This work was supported by the Key Social&#160;Development Foundation of&#160;Jinhua&#160;Municipality&#160;(No. 2020-3-071), Zhejiang College Student&#160;Innovation and&#160; Entrepreneurship Training&#160;Program (No: S202310345087,&#160;S202310345088) and Zhejiang Provincial&#160;College Students&#39;&#160;Science and Technology&#160;Innovation Activity Plan Project (2023R404044). The authors thank Miss Emma Ouyang (first-year student of Johns Hopkins University, Bachelor of Science, Baltimore, USA) for language editing the article.

All procedures involved in this protocol were performed under the Institutional Animal Care and Use Committee approval (Zhejiang Normal University, Permit Number, dw2019005) and in compliance with the ARRIVE and the NIH Guide for the Care and Use of Laboratory Animals. Technical specifications can be found in the Table of Materials.
1. Animal handling procedure
<ol>
	<li>House mice in a controlled environment with a temperature of 22-24 &#176;C, humidity ranging from 40%-60%, a 12 h light/dark cycle, and provide ad libitum access to water and standard mouse chow. For the purpose of this experiment, 25 ICR male mice (25-30 g, 8 weeks old) were used.</li>
	<li>Allocate randomly the mice into either the control group (n=12) or the rmTBI group (n=13). To prevent potential aggression from sham mice towards mice those that underwent rmTBI, separate them in distinct cages.</li>
	<li>Give mice at least 1 week to acclimate to their cage environment before beginning the experiment. This acclimation period ensures that the mice become familiar with their surroundings and minimizes the potential impacts of stress or anxiety on physiological or behavioral responses during the study.</li>
</ol>
2. Preparation of TBI device
<ol>
	<li>Fabricate the rmTBI model in mice using a modified FPI device (see Table of Materials; Figure 1A)13. Before using the FPI device, carefully inspect all connections for signs of leakage or cracking, paying special attention to the junctions between the cylinder and tubing, and between the three-way connector and tubing. To determine impact pressures accurately, a&#160;pressure transducer was installed to the point&#160;of impact on the fluid percussion injury device13.</li>
	<li>Conduct a careful inspection of the FPI device to ensure that the piston moves smoothly inside the cylinder and that impacts can be conducted effectively (Figure 1B). Verify that there are no air bubbles present within the system. If air bubbles are detected, carefully add distilled water to the system using a 50 mL syringe and expel the air bubbles by quickly pushing the cylinder rod through a nearby 3-way connector and/or through the syringe. 
	NOTE: The FPI system consists of distilled water within the cylinder and connected tubing. It is crucial to calibrate the device prior to operation to ensure accuracy and reliability of results.</li>
</ol>
3. Thinned-skull preparation
NOTE: The animal surgery and thinned-skull preparation should not be performed in view of other mice. The thinned-skull window is useful for evaluating cerebral vessel damage following an FPI procedure.
<ol>
	<li>Adhere to the requirements of the animal center by instructing the experimental operator to wear a sterile gown and mask during all animal handling procedures.</li>
	<li>Sanitize the lab bench top, FPI device, anesthetic tubing and adjacent counterspace before start experiment using a 70% ethanol spray.</li>
	<li>Weigh the mice in both the sham and rmTBI groups before surgery and compare their weights to those recorded 1 week prior to the experiment. Exclude from the study any mouse displaying poor health such as rough fur coat, diarrhea, weight loss, or lethargy. Additionally, exclude mice that weigh less than 20 g to ensure their ability to tolerate repeated impacts. These measures are crucial in maintaining animal welfare and ensuring reliable experimental outcomes.</li>
	<li>Anesthetize mice with 4%-5% isoflurane (in 100% oxygen at a flow rate of 1 L/min) for 3-4 min in an induction chamber. Administer an ophthalmic lubricant (see Table of Materials) to the animal&#39;s eyes to maintain lubrication throughout the surgery. To mitigate pain, administer buprenorphine subcutaneously (0.05 mg/kg) at the midpoint between their ears 20 min prior to anesthesia and subsequently every 6 h for a duration of 24 h. Furthermore, at the end of anesthesia, administer a single subcutaneous dose of 5 mg/kg carprofen to each animal.</li>
