The authors would like to thank Irene Ma, Rose Tobais, and Jong Rho for their technical assistance. Funding was provided to MJE by the Department of Pediatrics at the University of Calgary, the Alberta Children&#8217;s Hospital Foundation (ACHF) and the Alberta Children&#8217;s Hospital Research Institute (ACHRI). The Postdoctoral fellowship for RM was provided by ACHF.

NOTE: All experiments were carried out in accordance with the Canadian Council of Animal Care and were approved by the University of Calgary, Animal Care Ethics Committee.
1. Breeding and Animal Preparation
<ol>
	<li>Order pregnant rats from standard laboratory animal suppliers or breed pups in-house according to standard breeding protocols.</li>
	<li>House all animals with ad libitum access to food and water, in a temperature controlled rat housing room (21 &#176;C), that is maintained on a 12:12 hr&#160;light:dark cycle.</li>
	<li>When pups reach postnatal day 21 (P21), wean pups from their mothers and house in same-sex groups of 3 or 4.</li>
</ol>
2. Set-up of Mild Traumatic Brain Injury (mTBI) Apparatus
<ol>
	<li>Prior to commencement of procedure, mill weights to the desired mass (e.g. 150 g). Securely attach a metal loop to the top end of the weight allowing the fishing line to be fixed to the weight.</li>
	<li>Score tin foil with a sharp razor blade. Ensure that the scored tin foil supports the body weight of the rat but will not interfere with acceleration of the rat following head impact with the weight.</li>
	<li>Tape the scored tin foil to the U-shaped stage made of clear plastic (38 x 27 x 27 cm3) that contains a collection sponge (38 x 25 x 15 cm3) (Figure 1), so that it is taut.</li>
	<li>Place the U-shaped plastic stage in the correct position underneath the guide tube made of clear plastic.</li>
	<li>Hold the plastic guide tube (2.2 cm diameter x 1.5 m) in place with a clamp stand and position the guide tube so that it is 3.5 cm above the scored tin foil.</li>
	<li>Attach the fishing line through the metal loop to the weight ensuring that the bottom of the weight freely hangs 2.5 cm above the scored tin foil.</li>
	<li>Securely attach the fishing line to the clamp stand. 
	NOTE: Tethering the weight to the clamp stand at a height of 2.5 cm above the tin foil prevents re-hits while the rat is falling from the impact to the collection sponge).</li>
	<li>Pull the weight up through the plastic guide tube with the fishing line and hold it in place with an allen key pin at 1.0 m.</li>
</ol>
3. Induction of mTBI
<ol>
	<li>When rats reach P30 move cages into the procedural room.</li>
	<li>Place rats in an isoflurane chamber and lightly anesthetize the rat until it is non-responsive to a paw or tail pinch.</li>
	<li>Quickly place the rat chest-down on the scored tin foil with its head directly in the path of the falling weight. See Figure 2.&#160;If the rat begins to move or wake-up before it can be placed on the scored tin foil, return to the isoflurane chamber until non-responsive to a paw or tail pinch and begin again.</li>
	<li>Pull the Allen key pin, allowing the weight to fall vertically through the plastic guide tube and strike the rat on the head. The rat will rapidly undergo an 180&#176; rotation and land in the supine position.</li>
	<li>Immediately remove the rat from the collection sponge and apply topical lidocaine to the rat&#8217;s head with a cotton tipped applicator.</li>
	<li>Place the rat in the supine position in a clean cage that is heated by setting it upon a commercially available heating pad.</li>
	<li>Using a stopwatch or digital timer acquire the &#8220;time-to-right&#8221;. Time-to-right is the time taken by the rat to wake from the anesthetic and flip from the supine position to the prone position or begin walking.</li>
	<li>Return the rat to its home-cage after it has recovered normal behavior (grooming, walking, exploring, etc.).</li>
	<li>Repeat steps 3.2&#8211;3.8 for each additional rat requiring a mTBI.</li>
	<li>Repeat the procedure on the same rat at multiple time points for repetitive mTBI experiments.</li>
</ol>
4. Induction of the Sham Injury
<ol>
	<li>Lightly anesthetize the rat with isoflurane until it is non-responsive to a paw or tail pinch.</li>
	<li>Quickly place the rat chest-down on the scored tin foil with its head directly in the path of the falling weight.</li>
	<li>Remove the rat from the tin foil without pulling the allen key pin from the plastic guide tube and apply topical lidocaine to the head of the rat with a cotton tipped applicator.</li>
	<li>Place the rat in the supine position in a clean cage that is heated by setting it upon a commercially available heating pad.</li>
	<li>Use a stopwatch or digital timer to acquire the &#8220;time-to-right&#8221;.</li>
	<li>Return the rat to its home-cage after it has recovered normal behavior (grooming, walking, exploring, etc.).</li>
</ol>
5. Verification of mTBI with the Beam Walking Test 8
<ol>
	<li>24 hr after the induction of the mTBI or sham injury return animals to the procedural room.</li>
	<li>Transfer the rats to a clean holding cage and place the rat&#8217;s home-cage at the narrower end of the 165 cm tapered beam such that the open-ended portion of the home-cage faces the narrower end of the beam. 
