This work was supported by R01 NS067417 from the National Institute for Neurological Disorders and Stroke (MPB).

These studies were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes Health. The protocol was approved by the Institutional Animal Care and Use Committee at Georgetown University. Mice were housed in a temperature controlled animal facility and were kept on a 12 h light/12 h dark cycle. Food and water were available ad libitum.
1. Preparation of the mTBI Apparatus
NOTE: The mTBI apparatus includes a data acquisition (DAQ) box to control impact parameters, a high-velocity pneumatic impactor to perform the impaction, and a moulded gel filled base to decelerate head after impact.
<ol>
	<li>Turn on compressed air and initialize the high-velocity pneumatic impactor to a pressure setting of 861.85 kPa.</li>
	<li>Calibrate the DAQ control system to following parameters- piston velocity of 2.35 m/s, surface dwell time of 31.5 ms.</li>
	<li>Place the molded gel-filled base (compressibility 64 kPa/mm) such that the midline is perpendicular with the trajectory of impactor tip.</li>
</ol>
2. mTBI Impaction
<ol>
	<li>Record the weights of all mice to be used.</li>
	<li>Anesthetize mice with 3% isoflurane in oxygen for 120 s in an induction chamber.</li>
	<li>Transfer mice to mTBI apparatus, continuing anesthesia via a pliable, non-fixed nose cone.</li>
	<li>Whilst in nose cone, position mouse head on the gel pad so that the flat surface of the skull is presented perpendicular to the impactor tip.</li>
	<li>Place tape over mouse head to create a flat surface and hold ears away from the impact site.</li>
	<li>Lower the polytetrafluoroethylene impactor tip to align with the sagittal midline in the center of the head. The impactor tip used is 10 mm in diameter and will cover the area of the scalp from just behind the eyes to the midline of the ears. Adjust the impactor so it is just touching the surface of the mouse head.</li>
	<li>Retract the impactor tip and manually dial down the required deflection depth (7.5 mm).</li>
	<li>Arm the DAQ control system and depress trigger button to impact mouse head, either a singular or repetitive impacts. Repetitive impacts are delivered in succession with no delay between each impact.</li>
	<li>Immediately after the final impact, remove mouse from anesthesia and the mTBI apparatus and place in supine position.</li>
	<li>Using a stopwatch measure the latency of the return of righting reflex (supine to prone), to determine loss of consciousness time, as well as time to ambulation (unimpeded walking).</li>
	<li>Monitor mouse and upon recovery to normal behaviour, return to its home cage.</li>
	<li>Sham mice receive identical handling and anesthesia, but receive no impact.</li>
</ol>

