We thank Danye Jiang for editing the manuscript and insightful input. We thank Jhih Syuan Lin for assisting in manuscript preparation. This work was supported by the Ministry of Science and Technology of Taiwan (MOST 107-2320-B-002-063-MY2) to C.F.C.

All procedures described in this protocol were conducted under the approval of the Institutional Animal Care and Use Committee at Cheng Hsin General Hospital and National Taiwan University College of Medicine. Eight- to ten-week-old male C57BL/6 wild type mice were used in this protocol.
1. Anesthesia induction
<ol>
	<li>Anesthetize the mouse with ~4% isoflurane mixed with room air at ~0.2 L/min in an induction chamber connected to the isoflurane vaporizer.</li>
	<li>Ensure the respiratory pattern is smooth. Check the depth of anesthesia by confirming a lack of toe-pinch reflex in the animal.</li>
</ol>
2. Pre-surgical preparation
<ol>
	<li>Shave the mouse head with electrical clippers in a caudal to rostral direction. Do not trim the mouse whiskers. 
	NOTE: Loss of whiskers may influence the accuracy of subsequent behavioral test results.</li>
	<li>Place the mouse onto the stereotaxic frame. Carefully insert the ear bars into the ear canals. Ensure the mouse head is stabilized by both ear bars equally.</li>
	<li>Bring in the nose cone and maintain anesthesia at 1% - 2% isoflurane for the duration of the surgery.</li>
	<li>Apply ophthalmic ointment&#160;to both eyes to prevent drying out during the surgery. Keep the animal on a heating pad to maintain a body temperature of 37 &#730;C.</li>
	<li>Disinfect the shaved head with betadine followed by 70% alcohol using sterile cotton swabs. Repeat three times.</li>
</ol>
3. CCI surgery
<ol>
	<li>Administer 100 &#181;L of Bupivacaine (0.25%) subcutaneously using a 31 G insulin needle prior to the incision. Gently massage the injection site for better absorption. 
	NOTE: This local anesthetic provides pain relief directly at the site of surgery.</li>
	<li>Make a longitudinal incision (~1.5 cm) along the midline on the scalp with a scalpel or scissors. Use a hemostat to hold the skin off to the right side and allow the exposed skull to dry for 1 min. Use a sterile cotton swab to clean away any residual blood and tissues on the skull.</li>
	<li>Check that the mouse head is level in the horizontal plane.
	<ol>
		<li>Identify anatomical landmarks Bregma and Lambda and mark both locations with a sterile surgical marker/pencil.</li>
		<li>Ensure that the head of the animal is level in the rostral-caudal direction. Do this by measuring the Z coordinates of both Bregma and Lambda using a 31 G insulin needle attached to the stereotaxic frame. 
		NOTE: Adjust the ear bar vertically if necessary.</li>
		<li>Perform for the horizontal positioning of the animal head by following the same procedure of checking the Z coordinates at the midline along with two corresponding locations on the left and right side of the midline and adjust the ear bars if needed. 
		NOTE: A level and stable placement of the animal head is crucial for the reproducibility and reliability of the CCI model.</li>
	</ol>
	</li>
	<li>Use the same 31 G insulin needle to identify the craniectomy site. Set the XY origin to Bregma and laterally move the needle 3 mm to the right. Mark this position as the site of craniectomy and draw a circle 4 mm in diameter on the skull with a sterile surgical marker/pencil.</li>
	<li>Use a high-speed micro drill with a trephine (4 mm diameter) to cut along the pencil-outlined circle to create a 4 mm diameter open hole. Use a speed setting of 20,000 rpm. Avoid applying excess pressure. 
	NOTE: Perform this step quickly (usually within 30 s to 1 min) to prevent any thermal damage to the brain. Applying excess pressure while drilling may lead to accidental penetration that could compress and injure the brain surface.</li>
	<li>Carefully remove the bone flap with tweezer and temporally store it in ice cold normal saline. Gently rinse the hole with normal saline before applying pressure on the brain surface with the cotton swab tip to stop bleeding.</li>
	<li>Set the 2.5 mm diameter rounded impactor tip on the CCI device to an angle of 22.5&#730;. Zero the impact tip to the dural surface. Set the impact parameters on the control box to a velocity of 4 m/s and a deformation depth of 2 mm. Retract the metal tip. 
	NOTE: Zeroing the tip while it is statically and slightly pressed against the dural surface in the full stroke position improves the accuracy of the zero point and the reproducibility of the injury level.</li>
	<li>Discharge the piston to generate impaction on the brain. Place a sterile cotton swab onto the injured area to stop bleeding.</li>
	<li>Place the bone flap back to the mouse brain and secure with dental cement. Close the scalp with tissue adhesive (e.g., 3M Vetbond).</li>
</ol>
4. Postoperative recovery
<ol>
	<li>Place the mouse in a clean recovery cage with bedding under the heat lamp until full recovery.</li>
	<li>Provide moistened chow food and subcutaneously administer ketoprofen (5 mg/kg) for two consecutive days after surgery.</li>
	<li>Perform the above procedures except steps 3.7 and 3.8 for sham control animals.</li>
</ol>
5. Mouse euthanasia
<ol>
	<li>Euthanize mice on the day of study by isoflurane overdose and then decapitation. 
	NOTE: Several strategies can be used to euthanize the experimental animals prior to the sample collection.</li>
	<li>Collect brain samples for histological analysis.</li>
</ol>