	NOTE: The tapered beam is 165 cm long. The center platform of the beam has a width of 6 cm at the wide end and 1.75 cm at the narrow end. The center beam has ledges (2 cm wide and 2 cm below the center beam) that provide safety when the rat&#8217;s foot slips. Place foam padding below the beam to reduce risk of injury to rats that may fall off the beam during the test.</li>
	<li>Place a video camera at the wide end of the tapered beam and position/zoom the video camera to ensure that the investigator watching the video can clearly see the movements of the rat for the entire length of the tapered beam.</li>
	<li>Place the rat at the wide end of the tapered beam and encourage it to walk across the beam to its home-cage. Once the rat crosses the tapered beam, leave the rat in its home-cage for at least 60 sec to reinforce the target location. This is Trial #1 and not included in the analysis.</li>
	<li>Turn the video camera on and allow the rat to complete the beam-walking task 4 additional times (maintaining the 60 sec reinforcement periods in the home cage). Once the rat has completed all trials, return the rat to its home-cage.</li>
	<li>To score, record the number of hind leg foot-slips that occur and the time/duration to cross the beam, for each individual trial. Use this data to compute the average number of hind leg foot-slips and time to cross the beam for each rat.</li>
</ol>

<ol><li>DeWitt, D., Perez-Polo, R., Hulsebosch, C., Dash, P., Robertson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Challenges+in+the+development+of+rodent+models+of+mild+traumatic+brain+injury%5BTitle%5D)+AND+%22Journal+of+Neurotrauma%22%5BJournal%5D)">Challenges in the development of rodent models of mild traumatic brain injury.</a> Journal of Neurotrauma. 30, 688-701 (2013).</li>
<li>Xiong, Y., Mahmood, A., Chopp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+traumatic+brain+injury%5BTitle%5D)+AND+%22Nature+Reviews+Neuroscience%22%5BJournal%5D)">Animal models of traumatic brain injury.</a> Nature Reviews Neuroscience. 14, 128-142 (2013).</li>
<li>Zhang, Z., Lerner, S., Kobiessy, F., Hayes, R., Wang, K. Systems biology and the anostic approach to drug discovery and development to treat traumatic brain injury. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aZhang%2C+Z+%22Systems+Biology+in+Drug+Discovery+and+Development%3A+Methods+and+Protocols%2C+Methods+in+Molecular+Biology%22">Systems Biology in Drug Discovery and Development: Methods and Protocols, Methods in Molecular Biology</a>. Yan, Q. , Springer Science. Forthcoming.</li>
<li>Viano, D., Casson, I., Pellmen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Concussion+in+profession+football%3A+Biomechanics+of+the+struck+player+-+Part+14%5BTitle%5D)+AND+%22Neurosurgery%22%5BJournal%5D)">Concussion in profession football: Biomechanics of the struck player - Part 14.</a> Neurosurgery. 61, 313-327 (2007).</li>
<li>Marmarou, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+model+of+diffuse+brain+injury+in+rats%3A+Part+1+-+Pathophysiology+and+biomechanics%5BTitle%5D)+AND+%22Journal+of+Neurosurgery%22%5BJournal%5D)">A new model of diffuse brain injury in rats: Part 1 - Pathophysiology and biomechanics.</a> Journal of Neurosurgery. 80, 291-300 (1994).</li>
<li>Mychasiuk, R., Farran, A., Esser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+an+experimental+rodent+model+of+pediatric+mild+traumatic+brain+injury%5BTitle%5D)+AND+%22Journal+of+Neurotrauma%22%5BJournal%5D)">Assessment of an experimental rodent model of pediatric mild traumatic brain injury.</a> Journal of Neurotrauma. 31 (8), (2014).</li>
<li>Kane, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mouse+model+of+human+repetitive+mild+traumatic+brain+injury%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">A mouse model of human repetitive mild traumatic brain injury.</a> Journal of Neuroscience Methods. 203, 41-49 (2012).</li>