	<li>Disinfect the head after the mouse has lost its righting and pedal withdrawal reflexes. Trim the fur on the mouse&#39;s head using surgical scissors and use a shaver to remove the remaining fur. Disinfect the scalp with three sequential applications of 2% chlorhexidine, interspersed with 75% ethanol scrubs.</li>
	<li>Place a disposable sterile surgical pad under the animal and the surrounding area to ensure proper hygiene. Make a 1.5 cm incision using a fine small surgical scissor along the midline of the mouse&#39;s scalp to fully expose the surgical site (Figure 2A).</li>
	<li>Secure the mouse using nonterminal ear bars in a conventional stereotaxic frame (see Table of Materials). Adjust the mouse head position in a stereotaxic frame at a flat level or a slight tilted angle according to the specific target area that will be investigated. Clean fur from the surgical area to avoid inflammation later.</li>
	<li>Maintain the mouse&#39;s body temperature at 37 &#176;C using a conventional isothermal heating pad (see Table of Materials).</li>
	<li>During the surgical and impact hub (female Luer Lock) installation process, maintain mouse anesthesia (with no response to toe or tail pinch) with a nose cone that delivers a continuous 2% isoflurane concentration, regulated by a calibrated vaporizer.</li>
	<li>Clean the surgical area around the thinned-skull window carefully using a sterile saline-soaked cotton swab.</li>
	<li>Create a thinned-skull window, approximately 2.5 mm in diameter and 20 &#181;m in thickness, in the right frontal motor cortex using a flat-tipped micro-drill bit (see Table of Materials, Figure 2B) and a microsurgical blade. Locate the surgical site at 1.5 mm anterior to the bregma and 1.3-2.0 mm lateral to the midline (Figure 2C).
	<ol>
		<li>To prevent the micro-drill from penetrating the skull during the creation of the thinned-skull window, intermittently moisten the skull with saline while grinding with the micro-drill.</li>
		<li>Confirm the thickness of the skull by gently probing the thinned-skull with the flattened tip of a fine syringe needle and assessing its softness. Estimate the clarity of the exposed cortical micro-vessels visually to determine the thickness of the skull.</li>
		<li>To verify the thickness of the skull, apply sterile saline to the thinned area and visually inspect using a conventional dissecting microscope (see Table of Materials). This technique can help ensure that the skull has been adequately thinned. 
		NOTE: Lubricate the mouse eye throughout the entire thinned-skull preparation and rmTBI modeling procedure to prevent drying out. Thinning the skull to less than 15 &#181;m carries the risk of mild cortical trauma which may result in mild cortical inflammation14.</li>
	</ol>
	</li>
	<li>Attach an adjusted female Luer Lock (2.2 mm inside diameter, created from a 19G needle hub, as shown in Figure 2D) to the thinned-skull site. Secure the Luer Lock with glue and dental cement (Figure 2E). 
	NOTE: When using glue to secure the female Luer lock to the area around the thinned-skull window, it is crucial to thoroughly dry the skull area, and prevent the adhesive from entering the window itself. Adhesive within the window can significantly reduce the impact force of the FPI.</li>
</ol>
4. CHI associated rmTBI modeling procedure
<ol>
	<li>Introduce rmTBI using the lateral fluid percussion method with a modified FPI device as described previously13,15.</li>
	<li>After completing the thinned-skull window and impact hub procedures, transfer the mouse from the stereotaxic apparatus to the impactor platform.</li>
	<li>Given the potential effects of anesthesia on animal righting reflex time and injury severity after percussion9,16, monitor the anesthesia depth by evaluating the palpebral and paw withdrawal reflexes (Figure 2F).</li>
	<li>Connect the female Luer Lock, which was glued onto the thinned-skull window, to the male Luer Lock at the end of the FPI device tubing (Figure 2G). 
	NOTE: In the rmTBI modeling, the isoflurane-induced anesthesia in mice was prolonged due to the induction of apnea and unconsciousness by percussion.</li>
	<li>Introduce two mild TBI (48 h interval) with the modified device. Apply the first FPI impact immediately after completing the thinned-skull window surgery and installing the Luer Lock. Only administer the FPI impact once the mouse shows return of a withdrawal reflex to a paw pinch on each occasion (Figure 2H). Applying an FPI impact in deeply anesthetized mice can cause prolonged apnea and death.
	<ol>
		<li>To apply the FPI impact, raise the pendulum to the designated degree along the protractor on the device and release the pendulum using software control13,15. The impact should achieve a percussion intensity of 2.0 &#177; 0.1 atm, following established protocols used in rodent studies10,17,18. Exclude animals from further tests if the impact did not register between 1.9-2.1 atm or if skull fracture occurred during FPI.</li>
