Name: Development of an Uncomplicated Mild Traumatic Brain Injury Model Modified by Weight-Drop Method and Evidenced by Magnetic Resonance Imaging (Video) | JoVE Journal
Uploaded: 2025-04-11T12:20:00.000+00:00
Duration: 8 min 27 s
Description: Learn a closed-head injury rat model using weight-drop and MRI to study uncomplicated mild traumatic brain injury progression. This protocol enables longitudinal neuroimaging assessment of cortical atrophy and behavioral outcomes.

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This work was supported by a research grant from the National Science and Technology Council (NSTC) of Taiwan (NSTC 113-2314-B-A49-047). We thank the Small Animal Imaging Core Facility of the Brain Research Center at National Yang Ming Chiao Tung University and Taiwan Animal Consortium for the technical support in Bruker 7T PET/MR.

The authors have no potential conflicts of interest to disclose.

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 ...

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) models7,8,9,10,11,12,13,14. 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 models8,10,11,15,16,17. In addition, axial diffusivity (AD) and radial diffusivity (RD), indicating axonal and myelin integrity, changed after mTBI in preclinical studies10,15,16,18,19,20. 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 method8,11 and conducting longitudinal assessment following mTBI.

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

development of an uncomplicated mild traumatic brain injury model modified by weight-drop method and evidenced by magnetic resonance imaging

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.

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.
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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 ...

Our research focuses on developing a closed-head injury animal model that mimics neuroimaging outcomes of uncomplicated mild traumatic brain injury. We end to determine whether different impact parameters in RmTBI lead to distinct imaging, behavioral, and pathological changes. We developed an animal model that replicates the radiological changes of uncomplicated mTBI, demonstrating significant behavioral deficits and long-term brain atrophy.This work connects cross-sectional human studies with animal pathology research, providing translation and longitudinal neuroimaging assessment to better understand the mTBI progression. Our results open new avenues for investigating disease progression and outcome changes after uncomplicated mTBI. They also highlight the crucial role of injury parameters in shaping post-injury outcomes.Our lab focuses on pediatric and adolescent mTBI, the largest patient cohort. We'll apply our comfortable CHI model to study developmental brain injury and track long-term changes using MRI, behavioral, and neuropathological assessments. To begin, place the anesthetized rat on a heating pad and secure it in the stereotaxic frame using a tooth bar.Position the ear bars to stabilize the head. Ensure the rat is centered and symmetrical in the stereotaxic frame. Now, attach the sensor of the pulse oximeter to the hind paw to monitor respiration rate, heart rate, blood oxygen level, and body temperature.Apply hair removal cream to the head After three minutes, wipe off the cream using 70%isopropyl alcohol swabs. Clean the shaved area multiple times using a sterile cotton swab soaked in iodine. Then, remove the iodine residue with a cotton swab soaked in 70%ethanol.Using sterile surgical blade, create a midline incision approximately two to 2.5 centimeters in length to access the skull surface. Next, remove the soft tissue from the skull using a cotton pad. Clean the skull surface with a cotton swab soaked in 0.9%saline, followed by a dry cotton pad.Now, identify the bregma as the reference point to determine the impact area. After identifying the desired coordinates, cement a circular stainless steel helmet over the designated area using dental cement. Then, remove the heating pad and pulse oximeter.Move the stereotactic device and the rat onto the lift table under the closed head injury impactor. Elevate the rat's body using a foam sponge. Remove the rat from the ear bars while keeping it secured on the tooth bar connected to a nose cone delivering 2%isofluorine.Ensure the rat's head and body are level in the rostrocaudal direction. Adjust the lift table to ensure there is no gap between the CHI impactor and the helmet. Then, turn off isoflurane immediately before the impact.Now, drop a 600 gram brass weight from a height of one meter through a stainless steel tube to the secured impactor with a round tip, aiming at the metal helmet. After striking, lower the lift table. Remove the rat from the stereotaxic frame and place it in a supine position on a heating pad.Record the righting reflex time, which is the time when the rat attempts to move from the supine position to the prone position. Now, remobilize the rat in the stereotaxic frame. Remove the helmet and clean all the connective tissue before cementing from the skull surface.Now, cover the skull with dental cement and allow it to dry. Using the back of a tweezer, check that the cement is stiff and hard. Then, close the incision using 4o nylon surgical sutures with four to five independent knots and apply topical antibiotics to the surgical site to prevent infections.Inject one milliliter per kilogram body weight of carprofen subcutaneously into the neck as a post-surgery analgesic. Place the rat in a clean cage on a heating pad until it regains consciousness. Once the rat sits upright, return it to the home cage.Orally administer five milliliters of acetaminophen mixed in 200 milliliters of water daily for three consecutive days as an analgesic. Position the anesthetized rat in the head holder and connect to a nose cone for maintenance of anesthesia. Fixate the head with a small piece of tape to prevent movement during scanning.Then, place a pressure pad under the thorax to monitor respiration. Insert the electrodes and tape the oximeter clips to the hind limb to check the heart rate. Insert the rectal probe to measure the rectal temperature.Cover the rat with a heating blanket with circulating warm water and tissue wrap to maintain body temperature during the experiment. Use the laser positioning system of the PET MR scanner to mark the center of the head for precise alignment. Move the rat into the MRI bore using the motorized animal transport system until the center of the head aligns with the isocenter of the scanner.Finally, obtain the MRI images and analyze them. No significant skull fracture, brain contusion, white matter edema, or deformation was observed in T2 weighted images and fractional anisotropy maps at one and 50 days post-CHI, confirming minimal structural damage in the CHI model. A significant reduction in cortical volume was observed at 50 days post-CHI, and repetitive CHI resulted in greater cortical loss compared to single CHI.The most substantial cortical volume reduction was noted in slices at bregma minus four to plus zero and bregma minus five to plus one after repetitive CHI with different impact parameters. A significantly smaller cortical volume was observed at bregma zero after central brain CHI compared to SMCx CHI. Immunostaining at day 50 post-injury demonstrated astrocyte accumulation in the ipsilesional SMCx, irrespective of CHI severity and impact site.

