This work was supported by National Institutes of Health Grant GM117341 and The American College of Surgeons C. James Carrico Research Fellowship to S.J.S.

All procedures were approved by the Northwestern University Institutional Animal Care and Use Committee. C57BL/6 mice were purchased from the Jackson Laboratory and group housed at a barrier facility at the Center for Comparative Medicine at Northwestern University (Chicago, IL). All animals were housed in 12/12 h light/dark cycle with free access to food and water.
1. Induce anesthesia
<ol>
	<li>Anesthetize the mouse with ketamine (125 mg/kg) and xylazine (10 mg/kg) injected intraperitoneally.</li>
</ol>
2. Vital signs monitoring every 15 min
<ol>
	<li>Monitor temperature, respiratory rate, and skin color. The mouse should feel warm to the touch. The skin should appear pink and well perfused. Respiratory rate should range 50&#8211;70 breaths per minute.</li>
</ol>
3. Pre-surgical procedures
<ol>
	<li>Weigh all the mice on the day prior to injury induction.</li>
	<li>Sterilize one set of surgical instruments by autoclaving for each experimental subject. Sterilize the impacting device before use.&#160;</li>
	<li>Prepare a recovery cage by placing a clean cage over an electric heating pad set to &#8220;low&#8221; setting and positioned in a manner such that the mice can move away from the heat once ambulatory.</li>
	<li>Set up the operating theater within a sterilized laminar flow hood.
	<ol>
		<li>Position the stereotaxic operating frame.</li>
		<li>Attach the impacting device to the stereotaxic frame.</li>
		<li>Set the actuating device with the desired biomechanical parameters for velocity and dwell time. 
		NOTE: In this protocol a severe brain injury is described utilizing a 3 mm diameter impact tip via a 5 mm diameter craniectomy with the velocity set at 2.5 m/s and a dwell time of 0.1 s. A wide range of biomechanical parameters may be used to induce the full spectrum of TBI.</li>
	</ol>
	</li>
	<li>Don new personal protective equipment and sterile gloves.</li>
	<li>Shave the fur from the operative site using electric clippers.</li>
	<li>Apply protective opthalmic ointment to the eyes of the mouse to prevent corneal injury and drying.</li>
	<li>Place the mouse into the operating theater.</li>
	<li>Prep the skin with an iodine based surgical scrub alternated with alcohol three times.</li>
</ol>
4. Application of controlled cortical impact
<ol>
	<li>Incise the scalp 1 cm in the midline with a scalpel exposing the skull.</li>
	<li>Position the mouse within a stereotaxic operating frame by securing the bilateral temporal bones between miniature ear bars and locking the incisors within an incisor clamp creating a stable three-point-hold on the mouse head.</li>
	<li>Retract the scalp away from the operative site with a hemostat or locking forceps to ensure the scalp does not come in contact with the drill bit during craniectomy.</li>
	<li>Identify the sagittal and coronal sutures on the exposed skull. 
	NOTE: This protocol centers the craniectomy 2 mm left of the sagittal suture and 2 mm rostral to the coronal suture.</li>
	<li>Perform a craniectomy using a drill with a trephine drill bit.
	<ol>
		<li>To perform the craniectomy, first activate the drill at maximum speed and then apply the trephine drill bit perpendicular to the skull at the site of craniectomy.</li>
		<li>Apply gentle, even pressure to the drill once contact is made with the skull. A slight &#8220;give&#8221; will be felt once the drill penetrates through the skull. Do not penetrate the underlying dura. 
		NOTE: This protocol utilizes a 5 mm trephine drill bit to perform the craniectomy.</li>
	</ol>
	</li>
	<li>Use forceps and a small gauge hypodermic needle to remove the bone flap, fully exposing the underlying dura mater.</li>
	<li>Rotate the impactor tip into the operative field and lower it until it makes contact with the exposed dura mater. Once contact is made the instrument&#8217;s contact sensor will make an audible tone to alert the surgeon that contact has been made. This will mark the zero point from which the deformation depth is set. 
	NOTE: This protocol utilizes a 3 mm impacting tip to generate a severe injury. Tips as small as 1 mm may be used to apply more localized injury.</li>
	<li>Retract the impacting tip and set the desired impact depth by lowering the impactor position on the stereotaxic frame. 
	NOTE: In this protocol we describe a severe injury by setting the deformation depth to 2 mm.</li>
	<li>Apply the injury by activating impactor on the actuating device.</li>
	<li>Rotate the impact device out of the field and remove the animal from the stereotaxic frame.</li>
</ol>
5. Surgical site closure
<ol>
	<li>Control bleeding from the skull and injured cortical surface with direct pressure from a sterile cotton tipped applicator.</li>
	<li>Dry the skull with a sterile cotton tipped applicator.</li>
	<li>Close the scalp over the craniectomy using a commercially available surgical adhesive or monofilament suture. 
	NOTE: In this protocol a veterinary surgical adhesive is used to close the scalp. The bone flap is not replaced and is discarded.</li>
</ol>
6. Post-operative care and monitoring
<ol>
	<li>Administer post-operative analgesia (e.g., sustained release buprenorphine 0.1&#8211;0.5 mg/kg administered subcutaneously providing 72 h of sustained analgesia).</li>
	<li>Place the animal in the lateral decubitus recovery position in a clean pre-warmed cage.</li>
	<li>Observe the animals until awake and mobile, then return each mouse to its home cage.</li>
	<li>Ensure free access to food and water. Normal food and water intake typically resume within one to two hours after injury.</li>
	<li>Measure body weight every three days throughout the experiment.</li>
</ol>

