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

Summary

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.

Abstract

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.

Introduction

Mild traumatic brain injury (mTBI) is primarily observed in athletes engaged in contact sports, military veterans, and individuals involved in traffic accidents¹. It accounts for greater than 85 % of all reported head injuries². The vast etiology of mTBI and its increasing global incidence underscore the inclusion of mTBI as a tentative environmental risk factor of late-onset neurodegenerative disease³. Uncomplicated mild TBI is characterized by a Glasgow Coma Score (GCS) of 13-15, with no structural abnormalities observed in computer tomography (CT) or magnetic resonance imaging (MRI) scans. Common symptoms experienced by patients with uncomplicated mTBI include headaches, dizziness, nausea or vomiting, and fatigue. However, longitudinal assessment of outcomes following uncomplicated mTBI presents considerable challenges due to the high dropout rate in patients⁴.

The concerns of repetitive mTBI have increased, particularly within the National Football League (NFL) professional athlete community, subsequently raising awareness among non-professional athletes⁵. Brain vulnerability is presumed to increase following the initial mTBI, with subsequent insults potentially exacerbating injury outcomes. Recent findings from the largest donated brain cohort of football players not only implicated prior football participation in chronic traumatic encephalopathy (CTE) severity but also suggested a correlation between different football-related factors and the risk and severity of CTE⁶. Hence, the concern about the influence of the number of concussions and the repetitive regime on injury outcomes is growing. Preclinical research has explored neuropathological changes, neuroinflammatory cascade, and neuropsychological impairment after repetitive mTBI by using various closed-head injury (CHI) models^{7,8,9,10,11,12,13,14}. However, the investigation of impact parameters on the uncomplicated mTBI model, which may closely mimic sport-related repetitive concussive head impacts resulting in functional impairment in the acute phase and brain atrophy in the chronic phase, has not been well examined.

Diffusion tensor imaging (DTI), a technique assessing the diffusion of water molecules, has been commonly utilized in studies investigating the effects of mTBI. Fractional anisotropy (FA), a key metric derived from DTI, quantifies the degree of water diffusivity coherence and provides information regarding the structural organization of axons and nerve fiber bundles. Perturbation of FA values in the white matter (WM) has been proposed following mTBI in various models^{8,10,11,15,16,17}. In addition, axial diffusivity (AD) and radial diffusivity (RD), indicating axonal and myelin integrity, changed after mTBI in preclinical studies^{10,15,16,18,19,20}. However, discrepancies in DTI findings among previous studies are likely due to variations in mTBI severity, differences in impact parameters, diverse mTBI models, and inconsistent post-injury follow-up time points⁹.

The current protocol paper, thus, aims to establish an animal model of mTBI designed to evaluate the cumulative effects of single and repetitive mTBI. We incorporated comprehensive and longitudinal assessments, including evaluations of animal well-being, behavioral outcomes, DTI parameters, and cortical volume, to capture dynamic post-injury changes and explore the effects of different impact parameters. By demonstrating both acute functional impairment and long-term microstructural changes, this model effectively replicates the key features of uncomplicated mTBI that were not fully addressed in previous animal studies. Here, we provided a detailed protocol for developing an uncomplicated mTBI model using a modified closed-head weight-drop method^8,11 and conducting longitudinal assessment following mTBI.

Protocol

The study was performed in accordance with the recommendations of the National Institutes of Health Guidelines for Animal Research (Guide for the Care and Use of Laboratory Animals) and the Animal Research: Reporting In Vivo Experiments guidelines. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Yang Ming Chiao Tung University. Twenty animals were randomly assigned to 5 groups (n = 4 per group): (i) single impact at the sensorimotor cortex (SMCx/single), (ii) double impacts at SMCx with the 1-h interval (SMCx/2 hits/1 h), (iii) double impacts at SMCx with the 10-min interval (SMCx/2 hits/10 min), (iv) double impacts at the central brain with the 1-h interval (Central/2 hits/1 h), and (v) the sham group with surgery only but not impact directly to the head, for longitudinal outcome assessment (Figure 1). Of note, the inter-injury intervals selected for this study (1-h vs. 10-min intervals) were designed to mimic the repetitive subconcussive impacts^{8,10,11,13,21}, which can be up to a thousand times within a single season, experienced by the athletes engaging in contact sports^22,23.

