-made bed with a foam mattress of certain spring constant (see Table of Materials). Position the rat directly under a transparent plastic tube with a 450 g brass weight with the helmet as horizontal as possible. Detach the rat from anesthesia.</li>
	<li>Pull the weight up to 1 m and release when ready. Ensure that a second experimenter is present to move the rat away from the plastic tube immediately after impact to prevent a second impact. 
	NOTE: The sham injured rats receive the same experimental procedure (steps 1.1&#8211;2.7), except for step 2.2.</li>
	<li>Re-attach the rat to the anesthesia and inject 1 mL of physiological solution (0.9% NaCl) via the catheter to reduce the hemodynamic shock. 
	NOTE: It is possible that the rat briefly stops breathing because of the impact. Gently compress the thorax if the rat does not spontaneously breathe after 2 s to encourage the breathing reflex.</li>
	<li>Remove the helmet by gently pulling it from the skull. Remove any remaining glue from the skull and skin and close the incision with surgical suture. Apply local analgesia gel using a sterile applicator tip.&#160;</li>
	<li>Place the rat on the bed of the CT scanner. Confirm the correct position using a scout scan. Adjust the field of view to enable imaging of the whole head within one bed position. Administer a general purpose, low dose CT scan to rule out skull fractures. 
	NOTE: Skull fracture is a criterium for euthanasia.</li>
	<li>Place the rat in a clean cage on a heating pad (37 &#176;C). Monitor the time to regain consciousness. Once the rat is able to sit upright, the rat can be returned to the home cage.</li>
	<li>Administer a second dose of 0.05 mg/kg buprenorphine one day following TBI induction.</li>
</ol>
3. Diffusion magnetic resonance imaging (MRI)
NOTE: Diffusion-weighted imaging is performed before and 1 day following trauma induction.
<ol>
	<li>Anesthetize the rat in a small induction chamber filled with a mixture of isoflurane (5%) and O2. When the rat is non-responsive to a paw or tail pinch reduce the anesthesia to 2% with a flow rate of 500 mL/min. Transfer the animal to the scanner bed in head-first prone position.</li>
	<li>Position the rat in the head holder with the teeth bar and nose cone, delivering the anesthesia, and slide the head forwards until the center of the brain is at the level of the center of the quadrature volume MRI coil. Apply lubricating ointment to the eyes in small amounts to prevent any damage to the cornea. Fixate the head with a small piece of tape to avoid movement during scanning.</li>
	<li>Place a pressure pad under the thorax of the rat to monitor respiration and cover the rat with a circulating warm water heating blanket and bubble wrap to keep the rat warm. Before the scanning, check the respiratory monitor to ensure that the signal is clear without noise and that the respiratory cycle is consistent. Relocate the pressure pad, if necessary. 
	NOTE: The respiratory rate should be kept between 1 breath per 1,200&#8211;1,700 ms by adjusting the level of anesthesia between 1%&#8211;2%.</li>
	<li>Slide the quadrature volume coil over the head. Adjust the tuning and matching capacitors of the coil to the proper frequency and impedance according to the instructions provided by the coil vendor. Advance the scanner bed into the scanner bore to start scanning.</li>
	<li>Obtain a default three-plane scout scan (&#8220;tripilot&#8221;) to ensure correct positioning.
	<ol>
		<li>Load the tripilot sequence into the Scan Control by clicking New Scan and selecting the tripilot sequence from the protocol list. Next, click the traffic light button to start the scan.</li>
		<li>When the scan is finished, load the scan in the image display and ensure that 1) the head is lying straight and 2) the brain is positioned in the center of the magnet and coil. Adjust the position of the head and/or the scanner bed, if necessary, and acquire a new tripilot scan.</li>
	</ol>
	</li>
	<li>Adjust the local magnetic field using an automated second-order shimming protocol: load the second-order shim protocol into the Scan Control as described in step 3.5.1. Next, click on the Acq tab | Current Adjustments | Method specific adjustment for the Local Field Homogeneity in the Spectrometer Control Tool window to start automated shimming.</li>
	<li>Load a new T2 Rapid imaging with Refocused Echoes (RARE) sequence into the Scan control as described in step 3.5.1.
	<ol>
		<li>Acquire T2 weighted images using the default settings, except for the following parameters:</li>
		<li>Open the Edit Scan tab and adjust the repetition time (TR) and echo time (TE) to 5,500 ms and 37 ms, respectively. Also, modify the field of view and matrix size to allow for a higher in-plane resolution of 109 &#956;m x 109 &#956;m (default resolution = 156 &#956;m x 156 &#956;m). Make sure that the slice thickness is 600 &#956;m, number of slices is set to 45, and RARE factor is set to 8.</li>