		<li>For the sham mice, fix them onto the apparatus, but do not deliver the impact.</li>
	</ol>
	</li>
	<li>Following impact, immediately detach the Luer Lock connection, and transfer mice to an isothermal heating pad for recovery. After the mouse regains alertness and consciousness, return it to its home cage without removing the female Luer lock. Administer the second FPI impact, in the same manner, 48 h later.</li>
	<li>After the rmTBI, carefully remove the female Luer lock and dental cement. Suture the scalp using tissue adhesive and use flat forceps to pinch the scalp to facilitate the adhesive process (see Table of Materials; Figure 2I).</li>
	<li>To prevent inflammation, infection, and alleviate post-surgical pain and discomfort, apply a mixture of erythromycin and sodium diclofenac ointment in a 1:1 ratio (see Table of Materials) to the wound. Transfer mice to an isothermal heating pad for recovery.</li>
	<li>Record the duration of the righting reflex, which starts when the mouse is removed from the stereotaxic apparatus and placed laterally on the impactor platform for FPI and continues until the mouse can stand upright independently.</li>
	<li>After the mouse regains alertness and consciousness, return it to its home cage. Mice are typically fully conscious and able to walk within 1.5 h after the injury.</li>
	<li>In the days following TBI modeling, observe the mice for various signs including breathing patterns, presence of mucus around the nose and mouth, and redness, swelling, exudates, or reopening of the wound area. Exclude animals from the study with one or more of the above abnormal symptoms. 
	NOTE: Pre-microinjection of AAV-GCaMP6s allows for observation of underlying neuronal Ca2+ homeostasis and excitability in the injured cortex through the thinned-skull window using two-photon laser scanning microscopy15.</li>
</ol>
5. Morris water maze (MWM) test
NOTE: The MWM (see Table of Materials) is a widely recognized method for evaluating spatial learning and memory deficits in mice following TBI.
<ol>
	<li>Conduct the MWM test starting from 7 days post injury (DPI). The circular pool of the MWM had a diameter of 120 cm and a height of 50 cm, with the water temperature maintained at 25 &#176;C. Separate the circular pool into four quadrants, with the escape platform, a round platform with a diameter of 6 cm and a height of 30 cm, submerged 1 cm beneath the water surface in the northeast quadrant.</li>
	<li>Position a camera directly above the circular pool to record the movement trajectory of the mice. Mark the mice with black tape on their backs to facilitate recognition by the image acquisition software and for data recording, including the latency, swimming distance and the movement trajectory.</li>
	<li>Place the mice into the water, facing the interior wall of each of the four quadrants, one time for each quadrant. Once the mice find the platform, allow them to rest there for 10 s. If a mouse fails to find the platform within 60 s, ask the operator to guide the mouse to the platform, allow it to rest on the platform for 10 s, and then return the mouse to its home cage for rest.</li>
	<li>Repeat for each mouse the acquisition trial 4x daily. Following the acquisition trials, on 12 DPI, conduct a 60 s spatial probe experiment and record the number of times that the mice crossed the original platform area and the duration of the mouse stay in the quadrant where the platform was located.</li>
	<li>After each trial, quickly dry the mice with a towel or place under a warming lamp to maintain their body temperature and prevent hypothermia during the 60 s acquisition trial from DPI 7 to DPI 11.</li>
	<li>After the completion of the experimental procedures outlined above, anesthetize the mice with pentobarbital (45 mg/kg, i.p.) at 13 DPI. Perfuse isotonic saline transcardially, followed by perfusion with 4% paraformaldehyde in phosphate-buffered saline (pH 7.2). Retrieve the brains for conventional HE staining to evaluate gross cortical and hippocampal morphology alterations. A detailed description of the HE staining protocol can be found in prior publications13,15.</li>
	<li>After all experiments have been completed, euthanize mouse by injection of overdose pentobarbital (&#8805;100 mg/kg, i.p.) if there are no mouse samples needed. Before harvesting tissues or disposing of the carcass, monitor the mouse until there is no heartbeat for at least 60 s.</li>
</ol>