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The authors have nothing to disclose.

The CCI protocol produces highly reproducible mechanical injury to the brain for cerebral contusion research. The following steps are crucial for generating consistent brain injury in animals using this CCI protocol.
First, the mouse head should be stably mounted on the stereotaxic frame and the anatomical landmarks Bregma and Lambda always in the same horizontal plane. Unsteady or unlevel head placement oftentimes result in varied injury levels between animals. To ensure the animal head is safely secured, use a pair of forceps to gently nudge the mouse head laterally. The head needs to be laterally immobile and any movement is indicative of an incorrect placement of the ear bars. Last, gently suspend the mouse body by lifting its tail to confirm that the head is firmly fixed on the stereotaxic frame.
Next, avoid damaging the dura mater when drilling into the skull by applying even pressure along the circular path as mechanical damage to the dura mater can cause unnecessary injury to the brain. Use great care to inspect the depth of drilling by gently tapping the bone window with a pair of fine forceps. In addition, heat generated during prolonged drilling process also induces cortical injury. In our protocol we use trephine rather than the round tip drill bit seen in most other CCI protocols33,34 to shorten the duration of craniotomy. Ice cold saline can be applied onto the skull during the drilling step for additional protection against heat. After drilling, the bone flap must be gently lifted without damaging the brain tissue underneath. Keeping a consistent size of the drilled bone window between animals is crucial for ensuring a consistent level of intracranial pressure and degree of brain deformation.
Furthermore, the impactor tip must be properly positioned at the center of the bone window and slightly pressed against the dural surface in order to generate consistent brain damage. Depth variance of tip zeroing on the dural surface can affect the extent of CCI injury.
Lastly, the bone flap should be repositioned and well secured after impaction. In fact, removing the bone flap prior to the mechanical impact is not relevant to human cerebral contusion. Therefore, repositioning bone flap immediately after the CCI helps maintain a consistent intracranial pressure in the closed head between animals during the subsequent disease progression.
High reproducibility, low mortality, and ease of controlling severity are among the many strengths of using a CCI model14,15,16,18,34. The requirement of anesthesia and craniotomy during the procedure are the major limitations of CCI model. Meanwhile, the neurologic deficits are usually resolved within days to weeks in rodents whereas human patients&#8217; deficits can persist for months. However, no single animal model can mimic all clinical features. The CCI model is still highly reliable and oftentimes the preferred method to study short- and long-term pathophysiological events and to search for potential therapeutics for cerebral contusion14,15. Here we described the application of a CCI model to study hemorrhage contusion and secondary CNS inflammation after cerebral contusion. This protocol demonstrates a moderately severe CCI that produces focal brain injury in the primary sensory and motor cortical areas without damaging the hippocampus27,28. Depending on the research purpose, velocity and depth of impact, size of impactor tip, and position of the impaction can all be modified to create various severity of injury in different brain regions35.
Although we have applied this protocol in studying traumatic intraparenchymal hemorrhage, this CCI model can also be used to study other types of focal injury such as traumatic subarachnoid hemorrhage (tSAH), subdural hemorrhage (SDH), and epidural hemorrhage (EDH)1,7. While adult animal was used for the representative study, this CCI protocol with modification can also be applied to neonates to study the effect of cerebral contusion in the immature brain based on the goal of study36,37. A CCI model can not only facilitate study of single cerebral contusion but also repetitive mechanical cerebral impact that is relevant to people at high risk of multiple brain injuries such as military personnel19. Finally, we suggest that researchers perform pilot experiment to fine-tune the biomechanical parameters in the CCI model to better meet their research goal.