		<li>Open the Geometry editor and place the slice package in the correct position including the bulbus of the brain and the cerebellum.</li>
	</ol>
	</li>
	<li>Load three new echo-planar diffusion-weighted spin-echo sequence (DtiEpi) from the B_DIFFUSION folder into the Scan Control protocol as described in step 3.5.1. 
	NOTE: Using three different diffusion &#8220;shells&#8221;, the diffusion tensor imaging (DTI) model4,16, diffusion kurtosis imaging (DKI) model17, and white matter tract integrity (WMTI) model18 can all be estimated. It is recommended to use at least three different b-values, with the highest b-value having a maximum of 3000 s/mm2 with at least 15 evenly spaced directions per imaging shell17.
	<ol>
		<li>Acquire diffusion weighted images (DWIs) using default settings, apart from the following settings:</li>
		<li>Open the Edit Scan tab and adjust the geometrical parameters under the Geometry tab. Adjust the field of view and matrix size to 105 x 105 to ensure a resolution of 333 &#956;m x 333 &#956;m.</li>
		<li>Set the slice orientation to axial and the number of slices to 25, resulting in a slice thickness of 500 &#956;m and interslice distance of 600 &#956;m. Amend the readout direction into left-right.</li>
		<li>Click the Contrast tab to adjust the echo time to 24 ms and repetition time to 6,250 ms.</li>
		<li>Set the bandwidth to 250,000 Hz and turn the fat suppression on. Adjust the number of averages to one.</li>
		<li>Click on the Research tab and change the number of averages (EPI segments) to 4.</li>
		<li>Click on the Diffusion tab within the research tab. Perform this step separately for each of the three diffusion shells.
		<ol>
			<li>Adjust the number of diffusion directions to 32 for the first shell, 46 for the second shell, and 64 for the third shell.</li>
			<li>Adjust the gradient directions with custom gradient directions files.</li>
			<li>Change the number of B0 images to 5 for the first shell, 5 for the second shell, and 7 for the third shell.</li>
			<li>Adjust the b-value per direction to 800 s/mm2 for the first shell, 1500 s/mm2 for the second shell, and 2000 s/mm2 for the third shell. 
			NOTE: Adjusting the gradient directions with a custom gradient directions file can be done manually by setting Enter Diffusion Directions to yes or automatically by using the DTI_SET_DIRECTIONS macro.</li>
		</ol>
		</li>
		<li>Open the Geometry editor and place the field of view between the bulbus and cerebellum containing only the cerebrum to reduce artifact and scan time. Position six saturation bands of 5 mm outside the brain to reduce artifacts by clicking on Saturation and sliding the bands in the preferred position using the scroll bars. 
		NOTE: The bulbus and cerebellum can be identified based on anatomical landmarks and the three images of the tripilot scan.</li>
	</ol>
	</li>
	<li>Acquire the imported sequences by clicking on the traffic light symbol. Using the settings of the parameters described above, the acquisition time of the T2-RARE scan is 12 min, of the first DWI shell 15 min, of the second DWI shell 21 min and of the third shell 30 min. The total acquisition time is approximately 80 min (on a single receiver channel system).</li>
	<li>At the completion of the scanning protocol, remove the animal from the scanner bed, and place the animal in a clean cage with a heating pad at 37 &#176;C. Return the animal to the home cage when it regains consciousness.</li>
</ol>
4. Image processing
NOTE: In the following sections, the processing of the diffusion images is described in MRtrix3, ExploreDTI19 and Amide software20 which are open access toolboxes. However, the preprocessing steps can be performed in other toolboxes (e.g., FSL, MedInria, DTIStudio).
<ol>
	<li>Transfer the acquired data from the acquisition console by exporting the 2dseq file.</li>
	<li>Convert the 2dseq files (raw DWI files) into the .mif format, which is the standard formatting of MRtrix3, to allow for further preprocessing steps in MRtrix3. Moreover, concatenate the three diffusion shells using the following commands in the shell: 
	convert_bruker pdata/1/2dseq ratID_T2.mih (for the T2-weighted images) 
	convert_bruker pdata/1/2dseq ratID_dwi1.mih (for the first diffusion shell) 
	convert_bruker pdata/1/2dseq ratID_dwi2.mih (for the second diffusion shell) 
	convert_bruker pdata/1/2dseq ratID_dwi3.mih (for the third diffusion shell) 
	mrcat ratID_dwi1.mif ratID_dwi2.mif ratID_dwi3.mif ratID_dwi.mif</li>
	<li>Perform noise correction and Gibbs ringing correction on the DWIs in MRtrix321,22. Also, convert the corrected DWI images and T2 image to NIFTI format using the following commands: 
	dwidenoise ratID_dwi.mif ratID_dwi_denoised.mif 
	mrdegibbs ratID_dwi_denoised.mif ratID_dwi_denoised_gr.mif 
	mrconvert ratID_dwi_denoised_gr.mif ratID.nii 
	mrconvert ratID_T2.mif ratID_T2.nii</li>
	<li>Perform correction for EPI, motion and Eddy current distortions in ExploreDTI:
	<ol>