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<li>Belanger, H. G., Vanderploeg, R. D., McAllister, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subconcussive+Blows+to+the+Head%3A+A+Formative+Review+of+Short-term+Clinical+Outcomes%5BTitle%5D)+AND+%22Journal+of+Head+Trauma+Rehabilitation%22%5BJournal%5D)">Subconcussive Blows to the Head: A Formative Review of Short-term Clinical Outcomes.</a> Journal of Head Trauma Rehabilitation. 31 (3), 159-166 (2016).</li>
<li>Carman, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expert+consensus+document%3A+Mind+the+gaps-advancing+research+into+short-term+and+long-term+neuropsychological+outcomes+of+youth+sports-related+concussions%5BTitle%5D)+AND+%22Nature+Reviews+Neurology%22%5BJournal%5D)">Expert consensus document: Mind the gaps-advancing research into short-term and long-term neuropsychological outcomes of youth sports-related concussions.</a> Nature Reviews Neurology. 11 (4), 230-244 (2015).</li>
<li>Kramer, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Contribution+to+the+Theory+of+Cerebral+Concussion%5BTitle%5D)+AND+%22Annals+of+Surgery%22%5BJournal%5D)">A Contribution to the Theory of Cerebral Concussion.</a> Annals of Surgery. 23 (2), 163-173 (1896).</li>
<li>Lighthall, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Controlled+cortical+impact%3A+a+new+experimental+brain+injury+model%5BTitle%5D)+AND+%22Journal+of+Neurotrauma%22%5BJournal%5D)">Controlled cortical impact: a new experimental brain injury model.</a> Journal of Neurotrauma. 5 (1), 1-15 (1988).</li>
<li>Dixon, C. E., Clifton, G. L., Lighthall, J. W., Yaghmai, A. A., Hayes, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+controlled+cortical+impact+model+of+traumatic+brain+injury+in+the+rat%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">A controlled cortical impact model of traumatic brain injury in the rat.</a> Journal of Neuroscience Methods. 39 (3), 253-262 (1991).</li>
<li>Schwulst, S. J., Trahanas, D. M., Saber, R., Perlman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Traumatic+brain+injury-induced+alterations+in+peripheral+immunity%5BTitle%5D)+AND+%22Journal+of+Trauma+and+Acute+Care+Surgery%22%5BJournal%5D)">Traumatic brain injury-induced alterations in peripheral immunity.</a> Journal of Trauma and Acute Care Surgery. 75 (5), 780-788 (2013).</li>
<li>Trahanas, D. M., Cuda, C. M., Perlman, H., Schwulst, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+Activation+of+Infiltrating+Monocyte-Derived+Cells+After+Mild+and+Severe+Traumatic+Brain+Injury%5BTitle%5D)+AND+%22Shock%22%5BJournal%5D)">Differential Activation of Infiltrating Monocyte-Derived Cells After Mild and Severe Traumatic Brain Injury.</a> Shock. 43 (3), 255-260 (2015).</li>
<li>Makinde, H. M., Cuda, C. M., Just, T. B., Perlman, H. R., Schwulst, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonclassical+Monocytes+Mediate+Secondary+Injury%2C+Neurocognitive+Outcome%2C+and+Neutrophil+Infiltration+after+Traumatic+Brain+Injury%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">Nonclassical Monocytes Mediate Secondary Injury, Neurocognitive Outcome, and Neutrophil Infiltration after Traumatic Brain Injury.</a> Journal of Immunology. 199 (10), 3583-3591 (2017).</li>
<li>Thompson, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lateral+fluid+percussion+brain+injury%3A+a+15-year+review+and+evaluation%5BTitle%5D)+AND+%22Journal+of+Neurotrauma%22%5BJournal%5D)">Lateral fluid percussion brain injury: a 15-year review and evaluation.</a> Journal of Neurotrauma. 22 (1), 42-75 (2005).</li>
<li>Marmarou, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+model+of+diffuse+brain+injury+in+rats.+Part+I%3A+Pathophysiology+and+biomechanics%5BTitle%5D)+AND+%22Journal+of+Neurosurgery%22%5BJournal%5D)">A new model of diffuse brain injury in rats. Part I: Pathophysiology and biomechanics.</a> Journal of Neurosurgery. 80 (2), 291-300 (1994).</li>
<li>Reneer, D. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+multi-mode+shock+tube+for+investigation+of+blast-induced+traumatic+brain+injury%5BTitle%5D)+AND+%22Journal+of+Neurotrauma%22%5BJournal%5D)">A multi-mode shock tube for investigation of blast-induced traumatic brain injury.</a> Journal of Neurotrauma. 28 (1), 95-104 (2011).</li>
<li>Ma, X., Aravind, A., Pfister, B. J., Chandra, N., Haorah, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+Models+of+Traumatic+Brain+Injury+and+Assessment+of+Injury+Severity%5BTitle%5D)+AND+%22Molecular+Neurobiology%22%5BJournal%5D)">Animal Models of Traumatic Brain Injury and Assessment of Injury Severity.</a> Molecular Neurobiology. , (2019).</li>
<li>Makinde, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monocyte+depletion+attenuates+the+development+of+posttraumatic+hydrocephalus+and+preserves+white+matter+integrity+after+traumatic+brain+injury%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Monocyte depletion attenuates the development of posttraumatic hydrocephalus and preserves white matter integrity after traumatic brain injury.</a> PLoS One. 13 (11), e0202722(2018).</li>
<li>Osier, N. D., Dixon, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Controlled+Cortical+Impact+Model%3A+Applications%2C+Considerations+for+Researchers%2C+and+Future+Directions%5BTitle%5D)+AND+%22Frontiers+in+Neurology%22%5BJournal%5D)">The Controlled Cortical Impact Model: Applications, Considerations for Researchers, and Future Directions.</a> Frontiers in Neurology. 7, 134(2016).</li>
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The authors have no financial conflicts of interest.