1. Induction of closed-head injury (CHI)

NOTE: Adult male Sprague-Dawley rats aged 10 to 12 weeks and weighing over 250 g are housed under a 12/12 h light/dark cycle with ad libitum access to food and water.

1. Place the rat in a small induction chamber and anesthetize it with a mixture of isoflurane (5%) and medical air (2.5-3 L/min). Remove the rat from the chamber until it is non-responsive to a paw or tail pinch.
2. Place the rat on the heating pad.
   NOTE: Turn on the heating pad during the surgery to maintain the rats' body temperature.
3. Bring the rat onto a stereotaxic frame and secure it with a tooth bar. Administer isoflurane at 2% using the connected nose cone with medical air at a flow rate of 1.5-2 L/min for maintenance during the surgery.
4. Position the ear bars. Ensure the rat is centered and symmetrical on the stereotaxic frame.
   NOTE: All surgical procedures should be performed under aseptic conditions. The instruments were sterilized before surgery using a steam autoclave, and their tips were additionally sterilized with a bead sterilizer during the procedure. To prevent contamination, a surgical drape was placed over the animal. The surgeon wore a cap to cover their hair, a mask to cover their face, and was equipped with a lab coat and surgical gloves during the procedure²⁴.
5. Put on the sensor of the pulse oximeter to the hindpaw of the animal to monitor the respiration rate, heart rate, blood oxygen level, and body temperature of the animal.
6. Inject 1 mL/kg body weight of lidocaine (20 mg/mL) subcutaneously into the rat's neck as an analgesic.
7. Apply hair removal cream to the head of the animal and wait for 3 min. Wipe the cream out with 70% isopropyl alcohol swabs.
8. Clean the shaved area several times using a sterile cotton swab soaked in iodine. Remove the iodine residue using a cotton swab soaked in 70% ethanol.
9. Create a midline incision approximately 2-2.5 cm in length in the shaved skin using sterile surgical blade to access the surface of the skull.
10. Remove the tissue on the bone using a cotton pad to expose the skull. Clean the skull surface using a cotton swab soaked in 0.9% saline, then clean it with a dry cotton pad.
    NOTE: The skull sutures and both bregma and lambda can now be easily identified.
11. Identify the bregma as the reference point to further find out the impact area based on the coordinate.
    NOTE: In this protocol, two sets of coordinates are utilized for CHI induction: (-2.5,-2.0) (2.5 mm lateral 2.0 mm posterior to the bregma) on top of the sensorimotor cortex (SMCx), as well as (0,-3.0) on top of the central brain (central).
12. Identify the chosen coordinates on the skull surface and cement a circular stainless steel helmet (10 mm diameter and 1 mm thickness) over the designated area using dental cement. Remove the heating pad and pulse oximeter.
13. Move the stereotactic device and the rat on the lift table (14 cm length, 8 cm width, and 6.15 cm depth) under the CHI impactor.
14. Elevate the body of the rat using a foam sponge (19 cm in length, 10 cm in width, and 4 cm in depth, with a density of 18 kg/m³).
15. Remove the rat from the ear bars of the stereotaxic frame. Keep the rat still on the tooth bar connected with the nose cone, delivering 2% isoflurane. Ensure that the head and body are aligned level in the rostral-caudal direction.
16. Adjust the lift table to ensure no space between the CHI impactor and the helmet. Turn off isoflurane 5 s right before the impact.
    NOTE: To specify the righting reflex due to brain injury, temporary cessation of isoflurane was performed²⁵.
17. Drop a 600 g brass from a height of 1 m through a stainless steel tube (1 m height with an inner diameter of 20 mm for clearing a column of stainless brass weights) to the secured impactor with a round tip aiming at the metal helmet.
    NOTE: Animals in the sham group did not experience an impact, as the brass drop was released without making contact with the helmet on the rat's head.
18. Lower the lift table. Remove the rat from the stereotaxic frame and place the rat in the supine position on a heating pad.
19. Record the time of the righting reflex, which is the time when the animal attempts to change from the supine position into the prone position^26,27.
    NOTE: The animals subjected to the repetitive CHI were anesthetized again 3 min before the 2^nd impact. For the animals in the SMCx/double/10 min group who did not flip back to the prone position in time, the corresponding time of righting reflex was recorded as 420 s.
20. After the righting reflex recording, anesthetize the rat with isoflurane again using step 1.1.
21. Immobilize the rat with the stereotaxic frame using step 1.2.
    NOTE: After confirming the stability of the helmet on top of the stull, repeat steps 1.13-1.17 again to perform the 2^nd impact.
22. Remove the helmet. Remove all the connective tissue and cement on top of the skull.
23. Cover the skull with the dental cement and allow it to dry. Check that the dental cement is stiff and hard using the tweezer back.
    NOTE: The dental cement was applied atop the skull to eliminate susceptibility artifacts caused by skull-air or skull-blood interfaces between the skull and scalp post-surgery.
24. Close the incision using 4-0 nylon surgical sutures with 4-5 independent knots.
    NOTE: The wound is approximately 2-2.5 cm in length. Ensure that the surgical sutures have no capillary action and are made of silk or nylon material. Do not suture the incision using one single knot to prevent the opening of the wound by the scratching of the animal.
25. Apply topical antibiotics (Dermanest cream) to the surgical site to prevent infections.
26. Inject 1 mL/kg body weight of carprofen (50 mg/mL) subcutaneously into the neck as the post-surgery analgesics.
27. Place the rat in a clean cage on a heating pad till it regains consciousness. Once the rat sits upright, return it to the home cage.
28. Orally administer 5 mL of acetaminophen (24 mg/mL) mixed in 200 mL of water to the animal daily as an analgesic for 3 consecutive days after surgery.

