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.

<table><tbody><tr><td>75% ethanol&amp;nbsp;</td><td>Shandong XieKang Medical Technology Co., Ltd.&amp;nbsp;</td><td>220502</td><td></td></tr><tr><td>Buprenorphine hydrochloride</td><td>Tianjin Pharmaceutical Research Institute Pharmaceutical Co., Ltd</td><td>H12020272</td><td>Solution, Analgesic</td></tr><tr><td>Carprofen</td><td>Shanghai Guchen Biotechnology Co., Ltd</td><td>53716-49-7</td><td>Powder, Analgesic</td></tr><tr><td>Chlorhexidine digluconate</td><td>Shanghai Macklin Biochemical Co.,Ltd.</td><td>18472-51-0</td><td>19%-21% aqueous solution, Antimicrobial</td></tr><tr><td>Dental cement and solvent kit</td><td>Shanghai New Century Dental Materials Co., Ltd.</td><td>20220405, 3#</td><td>Powder reconsituted in matching solvent</td></tr><tr><td>Dissecting microscope</td><td>Shenzhen RWD Life Science Inc.</td><td>77019</td><td></td></tr><tr><td>Erythromycin ointment&amp;nbsp;</td><td>Wuhan Mayinglong Pharmaceutical Group Co.,Ltd.</td><td>220412</td><td>Antibiotic</td></tr><tr><td>Fiber Optic Cold Light Source</td><td>Shenzhen RWD Life Science Inc.</td><td>F-150C</td><td></td></tr><tr><td>Flat-tipped micro-drill bit&amp;nbsp;</td><td>Shenzhen RWD Life Science Inc.</td><td>HM31008</td><td>2 mm, steel</td></tr><tr><td>FPI device software</td><td>Jiaxing Bocom Biotech Inc.</td><td>Biocom Animal Brain Impactor V1.0</td><td></td></tr><tr><td>ICR mice</td><td>Jinhua Laboratory Animal Center&amp;nbsp;&amp;nbsp;</td><td>Stock#2023091</td><td>25 Male mice, 25-30g, 8 weeks old</td></tr><tr><td>Isoflurane</td><td>Shandong Ante Animal Husbandry Technology Co., Ltd.&amp;nbsp;</td><td>2023090501</td><td></td></tr><tr><td>Isothermal heating pad&amp;nbsp;</td><td>Wenzhou Repshop Pet Products Co., Ltd.&amp;nbsp;</td><td></td><td></td></tr><tr><td>Luer Loc hup</td><td></td><td></td><td>Custom made using a 19G needle hub</td></tr><tr><td>Micro hand-held skull drill</td><td>Shenzhen RWD Life Science Inc.</td><td>78001</td><td>Max: 38,000rpm</td></tr><tr><td>Modified FPI device</td><td>Jiaxing Bocom Biotech Inc.</td><td></td><td></td></tr><tr><td>Morris water maze</td><td>Shenzhen RWD Life Science Inc.</td><td>63031</td><td>Evaluate mouse spatial learning and memory abilities</td></tr><tr><td>Open field</td><td>Shenzhen RWD Life Science Inc.</td><td>63008</td><td>Evaluate mouse locomoation and anxiety</td></tr><tr><td>Ophthalmic lubricant&amp;nbsp;</td><td>Suzhou Tianlong Pharmaceutical Co., Ltd.&amp;nbsp;</td><td>SC230724B</td><td></td></tr><tr><td>Sodium diclofenac ointment&amp;nbsp;</td><td>Wuhan Mayinglong Pharmaceutical Group Co.,Ltd.</td><td>221207</td><td>nonsteroidal anti-inflammatory drug</td></tr><tr><td>Small animal anesthesia system-Enhanced&amp;nbsp;</td><td>Shenzhen RWD Life Science Inc.</td><td>R530IP</td><td></td></tr><tr><td>Smart video-tracking system</td><td>Panlab Harvard Apparatus Inc., MA, USA</td><td>V3.0</td><td>Animal tracking and analysis</td></tr><tr><td>Stereotactic frame&amp;nbsp;</td><td>Shenzhen RWD Life Science Inc.</td><td>68043</td><td></td></tr><tr><td>Vetbond Tissue Adhesive</td><td>3M, St Paul, MN, USA</td><td>202402AX</td><td>Suture the animal wound</td></tr><tr><td>Y maze</td><td>Shenzhen RWD Life Science Inc.</td><td>63005</td><td>Evaluate mouse spatial working memory</td></tr></tbody></table>

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.