		<li>Convert the NIFTI images to a .mat file by clicking Calculate DTI*.mat file | Convert raw data to DTI*.mat file. Change the diffusion tensor estimation to weighed linear and the b-value to NaN. Adjust the voxel size to 0.333 0.333 0.6, number of non-DWI images to 17, number of DWI images to 142, and matrix size to 105 105 25. 
		NOTE: By setting the b-value to NaN, ExploreDTI will regard the dataset as a kurtosis data set.</li>
		<li>Click on the Settings tab to adjust the settings for EPI correction (this is turned off by default). Select SM/EC/EPI correction, also register to other data? and click on Yes, to do the EPI correction (non-rigid). Specify the suffix of the anatomical T2 image corresponding to the diffusion data set. 
		NOTE: ExploreDTI corrects for EPI distortions using image registration between the undistorted anatomical image and the diffusion image.</li>
		<li>Click on the Plugins tab and select Correction for subject motion &#38; EC/EPI distortions and select the preprocessed diffusion data file from step 2.3. Make sure that the T2 image is in the same folder and has the same base as the diffusion data file name (e.g., rat1.nii for the DWI and rat1_T2.nii for the anatomical image). This step will generate a &#8220;native&#8221; (*native.mat) and &#8220;transformed&#8221; file (*trafo.mat).</li>
	</ol>
	</li>
	<li>Calculate the DTI metrics for each rat by clicking Plugins and Export stuff to *.nii and selecting the parametric maps of the DTI model: fractional anisotropy (FA), mean diffusivity (MD), radial diffusivity (RD), and axial diffusivity (AD; denoted as &#8220;largest eigenvalue L1&#8221;).</li>
	<li>Additionally, export the parametric maps for the kurtosis model (MK, AK, and RK) and WMTI model (AWF, AxEAD, RadEAD and TORT). Processing of the diffusion images will result in 12 parametric maps (Figure 1, Figure 2, Figure 3) that can be used for further microstructural analysis.</li>
	<li>Create a mask file for the hippocampus of each rat using MRtrix3.
	<ol>
		<li>Load the FA image of the rat in the MRtrix viewer by clicking Tool and ROI editor.</li>
		<li>Create a new ROI by clicking the &#8220;+&#8221; button and press Edit to draw the ROI on each slice that includes the hippocampus (Figure 4). To erase unwanted areas from the ROI drawn, press the right mouse button.</li>
		<li>When the drawing of the ROI is completed, save the mask image by clicking the Save button. 
		NOTE: This mask file will be a binary NIFTI image file with voxels of value 1 containing the hippocampal tissue, and remaining voxels will have values of 0. To standardize the region of the hippocampus across rats, the parametric maps can be co-registered with a study specific template with predefined regions of interest delineated23 or a rat brain atlas.</li>
	</ol>
	</li>
	<li>To extract the diffusion metrics of the hippocampus of the rat, use the created mask file of step 4.6 and open the Amide software.
	<ol>
		<li>Open the parametric maps and mask image of the rat.</li>
		<li>To add the ROI of the mask file into Amide, select the mask file image, click Edit | Add ROI | 3D Isocontour and click on the ROI displayed in the mask image. Give the ROI a meaningful name and confirm that this volume should only contain voxels having a value of one.</li>
		<li>To calculate the mean values of the diffusion metrics in the hippocampus, click Tools | Calculate ROI Statistics and indicate the images and ROI that should be included. After clicking Execute, another screen will pop up with computed values that can be used for further statistical analysis. This file can either be saved or copied into a preferred data format (e.g., .xlsx or .csv file).</li>
	</ol>
	</li>
</ol>
5. Statistical analysis
NOTE: In the following sections, we describe processing of the diffusion images in SPSS Statistics 24; however, the statistical analysis can be performed in other statistical toolboxes.
<ol>
	<li>Load the data in the wide format in an SPSS *.sav file.</li>
	<li>To test for statistical differences between the two groups for each timepoint (i.e., baseline or 1-day post-injury), click Analyze | Nonparametric tests | Legacy Dialogs | 2 Independent Samples Tests. Load the variables that need to be tested and specify the groups (i.e., TBI and sham groups). Indicate the Mann-Whitney U as the test type.</li>
	<li>To test for statistical differences between the 2 time point within each group the data file needs to be split. Go to Go to Data, Split File and indicate Compare groups. Next, click on Analyze, Nonparametric tests, Legacy Dialogs, 2 Related Samples Tests, load the variables that need to be compared and indicate Wilcoxon as test type. 
	NOTE: To correct for multiple comparisons, p-values are adjusted for each diffusion model using Bonferroni correction [i.e., p-value divided by the number of parameters compared (DTI 4, DKI 3, and WMTI 4)]. More specifically, p &#60; 0.0125 is considered significant for the DTI and WMTI models, and p &#60; 0.016 is considered significant for the DKI model.</li>
</ol>