2. Magnetic resonance imaging (MRI)

NOTE: T2-weighted image and diffusion-tensor imaging are performed using a sequential PET/MR 7T system before CHI, as well as on 1 and 50 days post-injury (Figure 1). A baseline MRI was performed within 1 week before the CHI procedure. For the evaluations on 1 and 50 days post-CHI, the behavioral assessments were conducted in the morning, followed by MRI scans in the afternoon on the same day.

1. Anesthetize the rat in a small induction chamber filled with a mixture of isoflurane (5%) and medical air (2.5-3 L/min).
2. Once the rat is non-responsive to a paw or tail pinch, temporarily suspend the anesthesia when transferring to the animal cradle in a head-first prone position.
3. Position the rat in the head holder connected with a nose cone, delivering 2% isoflurane with medical air at a flow rate of 1.5-2 L/min for maintenance during the image acquisition.
4. Fixate the head with a small piece of tape to avoid movement during scanning.
   NOTE: Apply some toothpaste to the rat's head on the first-day post-CHI to prevent magnetic susceptibility artifacts due to the scalp removal^28,29.
5. Place a pressure pad under the thorax of the rat to monitor respiration. Tape the oximeter clips to the hind limb to monitor heart rate.
6. Insert the rectal probe to measure rectal temperature. Cover the rat with a heating blanket with circulating warm water and tissue wrap during the experiment to maintain body temperature.
   NOTE: Throughout the experiment, monitor physiological conditions, including heart rate, respiratory rate, and rectal temperature. Before scanning, check all the physiological signals of the rat to ensure the quality of the vital sign monitor.
7. Use the laser positioning system of the PET/MR scanner to mark the center of the head for precise alignment.
8. Move the animal into the MRI bore automatically using the motorized animal transport system until the center of the head aligns with the iso-center of the scanner.
   NOTE: The motorized animal transport system integrated into the PET/MR system ensures accurate animal positioning and streamlines the workflow when transitioning between imaging modalities.
9. Obtain MRI sequence.
   1. Perform initial localization and overall adjustment.
   2. Use the middle sagittal slice and align the 8^th slice from the anterior with the decussation of the anterior commissure.
      NOTE: The coronal slices are positioned perpendicular to the horizontal plane defined by the line connecting the anterior commissure and the base of the cerebellum, corresponding to an approximately 15° angle relative to the long axis of the corpus callosum. The key parameters of the T2-RARE scan for the middle sagittal slice are as follows: repetition time (TR) = 2500 ms, echo time (TE) = 44 ms, field of view (FOV) = 3.5 cm, matrix size = 256 256, slice thickness = 1 mm, number of slices = 1, RARE factor = 8, bandwidth = 75 kHz, number of averages = 1, the acquisition time = 1 min 20 s.
   3. Use rapid acquisition with relaxation enhancement (RARE) with fat suppression and a saturation band underneath the brain to obtain T2-weighted images for anatomic reference (Figure 2).
      NOTE: The key parameters of the T2-RARE scan are as follows: repetition time (TR) = 3600 ms, echo time (TE) = 40 ms, field of view (FOV) = 2 cm, matrix size = 256 256, slice thickness = 1 mm, number of slices = 16, RARE factor = 8, bandwidth = 75 kHz, number of averages = 8, the acquisition time = 7 min 40 s.