rmTBI Induction in Mice via a Closed-Head Injury Method

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The authors have no conflicts of interest to disclose.

TBI refers to two primary types, closed and penetrating, with the latter characterized by a disruption of the skull and dura mater. Clinical data suggest that CHIs are more prevalent than penetrating injuries1,2. After a single mild TBI, most patients experience PCS symptoms that typically resolve in a short period of time, and there is controversy regarding the proportion of patients whose PCS develop into long-term sequelae23,...

Mild TBI, including concussion and sub-concussion, account for the majority of all TBI cases (&#62;80% of all TBI)1. Mild TBI commonly results from falls, traffic accidents, acts of violence, contact sports (e.g., football, boxing, hockey), and military combat2,3. Mild TBI can lead to neurobiological events that affect neurobehavioral functions throughout the patient&#39;s lifetime and increase the risk of neurodegenerative diseases4,5,6. Animal models provide an efficient and controlled means to study mild TBI, with the hope of further enhancing the diagnosis and treatment of mild TBI. Various models for mild TBI have been developed, such as the controlled cortical impact (CCI), weight drop (WD), fluid percussion injury (FPI), and blast-TBI models7,8. No single experimental model can mimic the entire complexity of TBI-induced pathology9,10. The heterogeneity of these models is advantageous for addressing the diverse features associated with mild TBI patients and investigating the corresponding cellular and molecular mechanisms. However, each animal model of TBI has its limitations3, limiting our current knowledge concerning animal mild TBI and their clinical relevance.
The WD and CCI models are utilized to replicate clinical conditions such as cerebral tissue loss, acute subdural hematoma, axonal injury, brain concussion, blood-brain barrier dysfunction, and even coma following TBI3,11,12. The WD model involves inducing brain damage by striking either the dura mater or skull with freely falling weights. The impact of a weighted object upon an intact skull can replicate mixed focal/diffuse injuries; however, this method is associated with poor accuracy and repeatability of the injury site, rebound injury, and a higher mortality rate due to skull fractures3,11,12. The CCI model involves applying air-driven metal to impact the exposed dura mater directly. Compared to the WD model, the CCI model is more accurate and reproducible, but it does not produce diffuse injury due to the small diameter of the impacting tip11. During FPI modelling, the brain tissue is briefly displaced and deformed by percussion. FPI can induce mixed focal/diffuse injury and replicate intracranial hemorrhage, brain swelling and progressive gray matter damage after TBI. However, FPI has a high mortality rate due to brainstem damage and prolonged apnea3,12. The craniotomy involved in conventional WD, CCI, and FPI models can lead to cortical contusion, hemorrhagic lesions, the damage of the blood-brain barrier, immune cell infiltration, glial cell activation, prolonged modeling time, and possible fatal outcomes3,12.
Mild TBI is characterized by a GCS (Glasgow coma scale, GCS) score within the range of 13 to 152. Mild TBI can be either focal or diffuse and is associated with both acute injuries, such as breakdown of cellular homeostasis, excitotoxicity, glucose depletion, mitochondrial dysfunction, blood flow disturbance, and axonal damage, as well as subacute injuries, including axonal damage, neuroinflammation, and gliosis2,3. Despite significant progress in delineating the intricate pathophysiology of TBI, the underlying mechanisms of mild TBI/rmTBI remain elusive and require further investigation9. Given that CHI is the most common type of TBI12, this protocol presents a novel approach to creating a more precisely controlled mouse model of rmTBI using a modified FPI device to perform impact in a thinned-skull window13. By avoiding craniotomy-induced injuries, variable skull thickness and shape-induced inaccuracies, and rebound injury, this approach aims to overcome the main disadvantages associated with the WD, CCI, and FPI models. Applying FPI impact on the thinned-skull window is convenient for evaluating cerebral vessel damage following rmTBI and helps minimize high mortality rates in some models, resulting in a closer resemblance to the clinical features of TBI patients.