There are several steps that are critical for applying a reliable and consistent injury. First, the mouse must reach a deep plane of surgical anesthesia ensuring no movement during the performance of the craniectomy. While numerous anesthetic regimens may be used to induce general anesthesia in rodents, anesthetics that induce respiratory depression such as inhalational anesthetics may result in respiratory arrest when combined with a severe TBI. This protocol utilizes ketamine (125 mg/kg) and xylazine (10 mg/kg) injected intraperitoneally. This combination of drugs produces a surgical plane of anesthesia within 5 min of administration for a duration of approximately 30&ndash;45 min. Furthermore, this combination of drugs does not result in respiratory depression. The next critical step is the performance of the craniectomy. The craniectomy should always be performed with a fresh trephine drill bit at high speed to ensure that minimal heat and vibration are transmitted to the mouse brain. Heat and vibration can result in damage to adjacent brain tissue outside the area of CCI leading to inconsistent size and mechanism of injury between subjects and experiments. Next, the mouse head must be firmly secured within the stereotaxic frame prior to the application of the CCI to ensure the depth and location of injury are consistent between injury applications. Miniature ear bars and an incisor clamp are essential components in properly securing the mouse head within the stereotaxic frame. Lastly, it is critical to utilize a device with a contact sensor. The sensor will indicate the exact point of contact between the impacting tip and the exposed dura mater. This allows investigator to note the exact zero point from which the depth of injury is can be set with the stereotaxic frame ensuring a precise and reproducible degree of injury.
To ensure that the incised scalp is outside the field at the time of CCI, it is often necessary to use a retractor such as clamp or forceps to pull to scalp away from the site of craniectomy. Should the scalp fall back into the CCI field as the injury is applied, the size and severity of injury will be unreliable. Additionally, although it is imperative to ensure the mouse head is immobilized within the stereotaxic frame, the investigator must ensure that the fixation does not impair respiration. Hypoxia at the time of injury secondary to restricted respiration will introduce a secondary form of injury making the degree, severity, and mechanism of injury unreliable between experimental subjects.
Given the ability to precisely specify multiple biomechanical parameters, CCI is one of the most consistent and reliable methods for inducing traumatic brain injury in rodent models15. However, there are a number of limitations that the investigator should be aware of when choosing which model of TBI is most appropriate to answer their scientific question22. CCI suffers from the same limitations as all preclinical models of brain injury in that it requires anesthesia and a surgical procedure (craniectomy) prior to the induction of injury. Both anesthesia and craniectomy are capable of generating an inflammatory response and must be considered as potential confounders during data analysis24. Additionally, although CCI produces a reliable and consistent injury, most TBI in human patients are diffuse and occur through multiple simultaneous mechanisms25. This may make direct translation to human TBI patients problematic as CCI produces a focal injury with varying degrees of diffuse effects depending on the severity of injury applied. Lastly, CCI requires the purchase and maintenance of several mechanical components that may prove to be cost prohibitive to some research groups. Without proper maintenance of the mechanical components, there may be substantial drift in the actual biomechanical parameters applied from experiment to experiment24.
Identifying appropriate controls for each experiment is critical. Sham-injured mice are an important control in every experiment. The sham injury group should receive anesthesia, scalp incision, placement into the stereotaxic frame, and post-operative analgesia. However, the sham-injury group should not undergo craniectomy. The vibration and heat transfer from craniectomy, even when performed quickly with expert precision, does result in a mild traumatic brain injury. Although this injury is difficult to see grossly, it is readily identified microscopically. Lastly, investigators should consider using a group of age-matched na&iuml;ve mice to rule out any normal changes that occur within the brain as the mice age.
Despite limitations, CCI remains the most consistent and reproducible model for inducing TBI in rodents. CCI is easy to standardize across subjects and experiments as compared to alternative methods of inducing TBI and allows investigators to apply the entire spectrum of TBI to precisely defined anatomic regions of the brain. The protocol above describes the application of a severe TBI to the left parietotemporal cortex in a mouse. This model utilizes a 5 mm craniectomy performed with a trephine drill bit at high speed. A 3 mm impacting tip is used with an injury depth of 2 mm at a velocity of 2.5 m/s and a dwell time of 0.1 s. When applied appropriately, and when the experimental subject is properly recovered, a long-term survival rate approaching 100% can be obtained allowing for short, intermediate, and long-term studies of murine TBI to be performed.