   4. Use a 4-shot spin-echo EPI to acquire diffusion tensor images (Figure 2).
      NOTE: The key parameters of the DTI scan are as follows: TR = 3000 ms, TE = 28 ms, field of view (FOV) = 2 cm, matrix size = 96 96, thickness = 1 mm, number of slices = 16, duration of the pulse (δ) = 5 ms, time between the two pulses (Δ) = 15 ms, number of B0 = 5, number of directions = 30, b-value = 1000 s/mm³, bandwidth =150 kHz, number of average = 4, the acquisition time = 14 min.
   5. Finish the scanning protocol. Slide the animal cradle out of the magnet. Remove the animal from the cradle.
   6. Transfer the rat to a clean cage with a heating pad underneath to maintain it's body temperature. Return the rat to its home cage once it regains consciousness.
10. Image preprocessing
    NOTE: Use MRtrix3, Statistical Parametric Mapping (SPM) software, and custom MATLAB scripts for data processing and analysis.
    1. Denoise the DTI images using the MRtrix3 command (dwidenoise)³⁰.
    2. Remove Gibb's ringing artifacts from the DTI images using the MRtrix command (mrdegibbs)³⁰.
    3. Coregister DTI images to T2-weighted images of the single subject across longitudinal scans using SPM functions (spm_coreg.m and spm_powell.m).
    4. Perform skull stripping on T2-weighted images by manually contouring the brain area slice-by-slice, followed by removing pixels with intensity below the computed threshold determined by Otsu's method³¹ (custom MATLAB script thr_otsu2.m).
    5. Perform cross-subjects coregistration among animals in the same experimental group using SPM functions (spm_coreg.m and spm_powell.m). Apply the brain mask to the corresponding DTI images.
       NOTE: Skull stripping is performed to decrease computer processing time.
    6. Calculate tensor maps based on DTI (custom MATLAB script, tensormap.m).
    7. Calculate FA maps (custom MATLAB script, calFA.m)
       NOTE: All the custom MATLAB scripts are available via the following database (https://doi.org/10.57770/9ZESXD).
11. Image analysis-FA
    1. Draw regions of interest (ROIs) in the cortex and corpus callosum (CC) for the three consecutive image slices underneath the coordinate of CHI.
       NOTE: All ROIs were manually drawn and visually inspected for gross errors by 2 experienced investigators blinded to the experimental groups.
    2. Extract and average the FA value from ROIs.
       NOTE: For the corpus callosum underneath the cortex, pixels with values of FA < 0.35 were excluded in the selected ROI to eliminate partial volume effects. For the cortex, all the pixels with values of FA < 0.35 in the ROI were recruited for analysis.
12. Image analysis-Volume
    1. Manually draw ROIs covering the cortical regions for 11 consecutive image slices at Bregma -7 to +3 mm.
       NOTE: All ROIs were manually drawn by 2 experienced investigators blinded to the experimental groups.
    2. Sum up the total pixels of the ROIs across slices and transform them into the volume by multiplying the slice thickness (1 mm).
    3. Normalize the cortical volume after CHI with the corresponding volume before CHI of each animal.
       NOTE: Normalize the data to eliminate individual differences in brain volume across animals before presentation.