Cerebral contusion is a form of traumatic brain injury that ranks high among the deadliest health issues in modern society1. It is primarily caused by accidental events such as traffic accident that results in external forces applying mechanical energy to the head. Traumatic brain injury affects an approximate of 3.5 million people and accounts for 30% of all acute injury-related deaths in the US each year2. Patients who survive cerebral contusion oftentimes suffer from long-term consequences including focal motor weakness, sensory dysfunction, and mental illness1.
The primary injury of cerebral contusion is induced by mechanical factors including stretching and tearing forces, leading to immediate parenchymal structure deformation and focal CNS cell death3. Hemorrhage contusion is a general term for brain hemorrhages due to vascular tear at the site of head trauma4. Specifically, intraparenchymal hemorrhage occurs immediately after a cerebral contusion leading to delayed hematoma formation. Within the hematoma, hemoglobin and free iron released from the lysed red blood cells can further trigger blood-related toxicity5,6 which cause herniation, brain edema, and intracranial pressure elevation5,6. The collaborative functions of neurons (axons), glia, blood vessels, and supportive tissue are also compromised by the mass effect of hematoma7. Additionally, persistent and diffuse neuroinflammation with progressive neurodegeneration continue for months and cause secondary damage in the brain8.
Microglia activation is one of many important pathological features of cerebral contusion9,10. After sensing the damage-associated molecular patterns (DAMPs) and leaked blood in the injured tissue, activated microglia trigger neuroinflammation which furthers secondary brain damage11. In addition, chemoattractant released from microglia promotes peripheral immune cell infiltration into the traumatic territory resulting in production of reactive oxygen species and pro-inflammatory cytokines. This creates a self-perpetuating pro-inflammatory environment which triggers progressive brain injury9,12. Meanwhile, microglia with an alternatively activated phenotype can contribute to tissue homeostatic restoration and brain repair through clearing debris from the injured tissue13. Prevention of secondary neuroinflammation by reducing detrimental microglial immune responses has been shown to be particularly useful for promoting brain recovery from cerebral contusion3,9,10,12.
Several preclinical models have been developed for studying traumatic brain injury including weight-drop model, lateral fluid percussion injury, and blast wave model14,15. However, these models each have their weakness including high mortality rate during the procedure, low reproducibility of histological results, and high variability of inflicted injury between laboratories16,17. In comparison, the controlled cortical impact (CCI) model is more adequate for studying focal cerebral contusion because of its precise control and high reproducibility14,15,18,19.
Furthermore, through manipulating the biomechanical deformation parameters such as velocity and depth of impact, the severity of the induced damage can be controlled to produce a wide range of injury magnitudes, allowing researchers to mimic different levels of impairment oftentimes seen in patients17. The preclinical model of CCI was first developed in 189620. Since then, CCI has been the broadest applicable model to be modified for the use in primates21, swine22, sheep23, rats24, and mice25. Together these features make CCI one of the most suitable experimental cerebral contusion models26.
Our laboratory uses a commercially available pneumatic CCI impact system and tested biomechanical deformation parameters to produce moderately severe focal cerebral contusion that territorializes the primary sensory and motor cortical areas without damaging the hippocampus27,28. We and others demonstrated that this CCI procedure can be used to study clinical features of human cerebral contusion including brain tissue loss, neuronal injury, intraparenchymal hemorrhage, neuroinflammation, and sensorimotor deficiency24,25,27,28,29,30. Here, we detail a standard protocol to perform mouse CCI which allows one to ask questions regarding CCI-induced myelin loss, iron deposition, CNS inflammation, hemorrhagic toxicity and the responses of microglia/macrophages in the aftermath of focal cerebral contusion.