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The authors have no conflicts of interest to disclose.

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>&lt;strong&gt;Induction of trauma&lt;/strong&gt;</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 &amp;mu;l injection</td></tr><tr><td>&lt;strong&gt;Diffusion MRI&lt;/strong&gt;</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>&lt;strong&gt;Anaesthesia&lt;/strong&gt;</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>&lt;strong&gt;Diffusion image processing&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amide</td><td>&lt;u&gt;http://amide.sourceforge.net&lt;/u&gt;</td><td>Version 1.0.5.</td><td>Medical Imaging Data Examiner Toolbox (Loening AM, Gambhir SS, &quot; AMIDE: A Free Software Tool for Multimodality Medical Image Analysis&quot;, Molecular Imaging, 2(3):131-137, 2003)</td></tr><tr><td>ExploreDTI</td><td>&lt;u&gt;http://www.exploredti.com&lt;/u&gt;</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>&lt;u&gt;http://www.mrtrix.org&lt;/u&gt;</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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.4K Views.  Ghent University. 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.

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

Assessing Spatial Memory Impairment in a Mouse Model of Traumatic Brain Injury Using a Radial Water Tread Maze

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and validated for rat use. The Radial Water Tread (RWT) maze test of spatial memory (designed specifically for mice and requiring no swimming) has been shown previously to successfully distinguish between controlled cortical impact-induced TBI mice and sham controls. Here, a detailed protocol for this task is presented. The RWT maze capitalizes on the natural tendency of mice to avoid open areas in favor of hugging the sides of an apparatus (thigmotaxis). The walls of the maze are lined with nine escape holes placed above the floor of the apparatus, and mice are trained to use visual cues to locate the escape hole that leads out of the maze. The maze is filled with an inch of cold water, sufficient to motivate escape but not deep enough to require that the mouse swim. The acquisition period takes only four training days, with a test of memory retention on day five and a long-term memory test on day 12. The results reported here suggest that the RWT maze is a feasible alternative to rat-validated, swimming-based cognitive tests in the assessment of spatial memory deficits in mouse models of TBI.