3. Behavior assessment

NOTE: The behavioral experiments are performed using the beam walk balance test and mNSS before CHI, as well as on 1 and 50 days post-CHI (Figure 1). All the assessment was performed by at least two observers to ensure the accuracy, consistency, and objectivity of the collected data.

1. Beam walk balance test
   1. Turn on the video camera and start the timer.
   2. Place the rats at one end of the balance beam (3 cm in depth, 3 cm in width, 80 cm in length, and 60 cm above the floor).
   3. Stop the timer once the rat completes one round trip, falls over, or freezes over 3 min.
      1. During the experiment, observe the animal condition for the assessment using mNSS^11,32,33,34. Follow these standards for evaluation:
      2. If the rat maintains balance with a steady posture on the beam, assign a score of 0.
      3. If the rat grasps the side of the beam, assign a score of 1.
      4. If the rat falls with one limb off the beam, assign a score of 2.
      5. If the rat falls with two limbs off or spins on the beam (>60 s), assign a score of 3.
      6. If the rat attempts to balance on the beam but falls off (>40 s), assign a score of 4.
      7. If the rat attempts to balance on the beam but falls off (>20 s), assign a score of 5.
      8. If the rat does not attempt to balance or hang on the beam and falls off within 20 s, assign a score of 6.
      9. If the rat fails to complete the task, consider it as having taken the maximum time of 3 min and assign a score of 6.
   4. Schedule testing days at specific time points.
      NOTE: Exclude rats not completing the beam walk round trip for two trials before CHI from the subsequent surgery and follow-up behavioral assessment.
2. Modified neurological severity score (mNSS)
   NOTE: mNSS assessment includes motor tests, sensory tests, absence of reflex, abnormal movements, beam balance, and walking on the floor ^32,33, which was quickly performed on a daily basis.
   1. Perform motor tests.
      1. Raise the rat by the base of the tail and observe the reflexes of its limbs for about 15 s to assess the proper flexion and extension.
      2. If normal flexion is observed in the forelimb, assign a score of 0. If no flexion is observed, assign a score of 1.
      3. If normal flexion is observed in the hindlimb, assign a score of 0. If no flexion is observed, assign a score of 1.
      4. If the head moves >10° to the vertical axis within 30 s after raising the rat by the tail, assign a score of 0. If not, assign a score of 1.
         NOTE: A maximum of 3 scores will be assigned in this session of the test.
   2. Perform limb placing tests.
      NOTE: The placing test is performed to evaluate the coordination between sensory (visual, tactile sensation, and proprioception) and motor function.
      1. Slowly lower the rat toward the surface of the table. Observe whether the paws of the rats reached and stretched toward the surface.
      2. If the rats reached the surface with both limbs stretched and forward, assign a score of 0. If there is a delay or no response, assign a score of 1.
      3. Place the rat on the surface and pull the paw against the table edge. Observe whether its paw returns to a normal position on the surface of the table.
      4. If immediate and normal placing responses are observed, assign a score of 0. If delayed placing responses are observed, assign a score of 1. If there is no response, assign a score of 2.
         NOTE: A maximum of 3 scores will be assigned in this session of the test.
   3. Observe reflect absence and abnormal movements.
      1. Observe for head shakes to assess the pinna reflex when touching the auditory meatus with the cotton end of a cotton swab.
      2. If a normal reflex is observed, assign a score of 0. If no reflex is observed, assign a score of 1.
      3. Assess the presence of the corneal reflex by touching the cornea with the cotton end of a cotton swab.
      4. If a normal response is elicited, assign a score of 0. If no eye blink response is elicited, assign a score of 1.
      5. Make a short and strong clap of the hands. Observe the presence of the startle reflex.
      6. If a reflex is observed, assign a score of 0. If no reflex is observed, assign a score of 1.
      7. Observe whether the rat has seizures, myoclonus, or myodystony.
      8. If any of them occur, assign a score of 1.
         NOTE: A maximum of 4 scores will be assigned in this session of the test.
   4. Perform the beam balance test, as previously described (step 3.1).
      NOTE: A maximum of 6 scores will be assigned in this session of the test.
   5. Perform walk on the floor test.
      1. Prepare the open field arena (75 cm length, 50 cm width, and 40 cm depth). Ensure it is clean and free from any previous odor cues.
      2. Place a rat in the center of the open field arena and observe how the rat walks in the arena.
      3. If the rat performs a regular walk, assign a score of 0.
      4. If the rat cannot walk straight, assign a score of 1.
      5. If the rat falls to the paretic side after placing it on the floor, assign a score of 3.
         NOTE: A maximum of 3 scores will be assigned in this session of the test.
   6. Sum up all the scores; the maximum possible score is 18.
      NOTE: The higher score indicates a worse outcome.