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			<td>75% ethanol&#160;</td>
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			<td>Shenzhen RWD Life Science Inc.</td>
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			<td>Wuhan Mayinglong Pharmaceutical Group Co.,Ltd.</td>
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			<td>Fiber Optic Cold Light Source</td>
			<td>Shenzhen RWD Life Science Inc.</td>
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			<td>Shenzhen RWD Life Science Inc.</td>
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			<td>FPI device software</td>
			<td>Jiaxing Bocom Biotech Inc.</td>
			<td>Biocom Animal Brain Impactor V1.0</td>
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			<td>ICR mice</td>
			<td>Jinhua Laboratory Animal Center&#160;&#160;</td>
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			<td>Shandong Ante Animal Husbandry Technology Co., Ltd.&#160;</td>
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			<td>Isothermal heating pad&#160;</td>
			<td>Wenzhou Repshop Pet Products Co., Ltd.&#160;</td>
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			<td>Luer Loc hup</td>
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			<td>Custom made using a 19G needle hub</td>
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			<td>Micro hand-held skull drill</td>
			<td>Shenzhen RWD Life Science Inc.</td>
			<td>78001</td>
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			<td>Modified FPI device</td>
			<td>Jiaxing Bocom Biotech Inc.</td>
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			<td>Morris water maze</td>
			<td>Shenzhen RWD Life Science Inc.</td>
			<td>63031</td>
			<td>Evaluate mouse spatial learning and memory abilities</td>
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			<td>Open field</td>
			<td>Shenzhen RWD Life Science Inc.</td>
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			<td>Evaluate mouse locomoation and anxiety</td>
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			<td>Suzhou Tianlong Pharmaceutical Co., Ltd.&#160;</td>
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			<td>Wuhan Mayinglong Pharmaceutical Group Co.,Ltd.</td>
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			<td>Small animal anesthesia system-Enhanced&#160;</td>
			<td>Shenzhen RWD Life Science Inc.</td>
			<td>R530IP</td>
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			<td>Smart video-tracking system</td>
			<td>Panlab Harvard Apparatus Inc., MA, USA</td>
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			<td>Shenzhen RWD Life Science Inc.</td>
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			<td>3M, St Paul, MN, USA</td>
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			<td>Shenzhen RWD Life Science Inc.</td>
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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

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

Traumatic brain injury is a significant health issue for the worldwide population. Our main focus is on the mechanism of TBI and the rehabilitation, with a particular focus on the mild TBI and the neuroprotective effects of access. No single animal models can adequately mimic all aspects of the human TBI due to the complexity of a clinical TBI.Each model has its own advantages and the disadvantages when implemented for TBI research. These challenges necessitate the optimization of animal models. The closed-head injury is a common message for making the rodent models of a repeated mTBI.However, this message is susceptible to many factors, including the impact methods used and the anatomy of the rodent scaffold. In our research, we combine the thinned-skull window and the fluid percussion measures to minimize the inflammation due to the craniotomy and the fluid percussion, and produce a more precise model of our repeated mTBI. Overall, the modified closed-head injury associated to repeated mTBI represents a valuable tour for researchers investigating the underlying mechanism that contribute to focal and diffuse pathological changes in repeated mTBI.