Here we present a protocol for a mouse-specific test of cognition that does not require swimming. This test can be used to successfully distinguish controlled cortical impact-induced traumatic brain injury mice from sham controls.

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and ...

Behavior

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, few studies have investigated the long-term effects of injury in rodent models of TBI. Shown here are behavioral measures that are well-established in TBI research for times early after injury, such as two weeks, until two months. Some of these methods have previously been used at later times after injury, up to one year, but by very few laboratories. The methods demonstrated here are a short neurological assessment to test reflexes, a Beam-Balance to test balance, a Beam-Walk to test balance and motor coordination, and a working memory version of the Morris water maze that can be sensitive to deficits in reference memory. Male rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid percussion injury (FPI) or sham injury. Rats can be tested on the short neurological assessment (neuroscore), the beam-balance, and the Beam-Walk multiple times, while testing on the water maze can only be done once. This difference is because rats can remember the task, thus confounding the results if repeated testing is attempted in the same animal. When testing from one to three days after injury, significant differences are detected in all three non-cognitive tasks. However, differences in the Beam-Walk task were not detectable at later time points (after 3 months). Deficits were detected at 3 months in the Beam-Balance and at 6 months in the neuroscore. Deficits in working memory were detected out to 12 months after injury, and a deficit in a reference memory first appeared at 12 months. Thus, standard behavioral tests can be useful measures of persistent behavioral deficits after FPI.

The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, ...

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 impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

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 ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Medicine

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...

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 closed-head mTBI in the preclinical setting to increase translatability to the clinical population. The awake closed-headed injury (ACHI) model uses a modified controlled cortical impactor to deliver closed-headed injury, inducing clinically relevant behavioral deficits without the need for a craniotomy or the use of an anesthetic.
This technique does not normally induce fatalities, skull fractures, or brain bleeds, and is more consistent with being a mild injury. Indeed, the mild nature of the ACHI procedure makes it ideal for studies investigating repetitive mTBI (r-mTBI). Growing evidence indicates that r-mTBI can result in a cumulative injury that produces behavioral symptoms, neuropathological changes, and neurodegeneration. r-mTBI is common in youths playing sports, and these injuries occur during a period of robust synaptic reorganization and myelination, making the younger population particularly vulnerable to the long-term influences of r-mTBI.
Further, r-mTBI occurs in cases of intimate partner violence, a condition for which there are few objective screening measures. In these experiments, synaptic function was assessed in the hippocampus in juvenile rats that had experienced r-mTBI using the ACHI model. Following the injuries, a tissue slicer was utilized to make hippocampal slices to evaluate bidirectional synaptic plasticity in the hippocampus at either 1 or 7 days following the r-mTBI. Overall, the ACHI model provides researchers with an ecologically valid model to study changes in synaptic plasticity following mTBI and r-mTBI.

Here, it is demonstrated how an awake closed-head injury model can be used for examining the effects of repeated mild traumatic brain injury (r-mTBI) on synaptic plasticity in the hippocampus. The model replicates important features of r-mTBI in patients and is used in conjunction with in vitro electrophysiology.

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

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful insights, developing a stable and reproducible animal model is essential. In this study, we report a detailed description of a closed-head mTBI model and a representative validation method using Sprague-Dawley rats to verify the modeling effect. The model involves dropping a 550 g mass weight from a height of 100 cm directly onto the head of a rat on a destructible surface, followed by a 180-degree turn. To assess the injury, rats underwent a series of neurobehavioral assessments 10 min post-injury, including time of loss of consciousness, first seeking-behavior time, escape ability, and beam balance ability test. During the acute and subacute stages following the injury, behavioral tests were conducted to assess motor coordination ability (Beam task), anxiety (Open Field test), and learning and memory abilities (Morris Water Maze test). The closed-head mTBI model produced a consistent injury response with minimal mortality and replicated real-life situations. The validation method effectively verified the model development and ensured the stability and consistency of the model.

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

Here, we present a closed-head mild traumatic brain injury (mTBI) rat model and its validation exhibiting remarkable similarity to human mTBI concerning behavioral manifestations during the acute and subacute stages.