<ol>
	<li>McMahon, P., 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=Symptomatology+and+functional+outcome+in+mild+traumatic+brain+injury:+results+from+the+prospective+TRACK-TBI+study.">Symptomatology and functional outcome in mild traumatic brain injury: results from the prospective TRACK-TBI study.</a> J Neurotrauma. 31 (1), 26-33 (2014).</li><li>Barkhoudarian, G., Hovda, D. A., Giza, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+pathophysiology+of+concussive+brain+injury.">The molecular pathophysiology of concussive brain injury.</a> Clin Sports Med. 30 (1), 33-48 (2011).</li><li>Blennow, K., Hardy, J., Zetterberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuropathology+and+neurobiology+of+traumatic+brain+injury.">The neuropathology and neurobiology of traumatic brain injury.</a> Neuron. 76 (5), 886-899 (2012).</li><li>Levin, H. S., Diaz-Arrastia, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis,+prognosis,+and+clinical+management+of+mild+traumatic+brain+injury.">Diagnosis, prognosis, and clinical management of mild traumatic brain injury.</a> Lancet Neurol. 14 (5), 506-517 (2015).</li><li>McCrory, P., 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=Consensus+statement+on+concussion+in+sport:+the+4th+International+Conference+on+Concussion+in+Sport,+Zurich,+November+2012.">Consensus statement on concussion in sport: the 4th International Conference on Concussion in Sport, Zurich, November 2012.</a> J Athl Train. 48 (4), 554-575 (2012).</li><li>Talavage, T. 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=Functionally-detected+cognitive+impairment+in+high+school+football+players+without+clinically-diagnosed+concussion.">Functionally-detected cognitive impairment in high school football players without clinically-diagnosed concussion.</a> J Neurotrauma. 31 (4), 327-338 (2014).</li><li>Angoa-Perez, 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=Animal+models+of+sports-related+head+injury:+bridging+the+gap+between+pre-clinical+research+and+clinical+reality.">Animal models of sports-related head injury: bridging the gap between pre-clinical research and clinical reality.</a> J Neurochem. 129 (6), 916-931 (2014).</li><li>Creed, J. 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=Concussive+brain+trauma+in+the+mouse+results+in+acute+cognitive+deficits+and+sustained+impairment+of+axonal+function.">Concussive brain trauma in the mouse results in acute cognitive deficits and sustained impairment of axonal function.</a> J Neurotrauma. 28 (4), 547-563 (2011).</li><li>Hamberger, 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=Concussion+in+professional+football:+morphology+of+brain+injuries+in+the+NFL+concussion+model--part+16.">Concussion in professional football: morphology of brain injuries in the NFL concussion model--part 16.</a> Neurosurgery. 64 (6), 1174-1182 (2009).</li><li>Kane, M. 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=A+mouse+model+of+human+repetitive+mild+traumatic+brain+injury.">A mouse model of human repetitive mild traumatic brain injury.</a> J Neurosci Methods. 203 (1), 41-49 (2012).</li><li>Prins, M. L., 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=Repeat+traumatic+brain+injury+in+the+juvenile+rat+is+associated+with+increased+axonal+injury+and+cognitive+impairments.">Repeat traumatic brain injury in the juvenile rat is associated with increased axonal injury and cognitive impairments.</a> Dev Neurosci. 32 (5-6), 510-518 (2010).</li><li>Tang, Y. P., 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+concussive-like+brain+injury+model+in+mice+(II):+selective+neuronal+loss+in+the+cortex+and+hippocampus.">A concussive-like brain injury model in mice (II): selective neuronal loss in the cortex and hippocampus.</a> J Neurotrauma. 14 (11), 863-873 (1997).</li><li>Winston, C. N., 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=Dendritic+Spine+Loss+and+Chronic+White+Matter+Inflammation+in+a+Mouse+Model+of+Highly+Repetitive+Head+Trauma.">Dendritic Spine Loss and Chronic White Matter Inflammation in a Mouse Model of Highly Repetitive Head Trauma.</a> Am J Pathol. 186 (3), 552-567 (2016).</li><li>Johnson, V. E., 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=Inflammation+and+white+matter+degeneration+persist+for+years+after+a+single+traumatic+brain+injury.">Inflammation and white matter degeneration persist for years after a single traumatic brain injury.</a> Brain. 136, 28-42 (2013).</li><li>Barrio, J. R., 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=In+vivo+characterization+of+chronic+traumatic+encephalopathy+using+[F-18]FDDNP+PET+brain+imaging.">In vivo characterization of chronic traumatic encephalopathy using [F-18]FDDNP PET brain imaging.</a> Proc Natl Acad Sci U S A. 112 (16), 2039-2047 (2015).</li></ol>

The authors have nothing to disclose.

