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>

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+patterns+in+the+epidemiology+of+traumatic+brain+injury.">Changing patterns in the epidemiology of traumatic brain injury.</a> Nature Reviews Neurology. 9 (4), 231-236 (2013).</li><li>Corso, P., Finkelstein, E., Miller, T., Fiebelkorn, I., Zaloshnja, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+and+lifetime+costs+of+injuries+in+the+United+States.">Incidence and lifetime costs of injuries in the United States.</a> Injury Prevention. 12 (4), 212-218 (2006).</li><li>Pearson, W. S., Sugerman, D. E., McGuire, L. C., Coronado, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergency+department+visits+for+traumatic+brain+injury+in+older+adults+in+the+United+States:+2006-08.">Emergency department visits for traumatic brain injury in older adults in the United States: 2006-08.</a> Western Journal of Emergency Medicine. 13 (3), 289-293 (2012).</li><li>Whitlock, J. A., Hamilton, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+outcome+after+rehabilitation+for+severe+traumatic+brain+injury.">Functional outcome after rehabilitation for severe traumatic brain injury.</a> Archives of Physical Medicine and Rehabilitation. 76 (12), 1103-1112 (1995).</li><li>Schwarzbold, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychiatric+disorders+and+traumatic+brain+injury.">Psychiatric disorders and traumatic brain injury.</a> Neuropsychiatric Disease and Treatment. 4 (4), 797-816 (2008).</li><li>Whelan-Goodinson, R., Ponsford, J., Johnston, L., Grant, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychiatric+disorders+following+traumatic+brain+injury:+their+nature+and+frequency.">Psychiatric disorders following traumatic brain injury: their nature and frequency.</a> Journal of Head Trauma Rehabilitation. 24 (5), 324-332 (2009).</li><li>Peskind, E. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expert+consensus+document:+Mind+the+gaps-advancing+research+into+short-term+and+long-term+neuropsychological+outcomes+of+youth+sports-related+concussions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Contribution+to+the+Theory+of+Cerebral+Concussion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+cortical+impact:+a+new+experimental+brain+injury+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+controlled+cortical+impact+model+of+traumatic+brain+injury+in+the+rat.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+brain+injury-induced+alterations+in+peripheral+immunity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Activation+of+Infiltrating+Monocyte-Derived+Cells+After+Mild+and+Severe+Traumatic+Brain+Injury.">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. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+fluid+percussion+brain+injury:+a+15-year+review+and+evaluation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+diffuse+brain+injury+in+rats.+Part+I:+Pathophysiology+and+biomechanics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multi-mode+shock+tube+for+investigation+of+blast-induced+traumatic+brain+injury.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+of+Traumatic+Brain+Injury+and+Assessment+of+Injury+Severity.">Animal Models of Traumatic Brain Injury and Assessment of Injury Severity.</a> Molecular Neurobiology. , (2019).</li><li>Makinde, H. 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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>
		</tr>
		<tr>
			<td>Buprenorphine SR Lab 0.5mg/mL</td>
			<td>Zoopharm-Wildlife Pharmaceuticals USA</td>
			<td>BSRLAB0.5-182012</td>
		</tr>
		<tr>
			<td>High Speed Rotary Micromotor KiT0</td>
			<td>Foredom Electric Company</td>
			<td>K.1070</td>
		</tr>
		<tr>
			<td>Imapact one for Stereotaxix CCI</td>
			<td>Leica Biosystems Nussloch GmbH</td>
			<td>39463920</td>
		</tr>
		<tr>
			<td>Ketathesia Ketamine HCl Injection USP</td>
			<td>Henry Schein, Inc</td>
			<td>56344</td>
		</tr>
		<tr>
			<td>Mouse Specific Stereotaxic Base</td>
			<td>Leica Biosystems Nussloch GmbH</td>
			<td>39462980</td>
		</tr>
		<tr>
			<td>Trephines for Micro Drill</td>
			<td>Fine Science Tools, Inc</td>
			<td>18004-50</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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Nanyang Technological University

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JoVE Journal

Neuroscience

9.4K 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 ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also demonstrate that traumatic brain injuries cause changes to the gut microbiota. However, mechanisms underlying the bidirectional regulation of the brain-gut axis remain unknown. Currently, few models exist for studying the changes in gut microbiota after traumatic brain injury. Therefore, the presented study combines protocols for inducing traumatic brain injury using a lateral fluid percussion device and analysis of caecum samples following injury for investigating alterations in the gut microbiome. Alterations of the gut microbiota composition after traumatic brain injury are determined using 16S-rDNA sequencing. This protocol provides an effective method for studying the relationships between enteric microorganisms and traumatic brain injury.

Presented here is a protocol to induce diffuse traumatic brain injury using a lateral fluid percussion device followed by the collection of the caecum content for gut microbiome analysis.

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also ...

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

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...

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

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of medical visits in the United States. There are currently no FDA-approved treatments available for TBI. The increased incidence of military-related, blast-induced TBI further accentuates the urgent need for effective TBI treatments. Therefore, new preclinical TBI animal models that recapitulate aspects of human blast-related TBI will greatly advance the research efforts into the neurobiological and pathophysiological processes underlying mild to moderate TBI as well as the development of novel therapeutic strategies for TBI.
Here we present a reliable, reproducible model for the investigation of the molecular, cellular, and behavioral effects of mild to moderate blast-induced TBI. We describe a step-by-step protocol for closed-head, blast-induced mild TBI in rodents using a bench-top setup consisting of a gas-driven shock tube equipped with piezoelectric pressure sensors to ensure consistent test conditions. The benefits of the setup that we have established are its relative low-cost, ease of installation, ease of use and high-throughput capacity. Further advantages of this non-invasive TBI model include the scalability of the blast peak overpressure and the generation of controlled reproducible outcomes. The reproducibility and relevance of this TBI model has been evaluated in a number of downstream applications, including neurobiological, neuropathological, neurophysiological and behavioral analyses, supporting the use of this model for the characterization of processes underlying the etiology of mild to moderate TBI.

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

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of ...

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...