<table><tbody><tr><td>4mm Short Trephine Drill</td><td>Salvin Dental Specialties, Inc.</td><td>TREPH-SHORT-4</td><td></td></tr><tr><td>anti-Iba1 antibody</td><td>Wako chemicals</td><td>#019-19741</td><td></td></tr><tr><td>anti-Ly76 antibody</td><td>abcam</td><td>ab91113</td><td></td></tr><tr><td>carboxylate cement</td><td>3M</td><td>70201136010</td><td></td></tr><tr><td>cortical contusion injury impactor</td><td>Custom Design &amp;amp; Fabrication, Inc.</td><td>S/N 49-2004-C, eCCI Model 6.3</td><td>CCI device (S/N 49-2004-C, eCCI Model 6.3)</td></tr><tr><td>cresyl violet acetate</td><td>Sigma-Aldrich</td><td>C5042</td><td></td></tr><tr><td>DAB staining kit</td><td>Vector</td><td>SK-4105</td><td></td></tr><tr><td>goat anti-rabbit IgG secondary antibody, Alexa Fluor 488</td><td>Invitrogen</td><td>A11034</td><td></td></tr><tr><td>goat anti-rat IgG secondary antibody, Alexa Fluor 594</td><td>Invitrogen</td><td>A11007</td><td></td></tr><tr><td>Mayer&#039;s Hematoxylin</td><td>ScyTek</td><td>HMM500</td><td></td></tr><tr><td>tweezers</td><td>fine science tools</td><td>11252-20 NO. 5</td><td></td></tr><tr><td>isoflurane</td><td>Panion &amp;amp; BF Biotech Inc.</td><td></td><td></td></tr><tr><td>Bupivacaine 0.25%</td><td>Hospira</td><td></td><td></td></tr><tr><td>lithium carbonate</td><td>Sigma-Aldrich</td><td>62470</td><td></td></tr><tr><td>steriotexic frame</td><td>stoelting</td><td></td><td></td></tr><tr><td>scissors</td><td>fine science tools</td><td>14068-12</td><td></td></tr><tr><td>solvent blue 38</td><td>Sigma-Aldrich</td><td>S3382</td><td></td></tr></tbody></table>

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.