In humans, mTBI is characterized by a functional impairment in the absence of structural injury. This can occur with, or without, a loss of consciousness1. Exposure to repeat concussions is currently thought to underlie the development and/or progression of neurodegenerative diseases such as CTE4. It is well documented that CTE is commonly found in boxers and football players and although exposure to repeat concussions (including those that do not result in a loss of consciousness) is known to be an essential element in developing CTE, we do not yet know why the mechanisms by which repeat concussion causes the distinct changes that occur in the brain.
A major hurdle to understanding these mechanisms is the difficulty in developing models that accurately recapitulate the key symptomologies observed in individuals that sustain an mTBI impact. This means the acute and chronic biological pathways underlying the concussive impacts are difficult to investigate and new treatments cannot be developed. In particular, existing animal models of repeat concussion are very severe, using only 2-3 repeat impacts that cause extensive neuronal cell loss, skull fracture and loss of brain tissue8,10,11,12. This extent of injury is not occurring after repeat concussion in humans, demonstrating the need for a new model of single and repetitive mild head impact.
Here we describe a novel model of mTBI, using a custom made diffuse injury device that has been specifically designed to transfer energy through the skull and brain and away from the mouse. Through both single impact and repetitive impact paradigms, the model allows for a means to begin investigating how concussion contributes neurological impairment later in life. The single hit paradigm recapitulates the hallmark characteristics of mild traumatic brain injury observed in humans, while the repetitive paradigm allows for the examination of how these seemingly mild injuries contribute to chronic and persistent degeneration over time. The device allows for repetitive mTBI head impacts without risk of skull fracture or structural brain damage.
As is the case with many techniques, certain aspects of this protocol are important to note for the generation of accurate, reliable results. During chamber induction and mTBI injury, it is essential to maintain a consistent level of anesthesia to each mouse. Due to the fact that righting and ambulation time is a key outcome measurement of this mTBI procedure, researchers should ensure that both mTBI injury and sham animals are exposed to comparable levels of anesthetic. Specifically, for the duration of the entire procedure, mice should only be under anesthesia for 3 min total (2 min in the induction chamber, 1 min with the mTBI nose cone device). This low level and duration of anesthesia is a major advantage over many other mTBI models, and yields consistent righting reflex response times (sham mice 20-40 s, mTBI mice 50-100 s). Additionally, when utilizing the repeat mTBI paradigm, it is important to maintain a record of mouse weights every day for the duration of the study. This is to ensure consistent monitoring for stress and general animal welfare throughout the entire time course of the mTBI procedures. This procedure does not result in mice becoming withdrawn or isolated. Normal grooming, eating and drinking should be observed within the first hour post procedure.
Post-injury there is no gross morphological pathology to the brain tissue resulting from either single or multiple impacts over a 1-week time course. Following a single mTBI, the only cellular response observed is a transient reduction in excitatory synapses; there is no inflammation, loss of grey or white matter, axonal injury or cell death. With 30 repeat mTBI (5 per day, over 6 days) there is chronic inflammation of the optic tract, at similar levels as previously quantitated1. Chronic white matter inflammation has been observed many years after a single mTBI14, and chronic inflammation can be detected in living athletes with a history of repeat mTBI15. The limitations of this model are that it cannot be used to study axonal injury outside of the optic tract or mechanisms of cell death, as these are absent in our model. Also based on our previously published data, this model does not induce changes in amyloid or tau pathology at 1-month post-mTBI in a mouse model of Alzheimer&#39;s disease13. We believe this lack of amyloid and tau pathology is related to the absence of axonal pathology in our model. Our model does provide a platform to investigate discrete changes in neuron networks, synaptic integrity and composition, and alterations in behavior following repeat concussive blows. Based on these outcomes, this novel model produces clinically relevant symptomology in a controlled, rigorous, and efficient manner. Further use of this model will allow for the investigation into the mechanisms that underlie the acute and chronic pathophysiology of mTBI and concussion.

Traumatic Brain Injury (TBI) is defined as a head injury sustained from an external physical force, resulting in the disruption of normal brain function. It represents a significant socio-economic and public health burden, with the Center for Disease Control and Prevention 2015 Report to Congress estimating that 2.5 million Americans sustain a TBI every year. This impacts not only the patient&#39;s quality of life, but also places an extremely high economic cost on the community, currently estimated at $76.5 billion annually. The amount of actual brain damage conveyed and the acute phase symptomologies is what defines mild, moderate and severe TBI.
Mild Traumatic Brain Injury (mTBI), also referred to as concussion, accounts for over 70% of TBIs reported each year1. It is most common among athletes who participate in high-risk contact sports including boxing and football2. Unlike moderate or severe forms of TBI, the immediate damage and symptoms associated with mTBI are sometimes not as pronounced3. By contrast, the long-term effects of mTBI can be just as debilitating as those seen in the moderate and severe forms. Those who suffer from repetitive mTBI have been shown to develop chronic traumatic encephalopathy (CTE), as well as other cognitive and degenerative illnesses4. Therefore, it is important to gain a greater understanding of the mechanisms that contribute to the short-term symptoms and overall long-term damage that arises following mTBI.
In humans, the definition of concussion as defined by the Fourth International Conference on Concussion in Sport (Zurich 2012)5 states that the level of injury for sports concussion is mild, does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Indeed, a recent study investigated the effect of mTBI on cognitive impairment in high school football players, using head impact telemetry systems. This study showed the amount of times players sustained helmet impacts &#62;20 g in a single season ranged from a low of 226 (average, 4.7 per session) to a high of 1855 (average, 38.6 per session)6. Most of these impacts did not result in the clinical diagnosis of a concussion; but evidence of functional changes in brain function could be observed using fMRI6. The brain changes that cause these functional changes are unknown, and therefore there is a pressing need to have a reliable and reproducible model to facilitate research into the effects of concussive and subconcussive mTBI.
Despite previous attempts to model mTBI in mice and rats7, many report adverse effects. In particular, most rodent models are limited in their repetitive nature using fewer than five mTBIs impacts, as well as having adverse pathological events including intracerebral bleeding, skull fractures, severe axonal injury, neuronal cell death, and increased mortality8,9,10,11,12. Herein, we describe a mouse model of mTBI that is closer to the true definition of concussion in humans. This model recapitulates many of the symptoms observed in human mTBI, such as the mechanical force that results in the transient loss of consciousness with no overt gross brain pathology. Furthermore, it is advantageous in the fact that it can be utilised for both single impact and repetitive impact paradigms over long time periods as reported previously13.