4. Immunohistology

1. Perform transcardiac perfusion³⁵.
   NOTE: Transcardiac perfusion is performed after the MRI scan 50 days post-CHI (Figure 1).
   1. Place the rat in a small induction chamber and anesthetize it with isoflurane (5%) until it is non-responsive to a paw or tail pinch.
   2. Administer 50 mg/kg body weight of zoletil (50 mg/mL) and 10 mg/kg body weight of Xylazine (Roumpun, 23.32 mg/mL) via intraperitoneal injection for deep anesthesia.
   3. Place the rat in the supine position.
   4. Make a transverse incision approximately 4-5 cm in length under the thorax using scissors.
   5. Locate the diaphragm and cut it to expose the heart.
   6. Use hemostatic forceps to clamp the pulmonary artery and then make an incision approximately 0.5-1 cm in length in the right atrium.
   7. Connect the needle to the pipeline attached to the infusion pump.
   8. Insert the needle into the left ventricle.
   9. Rinse the animal through transcardiac perfusion (40 mL/min) with 500 mL of 0.9% saline until the blood is cleared.
   10. Perfuse the animal through transcardiac perfusion (40 mL/min) with 500 mL of 4% paraformaldehyde (PFA) for fixation.
   11. Remove the rat's head, and carefully peel the brain tissue from the skull.
   12. Preserve the brain in approximately 20 mL of 4% PFA in the bottle for 48 h for post-fixation.
2. Perform tissue processing and IHC staining^36,37.
   NOTE: Perform immunohistochemical staining on formalin-fixed, paraffin-embedded tissue sections using the immunoperoxidase secondary detection system kit.
   1. Use formalin-fixed and paraffin-embedded tissue sections.
   2. Perform deparaffinization and treat the slides with 3% H₂O₂ to block endogenous peroxidase activity. Perform antigen retrieval using citrate buffer at 90 °C.
   3. Perform immunohistochemical staining using the immunoperoxidase secondary detection system kit.
      NOTE: Staining procedures are carried out following the manufacturer's recommendations.
   4. Use hematoxylin to counterstain specimens.
   5. Mount specimens with an antifade reagent.
   6. Use anti-glial fibrillary acidic protein (GFAP) antibodies for immunohistochemical staining.
   7. Acquire the images of ROIs using a light microscope slide scanner (Figure 6).

5. Statistical analysis of behavior and image outcomes

NOTE: In the current study, statistical analysis was performed in SPSS; however, the statistical analysis can be performed in other statistical toolboxes.

1. Load the data in the wide format in an SPSS *.sav file.
2. Conduct repeated measures analysis of variance (ANOVA) to compare the behavioral (normalized weight, mNSS, and beam walk duration) and image outcomes (FA values in the cortex and CC) over time among groups.
   1. Click Analyze > General Linear Model > Repeated Measures.
   2. Assign a name in the Within-Subject Factor Name box (e.g., time) and put '3' in the Number of Levels box (three levels with different follow-up time points). Assign a name in the Measure Name box (e.g., mNSS) in the Repeated Measures Define Factor(s) dialogue box.
   3. Load the Within-Subject Variables (data acquired at pre-, D1, and D50 post-CHI) that need to be tested and specify the between-subjects factor (e.g., animal groups with different impact parameters) in the Repeated Measures dialogue box.
   4. Select Bonferroni as a Post Hoc Test for Factor(s) (e.g., animal groups) in the post hoc multiple comparisons for Observed Means dialogue box.
      NOTE: To correct for multiple comparisons, the type I error was adjusted using Bonferroni corrections (0.05/3) for the comparisons over time. Statistical significance was defined as p < 0.05 (SPSS adjusted).
3. Conduct a one-way ANOVA analysis to compare the righting reflex and change of cortical volume among the groups.
   1. Click Analyze > Compare Means > One-Way ANOVA.
   2. Load the variables (righting reflex and change of cortical volume) in the Dependent List and the groups as the Factor in the One-Way ANOVA dialogue box.
   3. Select Bonferroni as a post hoc test in the One-Way ANOVA: Post Hoc Multiple Comparisons dialogue box.
      NOTE: To correct for multiple comparisons, type I error was adjusted using Bonferroni corrections (0.05/5) for the comparisons among groups. Statistical significance was defined as p < 0.05 (SPSS adjusted).