Illustration of stereotactic placement and craniotomy procedure.
The CCI model is known for its stability and reproducibility in producing injury ranging from mild to severe18. Proper stereotactic technique and craniotomy procedure are major determinants in producing stable and reproducible CCI-induced brain injury (Figure 1A,B). An ideal craniotomy procedure would cause minimal histological injury in the sham-operated brain27,28 as shown (Figure 1C).
CCI induces long-term brain damage, blood-related toxicity, and neuroinflammation.
To evaluate brain damage, axonal loss, hemorrhagic pathology, and microglia/macrophages activation induced by CCI, we performed a battery of histological analysis including Nissl (neurons), luxol fast blue (myelin), Ly76 (erythrocytes), Iba1 (microglia/macrophages), and Perls (ferric iron) staining on injured brain slices from acute to chronic phases of CCI25,27,28,29,30,31,32. Changes in brain damage (Figure 2A) and corpus callosum (axon) loss (Figure 2B) were observed at days 1, 3, 7, and 28 after CCI. In addition, unilateral brain atrophy on the contusion side was seen at day 28 post-CCI (Figure 2). A successful CCI procedure produces focal injury between Bregma +1.42 mm and -1.34 mm at the coronal level27,30. Injury severity peaks around days 3 to 5 post-CCI the core of which centers approximately around Bregma +0.02 mm at the coronal level along with corpus callosum and striatal injuries25,28,31,32. Neuroinflammation was revealed by Iba1-positive activated microglia/macrophages accumulation around the border of the brain contusion area where the intraparenchymal hemorrhage was also detectable by the Ly76-positive staining from days 1 to 7 post-CCI (Figure 3B). A mixture of red blood cells (RBCs) with ruptured and intact cell morphology was observed at day 7 post-CCI (Figure 3B) suggesting the RBCs were lysed at this stage. This phenomenon is consistent with the observation that ferric iron deposition was detectable in the contusion region from days 7 to 28 post-CCI (Figure 3C). As CCI also induces corpus callosum and striatal injuries, activated microglia/macrophages are observed in the striatum far away from the contusion site (Figure 3D) from days 3 to 28 post-CCI. These observations indicate that the CCI method can reproduce the pathological features closely resembling human cerebral contusion including neuronal/axonal injury, intraparenchymal hemorrhage, and CNS inflammation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61393/61393fig01.jpg" /> 
Figure 1: Representative images of stereotactic and craniotomy procedures and histology of sham-operated brains. 
(A) The mouse head was levelly and stably placed on the stereotactic frame. Notice the readings on the horizontal and vertical scales of the ear bars are the same. (B) The bone flap was removed after craniotomy (left) and representative images of brain surface after proper (middle) and faulty (right) craniotomy are shown. (C) Representative images of cresyl violet (left) and luxol fast blue (right) stained sham-operated brain sections at day 3.&#160;Drapes are omitted for demonstration purpose to show the horizontal and vertical scales of ear bars in A and B.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61393/61393fig01large.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/61393/61393fig02.jpg" /> 
Figure 2: Brain tissue damage and myelin loss in CCI-induced cerebral contusion. 
(A) Representative images of cresyl violet-stained brain sections after CCI at days 1, 3, 7, and 28. The dotted line indicates the contusion area. Scale bar = 1 mm. (B) Representative luxol fast blue-stained CCI brain sections at days 1,3,7, and 28. The residual myelin at corpus callosum are outlined by dotted line. Scale bar = 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61393/61393fig02large.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/61393/61393fig03.jpg" /> 
Figure 3: CCI-induced intraparenchymal hemorrhage and neuroinflammation. 
(A) Representative cresyl violet stained CCI brain section shows boxed regions selected for Iba1/Ly76 (red), Perls (yellow), and Iba1 (green) imaging. (B) Representative immunostaining of activated microglia/macrophage marker Iba1 (green), RBC marker Ly76 (red), and nuclear marker DAPI (blue). The dotted line indicates the border of contusion region. Scale bar = 100 &#181;m in the full images and 50 &#181;m in the inset images. (C) Representative Perls staining show iron deposition in the contusion region at days 1, 3, 7, and 28 post-CCI. Scale bar = 100 &#181;m. (D) Representative images of activated microglia/macrophage marker Iba1 (green) expression at days 1, 3, 7, and 28 post-CCI. Scale bar = 100 &#181;m in the full images and 50 &#181;m in the inset images. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61393/61393fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

1.8K Views.  Cheng Hsin General Hospital. Intraparenchymale bloeding en neuro-inflammatie vergezeld van hersenkneuzing kunnen ernstig secundair hersenletsel veroorzaken. Dit protocol beschrijft een muisgecontroleerd corticaal impactmodel (CCI), waarmee onderzoekers bloedingen, kneuzingen en posttraumatische immuunresponsen kunnen bestuderen en mogelijke therapieën kunnen onderzoeken.

Video: Een preklinisch gecontroleerd corticaal impactmodel voor traumatische bloedingen, kneuzingen en neuro-inflammatie

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, ...

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

Persistent cognitive and motor symptoms are known consequences of concussions/mild traumatic brain injury (mTBIs) that can be partly attributable to altered neurotransmission. Indeed, cerebral microdialysis studies in rodents have demonstrated an excessive extracellular glutamate release in the hippocampus within the first 10 min following trauma. Microdialysis offers the clear advantage of in vivo neurotransmitter continuous sampling while not having to sacrifice the animal. In addition to the aforementioned technique, a closed head injury model that exerts rapid acceleration and deceleration of the head and torso is needed, as such a factor is not available in many other animal models. The Wayne State weight-drop model mimics this essential component of human craniocerebral trauma, allowing the induction of an impact on the head of an unrestrained rodent with a falling weight. Our novel and translational rat model combines cerebral microdialysis with the Wayne State weight-drop model to study, in lightly anesthetized and unrestrained adult rats, the acute changes in extracellular neurotransmitter levels following concussion. In this protocol, the microdialysis probe was inserted inside the hippocampus as region of interest, and was left inserted in the brain at impact. There is a high density of terminals and receptors in the hippocampus, making it a relevant region to document altered neurotransmission following concussion. When applied to adult Sprague-Dawley rats, our combined model induced increases in hippocampal extracellular glutamate concentrations within the first 10 min, consistent with the previously reported post-concussion symptomology. This combined weight-drop model provides a reliable tool for researchers to study early therapeutic responses to concussions in addition to repetitive brain injury, since this protocol induces a closed-head mild trauma.