<table border="0" cellpadding="0" cellspacing="0" width="811">
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			<td height="21" style="height:21px;width:323px;">Powerlab 8SP data acquisition (DAQ) control box&#160;</td>
			<td style="width:203px;">(AD instruments)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:323px;">VIP 3000 calibrated vaporizer</td>
			<td style="width:203px;">Matrx</td>
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			<td height="21" style="height:21px;">Isoflurane</td>
			<td style="width:203px;">Henry Schein Animal Health</td>
			<td style="width:119px;">29405</td>
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			<td height="21" style="height:21px;width:323px;">Oxygen</td>
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			<td>Commercially available</td>
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			<td height="21" style="height:21px;width:323px;">Compressed Air</td>
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			<td>Commercially available</td>
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			<td height="21" style="height:21px;">Masking Tape</td>
			<td style="width:203px;"></td>
			<td style="width:119px;"></td>
			<td>Commercially available</td>
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			<td height="21" style="height:21px;">Stop Watch</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>02-261-840</td>
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			<td height="21" style="height:21px;">C57 Bl/6 Mice</td>
			<td style="width:203px;">Jackson Laboratories</td>
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			<td height="21" style="height:21px;width:323px;">Digital Scale and weigh container</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>20031</td>
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			<td height="21" style="height:21px;width:323px;">anti-Iba1 antibody</td>
			<td style="width:203px;">Wako</td>
			<td style="width:119px;">019-19741</td>
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			<td height="21" style="height:21px;width:323px;">HRP labelled secondary&#160;</td>
			<td style="width:203px;">Jackson Immunoresearch</td>
			<td style="width:119px;">111-035-003</td>
			<td></td>
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a mouse model of single and repetitive mild traumatic brain injury