The Centers for Disease Control and Injury Prevention estimate that approximately 2 million Americans sustain a traumatic brain injury (TBI) every year1,2. In fact, TBI contributes to over 30% of all injury related deaths in the United States with healthcare costs nearing $80 billion annually and almost $4 million per person per year surviving a severe TBI3,4,5. The impact of TBI is highlighted by the significant long-term neurocognitive and neuropsychiatric complications suffered by its survivors with the insidious onset of behavioral, cognitive, and motor impairments termed Chronic Traumatic Encephalopathy (CTE)6,7,8,9,10. Even subclinical concussive events&#8212;those impacts that do not result in clinical symptoms&#8212;can lead to long-term neurologic dysfunction11,12.
Animal models for the study of TBI have been employed since the late 1800&#8217;s13. In the 1980s, a pneumatic impactor for the purpose of modeling TBI was developed. This method is now referred to as controlled cortical impact (CCI)14. The control and reproducibility of CCI led researchers to adapt the model for use in rodents15. Our laboratory uses this model to induce TBI via a commercially available impactor and electronic actuating device16,17. This model is capable of producing a wide range of clinically applicable TBI states depending on the biomechanical parameters used. Histologic evaluation of TBI brains after a severe injury induced in our laboratory demonstrates significant ipsilateral cortical and hippocampal loss as well as contralateral edema and distortion. Additionally, CCI produces a consistent impairment in motor and cognitive function as measured by behavioral assays18. Limitations to CCI include the need for craniotomy and the expense of acquiring the impactor and actuating device.
Several additional models of TBI exist and are well established in the literature including the lateral fluid percussion model, weight drop model, and blast injury model19,20,21. While each of these models have their own distinct advantages their main drawbacks are mixed injury, high mortality and lack of standardization, respectively22. Furthermore, none of these models offer the accuracy, precision, and reproducibility of CCI. By adjusting the biomechanical parameters input into the actuating device, the CCI model allows the investigator precise control over size of the injury, depth of the injury, and kinetic energy applied to the brain. This gives investigators the ability to apply the entire spectrum of TBI to specific areas of the brain. It also permits the greatest reproducibility from experiment to experiment.