The protocol described in this study outlines a method for inducing rmTBI through a thinned-skull window, which offers a solution to the brain injury caused by craniotomy preparation during conventional percussion TBI modeling. By utilizing this modified fluid percussion procedure with the modified device, improved precision and reproducibility of FPI impact were achieved13. The modified impactor has the versatility to be used for both CHI and FPI modeling, with or without a skull craniotomy. Furt...

Read this Scientific Article Protocol about Author Spotlight: Optimizing Rodent Models for Investigating Mechanisms and Rehabilitation in Repeated Mild Traumatic Brain Injury at JoVE.com

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 ...

modified-mouse-model-repetitive-mild-traumatic-brain-injury

Research

JoVE Journal

Neuroscience

232 Views.  Zhejiang Normal University. Traumatic brain injury is a significant health issue for the worldwide population. Our main focus is on the mechanism of TBI and the rehabilitation, with a particular focus on the mild TBI and the neuroprotective effects of access. No single animal models can adequately mimic all aspects of the human TBI due to the complexity of a clinical TBI.Each model has its own advantages and the disadvantages when implemented for TBI research. These challenges necessitate the optimization of anim...

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

Inducing and Monitoring Repetitive Mild Traumatic Brain Injury (rmTBI) in Mice Using a Modified Fluid Percussion Device

To begin, randomly allocate the mice into either the control or repetitive mild traumatic brain injury group. Acclimate the mice in separate cages one week before the experiment. Carefully inspect the modified fluid percussion device for signs of leakage and ensure that there are no bubbles present within the system.After anesthetizing and trimming the fur on the mouse's head, disinfect the scalp with 75%ethanol. Secure the mouse using non-terminal ear bars in a stereotaxic frame and place an isothermal heating pad underneath to maintain body temperature at 37 degrees Celsius. Carefully clean the surgical area using a saline-soaked cotton swab.After making a 1.5-centimeter incision along the midline of the scalp, use a flat-tipped micro drill bit and a microsurgical blade to create a thin skull window in the right frontal motor cortex. Locate the surgical site at 1.5 millimeters anterior to the bregma and 1.3 to 2.0 millimeters lateral to the midline. Attach an adjusted female Luer lock to the thin skull site.Transfer the mouse from the stereotaxic apparatus to the Impactor platform. Connect the female Luer lock to the male Luer lock at the end of the fluid percussion injury device tubing. To apply fluid percussion injury impact, raise the pendulum to the designated degree along the protractor on the device and release the pendulum using software control, achieving a percussion intensity of approximately two atmospheres.Following the impact, immediately detach the Luer lock connection and transfer the mouse to an isothermal heating pad for recovery. After the second traumatic brain injury, carefully remove the female Luer lock and dental cement. Suture the scalp with tissue adhesive and use flat forceps to pinch the scalp to facilitate the adhesive process.To alleviate post-surgical pain and discomfort, apply a one-to-one mixture of erythromycin and sodium diclofenac ointment to the wound. Finally, transfer the mouse to an isothermal heating pad for recovery. Analysis at seven to 12 days post-injury using the Morris water maze showed that repeated mild traumatic brain injury led to impairments in spatial learning and memory in mice.The open field test conducted at six days post-injury showed no significant impact on the mice's general locomotive abilities, but induced significant anxiety and altered exploratory behavior in repeated mild traumatic brain injury mice. The Y-maze test performed at eight days post-injury revealed impairments in both spatial working and reference memory in repeated mild traumatic brain injury mice.