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

Persistent cognitive and motor symptoms are known consequences of concussions/mild traumatic brain injury (mTBIs) that can be partly attributable to ...

Neuroscience

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.

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 ...

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 ...

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 ...

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

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of closed-head mTBI in the preclinical setting to increase translatability to the clinical population. The awake closed-headed injury (ACHI) model uses a modified controlled cortical impactor to deliver closed-headed injury, inducing clinically relevant behavioral deficits without the need for a craniotomy or the use of an anesthetic.
This technique does not normally induce fatalities, skull fractures, or brain bleeds, and is more consistent with being a mild injury. Indeed, the mild nature of the ACHI procedure makes it ideal for studies investigating repetitive mTBI (r-mTBI). Growing evidence indicates that r-mTBI can result in a cumulative injury that produces behavioral symptoms, neuropathological changes, and neurodegeneration. r-mTBI is common in youths playing sports, and these injuries occur during a period of robust synaptic reorganization and myelination, making the younger population particularly vulnerable to the long-term influences of r-mTBI.
Further, r-mTBI occurs in cases of intimate partner violence, a condition for which there are few objective screening measures. In these experiments, synaptic function was assessed in the hippocampus in juvenile rats that had experienced r-mTBI using the ACHI model. Following the injuries, a tissue slicer was utilized to make hippocampal slices to evaluate bidirectional synaptic plasticity in the hippocampus at either 1 or 7 days following the r-mTBI. Overall, the ACHI model provides researchers with an ecologically valid model to study changes in synaptic plasticity following mTBI and r-mTBI.

Here, it is demonstrated how an awake closed-head injury model can be used for examining the effects of repeated mild traumatic brain injury (r-mTBI) on synaptic plasticity in the hippocampus. The model replicates important features of r-mTBI in patients and is used in conjunction with in vitro electrophysiology.

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of ...

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 ...

Stab-Wound Mouse Model for Studying Hemorrhage and Inflammation in Traumatic Brain Injury

Traumatic brain injury (TBI) results from physical damage, often caused by accidents or sports-related incidents. The causes of TBI are diverse, including concussions, brain contusions, hematomas, and skull fractures. To replicate these different causes, various TBI mouse models have been developed using distinct protocols. Physical brain injury leads to both primary and secondary brain injuries, which exacerbate neuronal loss. Primary injury occurs immediately after the damage, often due to hemorrhage, and subsequently triggers secondary injuries, including inflammation around the lesion. Developing a TBI model suitable for assessing hemorrhage extension and inflammatory severity is therefore crucial. This protocol introduces a method for mimicking penetrating brain injury, referred to as the stab-wound TBI mouse model, to study mechanisms of hemorrhage, inflammation, and neuronal loss associated with TBI pathology. This model is created by puncturing the skull and brain with needles and is simple to execute without the need for specialized experimental equipment. Additionally, the minor injury inflicted on the mouse cerebral cortex using a needle does not affect the animal's behavior post-surgery. This feature allows researchers to study the localized effects of brain injury without concerns about broader behavioral consequences. Sample data from stab-wounded mouse cerebral cortices demonstrate the model's effectiveness in assessing blood leakage into the parenchyma, glial activation, and inflammatory cytokine production. Furthermore, this protocol facilitates the evaluation of blood coagulants and anti-inflammatory compounds, aiding in the development of therapeutic agents for TBI.

Simplified traumatic brain injury (TBI) models have facilitated the development of therapeutic approaches. This protocol outlines the creation of a stab-wound mouse cortex using needles, enabling the analysis of hemorrhage and inflammation. The stab-wound TBI mouse model offers the advantage of being performed without requiring specialized equipment.

Traumatic brain injury (TBI) results from physical damage, often caused by accidents or sports-related incidents. The causes of TBI are diverse, including ...

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

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