Results

Figure 2 shows longitudinal MRIs from representative animal with sham and repetitive CHI at the SMCx. No significant skull fracture or brain contusion was found in T2-weighted images on 1 and 50 days post-CHI. No significant edema or deformation of WM was found in FA maps on 1 and 50 days post-CHI. All animals subjected to CHI in this study survived the entire experimental duration of 50 days, demonstrating low mortality (0-5%)⁷ of the CHI model.

Discussion

This study aimed to establish an animal model of uncomplicated mild traumatic brain injury (mTBI) to evaluate the cumulative effects of single and repetitive injuries, as well as the outcomes of impacts on different brain regions. The closed-head injury (CHI) model, adapted from the closed-head weight-drop injury paradigm, was designed to mimic concussions commonly experienced by athletes and individuals with helmet protection. This model minimizes focal brain damage while enabling the precise manipulation of key impact ...

Materials

Name	Company	Catalog Number	Comments
Acetaminophen	Center Laboratories Inc	N02BE01
Antibiotics (Dermanest cream)	Commwell Pharmaceutial Co., Ltd	49391
Antigen Retrival buffer (100x Citrate buffer)	Abcam	AB93678
Anti-glial fibrillary acidic protein (GFAP) antibody	Bioworld Technology, Inc	BS6460
Balance beam	Custom made	Custom made	3 cm depth, 3 cm width, 80 cm length, and 60 cm above the floor
Behavior apparatus
Circular helmet	Custom made	Custom made	Stainless steel, 10-mm diameter, 1-mm thickness
Closed-head injury
Closed-Head injury impactor	Custom made	Custom made	A stainless steel tube (1-m height with 20-mm inner diameter), a secured impactor with a round tip (stainless steel, 10-mm tip diameter) at the bottom of the tube, a weight (stainless steel, 600 g).
Formalin	Bioworld Technology, Inc	C72
Gas Anesthesia Instrument (Vaporizer)	RWD Life Science Co.	R580S Animal Anesthesia Vaporizers and Accessories
Hematoxylin	Bioman Scientific Co., Ltd	17372-87-1
Immunohistology
Immunoperoxidase Secondary Detection system kit	Bio-Check Laboratories Ltd	K5007
Isoflurane	Panion & BF Biotech Inc.	8547
Lidocaine	Step Technology Co., Ltd	N01BB02
light microscope slide scanner	Olympus	BX63
MR-compatible small animal monitoring and gating system	SA Instruments	Model 1025	The monitoring kit with the respiratory pillow, ECG electrodes, and rectal probe
MRI
MRI operating council	Bruker	Biospec	Paravision 360 software.
MRI System	Bruker	Biospec	PET/MR scanner (PET inline), 7 T, 105 cm  inner bore diameter with gradient set.
Open field arena	Custom made	Custom made	75 cm length, 50 cm width, and 40 cm depth
Pulse oximeter	STARR Life Sciences Corp.	MouseOx Plus	Mouse & Rat Pulse Oximeter
Rat Adaptors	RWD Life Science Co.	68021
SPSS Statistics 29	IBM	Version 29.0
Stereotaxic frame	RWD Life Science Co.	G1124901-001
Volume coil	Bruker	Biospec	40-mm inner diameter, transceiver for radiofrequency excitation and signal receiving.
Xylazine	Bayer Taiwan Company Ltd
Zoletil	Virbac	BN8M3YA