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

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.

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 ...

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

Research

JoVE Journal

Neuroscience

313 Views.  Zhejiang Normal University. 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.

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

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

A Repetitive Concussive Head Injury Model in Mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

Medicine

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal neurological deficits and even amnesia. Athletes participating in contact sports are at high risk of exposure to large number of mTBIs. In terms of the level of injury in a sporting athlete, a mTBI is defined as a mild injury that does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Despite previous attempts to model mTBI in mice and rats, many have reported gross adverse effects including skull fractures, intracerebral bleeding, axonal injury and neuronal cell death. Herein, we describe our highly reproducible animal model of mTBI that reproduces clinically relevant symptoms. This model uses a custom made pneumatic impactor device to deliver a closed-head trauma. This impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI.

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.

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal ...

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

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

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.

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

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 ...

Modeling Repetitive Concussive Head Injuries in a Mouse Model

<a href='https://app.jove.com/v/54530/a-repetitive-concussive-head-injury-model-in-mice'>Source: Yang, Z., et.al.&#160;A Repetitive Concussive Head Injury Model in Mice. J. Vis. Exp. (2016).</a>This video demonstrates the induction of repetitive concussive head injuries in a mouse using an electromagnetic impact device. The procedure involves positioning the impact tip between the interfrontal and lambdoid sutures on the skull of an anesthetized mouse and using a probe tip to precisely set the impact depth. The impact is delivered at a controlled velocity and depth to simulate mild traumatic brain injuries, followed by a recovery period, with the injuries repeated at specified intervals.

Source: Yang, Z., et.al.&#160;A Repetitive Concussive Head Injury Model in Mice. J. Vis. Exp. (2016).This video demonstrates the induction of repetitive ...

JoVE Encyclopedia of Experiments

Neuropathology

Trauma

Generating a Closed-Head Mild Traumatic Brain Injury in a Murine Model

<a href='https://app.jove.com/v/61244/low-intensity-blast-wave-model-for-preclinical-assessment-closed-head'>Source: Logsdon, A. F., et al. Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents. J. Vis. Exp.&#160;(2020)</a>This video demonstrates the induction of closed-head mild traumatic brain injury in a mouse model using a pressurized gas shock tube setup. The generated pressure wave ruptures the polyester membrane, delivering a shockwave to the mouse&#8217;s head that results in rapid brain movement within the skull, causing a mild traumatic brain injury.

Source: Logsdon, A. F., et al. Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents. J. Vis. ...

Induction of Mild Traumatic Brain Injury in a Mouse Model

<a href='https://app.jove.com/v/64556/electromagnetic-controlled-closed-head-model-mild-traumatic-brain'>Source: Macheda, T., et. al. Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice. J. Vis. Exp. (2022)</a>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.

Source: Macheda, T., et. al. Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice. J. Vis. Exp. (2022)This video ...

Generating a Mouse Model of Traumatic Brain Injury Using a Lateral Fluid Percussion Device

For traumatic brain injury induction, confirm a lack of response to toe pinch in an anesthetized mouse, and apply ointment to the animal&#39;s eyes. After shaving, disinfect the exposed scalp skin with 70% ethanol before incising the scalp in a sagittal plane. Using forceps, retract the incision on both sides and separate the periosteum slightly.
Use a marker to draw a 3 millimeter diameter circle on the right parietal area of the skull, 2 millimeter away from the midline. Use an electric drill to carefully penetrate the skull without damaging the dura. Remove the bone flap to expose a 3 millimeter bone window, and place a plastic injury cannula over the craniotomy.
After cementing the cannula to the skull with dental acrylic, use a 5 milliliters syringe to fill the cannula with sterile 0.9% normal saline, taking care to avoid bubbles. Turn on the oscilloscope and amplifier. Confirm that the high pressure tube of the lateral fluid percussion entry device is free of air bubbles, and deliver about 10 test pulses until the device gives a steady signal.
Adjust the angle of the pendulum starting position to reach a pulse intensity of about two standard atmosphere, and connect the entry cannula to the lateral fluid percussion injury device. Pull the trigger to release the pendulum, inducing the brain injury. Then obtain and transmit a pulse to the dura through the entire closed fluid filled tubing system.
After brain injury induction, remove the cannula and suture the incision. Then, place the mouse on a heating pad with monitoring until it is ambulatory, before returning the animal to its home cage with ad libitum access to food and water.