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

The use of this novel mTBI device allows for single and repetitive mild head injuries without risk of skull fracture or structural brain damage. The model uses a custom made pneumatic Teflon impactor device to deliver a closed head mechanical energy impact. The impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI (Figure 1).
The presence or absence of LOC is a useful tool in grading concussion severity in animal models. The return of the righting reflex time is an acute neurological evaluation of injury severity that we used to quantify the LOC after a single and repeat mTBIs (7.5 mm depth, Figure 2). During the procedure mice receive a total of 3 min of isoflurane in oxygen, and thus all mice including shams have a LOC period after anaesthesia withdrawal. Single mTBI results in significantly increased LOC compared to sham mice (36.4 &#177; 1.6 s v 64.2 &#177; 7.7 s, n = 5, **p &#60;0.01, Figure 2A). This also correlated with increased ambulation times following a single mTBI (52.0 &#177; 4.5 s v 140.0 &#177; 21.1 s, n = 5, **p &#60;0.01, Figure 2B). In repeat injury paradigms (a total of 30 impacts, 5 impacts per day for 6 days), there was significantly elevated LOC and ambulation times on all testing days (repeated measures two-way analysis of variance injury effect F1,14= 22.92, p &#60;0.0003). During the entire study, the average LOC over the 6 days was Sham: 35.5 &#177; 1.4 s v mTBI: 64.9 &#177; 1.7 s, n = 8, p &#60;0.01, Figure 2C, and average ambulation times Sham: 64.3 &#177; 3.3 s v mTBI: 160.8 &#177; 5.3 s, n = 8, p &#60;0.01, Figure 2D. Iba1 staining for microglia/macrophages revealed no change between sham and single mTBI mice, but extensive Iba1 immunoreactivity in the optic tract in repeat mTBI mice (Figure 3). Repeat mTBI mice showed no evidence of grey matter inflammation in the cortex (Figure 3) or other brain areas.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55713/55713fig1.jpg" /> 
Figure 1: Illustrative Representation of the Mouse Model of mTBI. (A) The setup of all materials required to perform mTBI. (B) Mouse placed in isoflurane induction chamber for 2 min. (C) On the mTBI apparatus, mouse placed in a non-fixed nose cone to continue anesthesia. (D) Gently tape mouse head to create a flat surface and hold ears back. Impactor tip lowered to just touch the surface of the head. (E) Impactor tip retracted and required deflection depth is lowered using the dial (inset). mTBI performed by depressing trigger button. (F) Loss of consciousness measured by the time taken for the return of righting reflex (supine to prone). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55713/55713fig1large.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/55713/55713fig2.jpg" /> 
Figure 2: Single and Repeat mTBIs Increases Transient Loss of Consciousness and Return to Ambulation Times. Mice that receive a single mTBI have significantly increased (A) loss of consciousness (LOC) and (B) ambulation times compared to sham control mice (n = 5 per group, **p &#60;0.01). (C-D) Repetitive mTBI (5 impacts per day for 6 days) significantly increases daily LOC and ambulation times compared to sham mice (n = 8 per group, *p &#60;0.05, **p &#60;0.01, ***p &#60;0.001). Data expressed as mean &#177; S.E.M, analyzed by Two-way repeated measures ANOVA with a Bonferroni post-hoc test. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55713/55713fig2large.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/55713/55713fig3.jpg" /> 
Figure 3: Repeat mTBI Induces Microglia/Macrophages Activation in the Optic Tract. (A) Iba1 staining in sham mice reveals quiescent microglia/macrophages in the cortex (a1 - a3) and limited staining in the optic tract (a4-a6). (B) Mice that receive a single mTBI (7.5 mm) have similar Iba1 staining profiles as sham mice in both the cortex (b1-b3) and optic tract (b4-b6), 24 h post-impact. (C) Repeat mTBI mice (5 impacts per day for 6 consecutive days) display a strong inflammatory response in the optic tract (c4-c6) but not in the cortex (c1-c3), 24 h after the last impact (Day 7). Enlarged images demonstrating the morphology of Iba-1 positive microglia/macrophages in the cortex (a3, b3, c3) and optic tract (a6, b6, c6) of sham, single and repetitive mTBI mice, respectively. Scale bars = 200 &#181;m (A, B, C); 50 &#181;m (a1-a5, b1-b5, c1-c5); 20 &#181;m (a3, a6, b3, b6, c3, c6, magnified panels). Images representative of n = 6 per group. Cortex (cx), optic tract (opt), mild traumatic brain injury (mTBI). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55713/55713fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury at JoVE.com

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

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

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Nanyang Technological University

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11.0K Views.  Georgetown University Medical Center. Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

Video: A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

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, ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor vehicle accidents, and falls, a reliable animal model of mTBI had previously not been developed for this important aspect of development. The modified weight-drop technique employs a glancing impact to the head of a freely moving rodent transmitting acceleration, deceleration, and rotational forces upon the brain. When applied to juvenile rats, this modified weight-drop technique induced clinically relevant behavioural outcomes that were representative of post-concussion symptomology. The technique is a rapidly applied procedure with an extremely low mortality rate, rendering it ideal for high-throughput studies of therapeutics. In addition, because the procedure involves a mild injury to a closed head, it can easily be used for studies of repetitive brain injury. Owing to the simplistic nature of this technique, and the clinically relevant biomechanics of the injury pathophysiology, the modified weight-drop technique provides researchers with a reliable model of mTBI that can be used in a wide variety of behavioural, molecular, and genetic studies.

The modified weight-drop technique is an easy, cost-effective procedure used for the induction of mild traumatic brain injury in juvenile rats. This novel technique produces clinically relevant symptomology that will advance the study of mild traumatic brain injury (mTBI) and concussion.

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

A Repetitive Concussive Head Injury Model in Mice

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

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

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

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...