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Source: Macheda, T., et. al. Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice. J. Vis. Exp. (2022)This video demonstrates the procedure to induce a closed-head injury in a mouse model using a stereotaxic frame. The controlled impact replicates mild traumatic brain injury, allowing researchers to study neurological effects and recovery by measuring post-injury reflexes and behavior.

Induction of Mild Traumatic Brain Injury in a Mouse Model

The modified weight-drop technique is an easy, cost-effective procedure used for the induction of mild traumatic brain injury in juvenile rats. This novel technique produces clinically relevant symptomology that will advance the study of mild traumatic brain injury (mTBI) and concussion.

A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats

Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Neurotransmitter alteration is a mechanism of neural dysfunction that occurs after concussion and contributes to the sometimes-catastrophic long-term consequences. This rat model combines microdialysis, allowing in vivo neurotransmitter quantification, with a weight-drop technique exerting rapid acceleration and deceleration of the head and torso, an important factor of human craniocerebral trauma.

A Novel and Translational Rat Model of Concussion Combining Force and Rotation with In Vivo Cerebral Microdialysis

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.

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

We present here a protocol of a blast wave model for rodents to investigate neurobiological and pathophysiological effects of mild to moderate traumatic brain injury. We established a gas-driven, bench-top setup equipped with pressure sensors allowing for reliable and reproducible generation of blast-induced mild to moderate traumatic brain injury.

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

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

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

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.

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

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.

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

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.

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

Learn a closed-head injury rat model using weight-drop and MRI to study uncomplicated mild traumatic brain injury progression. This protocol enables longitudinal neuroimaging assessment of cortical atrophy and behavioral outcomes.

Development of an Uncomplicated Mild Traumatic Brain Injury Model Modified by Weight-Drop Method and Evidenced by Magnetic Resonance Imaging (Video) | JoVE Journal

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

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