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

rmTBI Induction in Mice via a Closed-Head Injury Method

Modified Mouse Model of Repetitive Mild Traumatic Brain Injury Incorporating Thinned-Skull Window and Fluid Percussion

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models with well-defined pathologies are urgently needed for studying the mechanisms of neuropathology after mild TBI and testing therapeutics. Replicating the entire sequelae of TBI in animal models has proven to be a challenge. Therefore, the availability of multiple animal models of TBI is necessary to account for the diverse aspects and severities seen in TBI patients. CHI is one of the most common methods for fabricating rodent models of rmTBI. However, this method is susceptible to many factors, including the impact method used, the thickness and shape of the skull bone, animal apnea, and the type of head support and immobilization utilized. The aim of this protocol is to demonstrate a combination of the thinned-skull window and fluid percussion injury (FPI) methods to produce a precise mouse model of CHI-associated rmTBI. The primary objective of this protocol is to minimize factors that could impact the accuracy and consistency of CHI and FPI modeling, including skull bone thickness, shape, and head support. By utilizing a thinned-skull window method, potential inflammation due to craniotomy and FPI is minimized, resulting in an improved mouse model that replicates the clinical features observed in patients with mild TBI. Results from behavior and histological analysis using hematoxylin and eosin (HE) staining suggest that rmTBI can lead to a cumulative injury that produces changes in both behavior and gross morphology of the brain. Overall, the modified CHI-associated rmTBI presents a useful tool for researchers to explore the underlying mechanisms that contribute to focal and diffuse pathophysiological changes in rmTBI.

Author Spotlight: Optimizing Rodent Models for Investigating the Mechanisms and Rehabilitation in Repeated Mild Traumatic Brain Injury

This protocol presents a modified mouse model of repetitive mild traumatic brain injury (rmTBI) induced via a closed-head injury (CHI) method. The approach features a thinned-skull window and fluid percussion to reduce the inflammation commonly caused by meninges exposure, along with improved reproducibility and accuracy in modeling rmTBI in rodents.

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models ...

Development of an Uncomplicated Mild Traumatic Brain Injury Model Modified by Weight-Drop Method and Evidenced by Magnetic Resonance Imaging

Mild traumatic brain injury (mTBI), known as concussion, accounts for more than 85% of brain injuries globally. Specifically, uncomplicated mTBI showing negative findings in routine clinical imaging in the acute phase hinders early and appropriate care in these patients. It has been acknowledged that different impact parameters may affect and even accelerate the progress of subsequent neuropsychological symptoms following mTBI. However, the association of impact parameters during concussion to the outcome has not been extensively examined. In the current study, an animal model with closed-head injury (CHI) modified from the weight-drop injury paradigm was described and demonstrated in detail. Adult male Sprague-Dawley rats (n = 20) were randomly assigned to CHI groups with different impact parameters (n = 4 per group). Longitudinal MR imaging studies, including T2-weighted imaging and diffusion tensor imaging, and sequential behavioral assessments, such as modified neurological severity score (mNSS) and the beam walk test, were conducted over a 50-day study period. Immunohistochemical staining for astrogliosis was performed on day 50 post-injury. Worse behavioral performance was observed in animals following repetitive CHI compared to the single injury and sham group. By using longitudinal magnetic resonance imaging (MRI), no significant brain contusion was observed at 24 h post-injury. Nevertheless, cortical atrophy and alteration of cortical fractional anisotropy (FA) were demonstrated on day 50 post-injury, suggesting the successful replication of clinical uncomplicated mTBI. Most importantly, changes in neurobehavioral outcomes and image features observed after mTBI were dependent on impact number, inter-injury intervals, and the selected impact site in the animals. This in vivo mTBI model combined with preclinical MRI provides a means to explore brain injury on a whole-brain scale. It also allows the investigation of imaging biomarkers sensitive to mTBI across varying impact parameters and severity levels.

Here, we present a protocol to establish a closed-head injury animal model replicating the neuroimage outcome of uncomplicated mild traumatic brain injury with the preserved brain structure in the acute phase and long-term brain atrophy. Longitudinal magnetic resonance imaging is the primary method used for evidence.

Mild traumatic brain injury (mTBI), known as concussion, accounts for more than 85% of brain injuries globally. Specifically, uncomplicated mTBI showing ...