<table><tbody><tr><td>AnaSed Injection Xylazine Sterile Solution</td><td>LLOYD, Inc.</td><td>5939911020</td><td></td></tr><tr><td>Buprenorphine SR Lab 0.5mg/mL</td><td>Zoopharm-Wildlife Pharmaceuticals USA</td><td>BSRLAB0.5-182012</td><td></td></tr><tr><td>High Speed Rotary Micromotor KiT0</td><td>Foredom Electric Company</td><td>K.1070</td><td></td></tr><tr><td>Imapact one for Stereotaxix CCI</td><td>Leica Biosystems Nussloch GmbH</td><td>39463920</td><td></td></tr><tr><td>Ketathesia Ketamine HCl Injection USP</td><td>Henry Schein, Inc</td><td>56344</td><td></td></tr><tr><td>Mouse Specific Stereotaxic Base</td><td>Leica Biosystems Nussloch GmbH</td><td>39462980</td><td></td></tr><tr><td>Trephines for Micro Drill</td><td>Fine Science Tools, Inc</td><td>18004-50</td><td></td></tr></tbody></table>

murine model of controlled cortical impact for the induction of traumatic brain injury

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the United States. In fact, TBI is a contributing factor to over a third of all injury-related mortality. Nonetheless, the cellular and molecular mechanisms underlying the pathophysiology of TBI are poorly understood. Thus, preclinical models of TBI capable of replicating the injury mechanisms pertinent to TBI in human patients are a critical research need. The controlled cortical impact (CCI) model of TBI utilizes a mechanical device to directly impact the exposed cortex. While no model can full recapitulate the disparate injury patterns and heterogeneous nature of TBI in human patients, CCI is capable of inducing a wide range of clinically applicable TBI. Furthermore, CCI is easily standardized allowing investigators to compare results across experiments as well as across investigative groups. The following protocol is a detailed description of applying a severe CCI with a commercially available impacting device in a murine model of TBI.

The impactor mounts directly on the stereotaxic frame allowing for as much as 10 &#181;m resolution for control of the point of impact, depth and penetration. The electromagnetic forces employed can impart impact velocities ranging 1.5&#8211;6 m/s. This allows for unparalleled precision and reproducibility over the entire range of clinically relevant TBI. Investigators can run pilot experiments changing the injury parameters such as impactor tip size, impact velocity, and impact depth to determine the parameters that best produce the desired degree of injury. This protocol describes a severe TBI to the left parietotemporal region by performing a 5 mm craniectomy 2 mm left of the sagittal suture and 2 mm rostral to the coronal suture (Figure 1A). A controlled cortical impact is delivered with a 3 mm impacting tip at 2.5 m/s and a deformation depth of 2 mm (Figure 2). Injury consists of subdural, intraparenchymal, and subarachnoid hemorrhage (Figure 3). Neurocognitive testing one month after this injury demonstrates persistent deficits in working memory, skill acquisition, and motor coordination18. Histologic evaluation of TBI brains after a severe injury induced in our laboratory demonstrates significant ipsilateral cortical and hippocampal loss as well as contralateral edema and distortion. MRI examination of severely injured brains using this model demonstrates progressive tissue loss and replacement by cerebrospinal fluid (Figure 4)23. Lastly, flow cytometric analysis of injured and sham brains demonstrates a marked difference in infiltrating inflammatory cells throughout the course of injury17,18.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60027/60027fig01.jpg" /> 
Figure 1: Equipment setup for the murine model of controlled cortical impact. 
(A) The actuating device is set a velocity of 2.5 m/s and a dwell time of 0.1 s. (B) The impactor with a 3 mm impacting tip is secured to the stereotaxic frame. (C) A mouse with 5 mm craniectomy is secured into the stereotaxic operating frame with ear bars and an incisor bar. <a href="https://www.jove.com/files/ftp_upload/60027/60027fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60027/60027fig02.jpg" /> 
Figure 2: Severe TBI via open-head controlled cortical impact. 
(A) The grounding cable is clipped to the mouse&#8217;s hind region and the impacting tip is lowered onto the dura mater until the contact sensor alarms. This is the zero point. (B) The impacting tip is retracted, a 2 mm depth of injury is dialed into the stereotaxic frame, and the impact is applied. (C) After the CCI is applied, the impacting tip is rotated out of the field and the mouse is recovered from the stereotaxic frame. <a href="https://www.jove.com/files/ftp_upload/60027/60027fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60027/60027fig03.jpg" /> 
Figure 3: Gross examination of mouse brains after severe TBI induced by controlled cortical impact. 
(A) Brain from a 12-week-old na&#239;ve mouse. (B) Brain from a 12-week-old mouse 24 h after sustaining a severe TBI via controlled cortical impact. (C) Brain from a 12-week-old mouse 7 days after sustaining a severe TBI via controlled cortical impact. <a href="https://www.jove.com/files/ftp_upload/60027/60027fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60027/60027fig04.jpg" /> 
Figure 4: Histologic and MRI evaluation of severe TBI after controlled cortical impact. 
Hematoxylin and eosin (H&#38;E) stained coronal sections and representative coronal T1-weighted MR images. (A) Sham injury, consisting of craniotomy only. (B) CCI results in a severe TBI with large volume loss of cortex (Ctx) at the site of impact as well as loss and distortion of the underlying hippocampal formation (HF) and thalamus (TH). (C) MRI at 1-day post-TBI demonstrates tissue trauma and edema over the left parietotemporal cortex. (D&#8211;E) Representative images from post-injury days 7 and 14 demonstrate increased areas of hyperattenuation representing progressive replacement of devitalized tissue with cerebrospinal fluid. Figure has been adapted from Makinde, et al.23. <a href="https://www.jove.com/files/ftp_upload/60027/60027fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury at JoVE.com

Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury

Here we describe a protocol for the induction of murine traumatic brain injury via an open-head controlled cortical impact.

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the ...

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10.3K Views.  Northwestern University. Here we describe a protocol for the induction of murine traumatic brain injury via an open-head controlled cortical impact.

Video: Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury

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 ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Developmental Biology

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

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 ...

A Preclinical Controlled Cortical Impact Model for Traumatic Hemorrhage Contusion and Neuroinflammation

Cerebral contusion is a severe medical problem affecting millions of people worldwide each year. There is an urgent need to understand the pathophysiological mechanism and to develop effective therapeutic strategy for this devastating neurological disorder. Intraparenchymal hemorrhage and post-traumatic inflammatory response induced by initial physical impact can aggravate microglia/macrophage activation and neuroinflammation which subsequently worsen brain pathology. We provide here a controlled cortical impact (CCI) protocol that can reproduce experimental cortical contusion in mice by using a pneumatic impactor system to deliver mechanical force with controllable magnitude and velocity onto the dural surface. This preclinical model allows researchers to induce moderately severe focal cerebral contusion in mice and to investigate a wide range of post-traumatic pathological progressions including hemorrhage contusion, microglia/macrophage activation, iron toxicity, axonal injury, as well as short-term and long-term neurobehavioral deficits. The present protocol can be useful for exploring the long-term effects of and potential interventions for cerebral contusion.

Intraparenchymal hemorrhage and neuroinflammation accompanied by cerebral contusion can trigger severe secondary brain injury. This protocol details a mouse controlled cortical impact (CCI) model allowing researchers to study hemorrhage contusion and post-traumatic immune responses and explore potential therapeutics.

Cerebral contusion is a severe medical problem affecting millions of people worldwide each year. There is an urgent need to understand the ...

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 ...

rmTBI Induction in Mice via a Closed-Head Injury Method

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

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

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

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

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

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

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

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

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

Generating a Traumatic Brain Injury Mouse Model Using an Impactor Probe

Source: Furmanski, O., et al. <a href="https://app.jove.com/v/59561">Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells.</a> J. Vis. Exp. (2019).
This video demonstrates the procedure for inducing an open-skull traumatic brain injury in a mouse by making a midline skull incision, drilling through the skull and removing the bone flap to expose the brain cortex, and applying high pressure to induce trauma. The wound is then sutured to prepare the mouse model for subsequent experiments.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

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Trauma

Generating a Mouse Model of Traumatic Brain Injury Using an Impactor Device

Source: Schwulst, S. J. et al. <a href="https://app.jove.com/v/60027">Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury.</a> J. Vis. Exp. (2019)
This video demonstrates the procedure for inducing traumatic brain injury (TBI) in a mouse model. An anesthetized mouse with an exposed skull is taken to create an opening in the skull, and then a controlled cortical injury is induced using an impact device. The injury damages the brain tissue, causing impaired cognitive function and motor coordination.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Induce ...