<li>Schallert, T., Woodlee, M., Fleming, S. Disentangling multiple types of recovery from brain injury. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSchallert%2C+T+%22Pharmacology+of+Cerebral+Ischemia%22">Pharmacology of Cerebral Ischemia</a>. Krieglstein, J., Klumpp, S. , Medpharm Scientific Publishers. Stuttgart. (2002).</li>
<li>Mychasiuk, R., Hehar, H., Farran, A., Esser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mean+Girls%3A+Sex+differences+in+the+effects+of+mild+traumatic+brain+injury+on+the+social+dynamics+of+juvenile+rat+play+behaviour%5BTitle%5D)+AND+%22Behavioural+Brain+Research%22%5BJournal%5D)">Mean Girls: Sex differences in the effects of mild traumatic brain injury on the social dynamics of juvenile rat play behaviour.</a> Behavioural Brain Research. 259, (2014).</li>
<li>Centers for Disease Control and Prevention. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Report+to+congress+on+mild+traumatic+brain+injury+in+the+United+States%3A+Steps+to+prevent+a+serious+public+health+problem%22">Report to congress on mild traumatic brain injury in the United States: Steps to prevent a serious public health problem</a>. , Centers for Disease Control and Prevention. Atlanta, GA. 1-47 (2003).</li>
</ol>

No competing financial interests exist.

Reliable modelling systems are needed to effectively cultivate basic science research that has significant translational validity. In response to rising occurrences and popular media, the investigation of mTBI and concussion has become a priority in many disciplines. However, despite increased research, there have been only incremental improvements in therapeutic strategies and treatment options 3. This lack of progress may be partially due to a discrepancy between the modeling systems employed and actual injury etiology. The majority of studies utilized rodent models that failed to reproduce the important biomechanical forces and appropriate post-injury symptomology. The current human definition of mTBI specifies that the injury results from acceleration and deceleration forces associated with a blunt trauma 10. The modified weight drop technique described here is therefore an ideal model for the study of mTBI and concussion because it uses a glancing impact to cause rapid rotational acceleration and deceleration to the head of an unrestrained animal, mimicking the biomechanical forces identified in sports-related injuries and automobile accidents. In addition, this model is easily adapted to examine repetitive mTBI, a phenomena that is emerging as a serious medical and socioeconomic issue. Studies indicate that rodents may be exposed to a series of 10 distinct mTBIs with minimal mortality 7. Finally, the method is inexpensive and can be carried out rapidly, allowing for high-throughput examination of a many therapeutic compounds and treatment regiments. 
Just as with any procedural technique, certain aspects of the protocol are particularly important to the generation of reliable results. First, the tin foil needs to be scored effectively. If the tin foil is not properly scored, the force imparted by the weight during the glancing impact will not be enough to propel the juvenile rat through the tin foil onto the collection sponge. In these situations, the rat will remain in the starting position (chest down on the tin foil) and the mTBI will result from the blunt trauma from the weight impacting the stationary head, not the rotational acceleration and deceleration desired. Second, during the induction of the mTBI and the sham injury, the level of anesthetic applied to each rat should be consistent. Owing to the fact that time-to-right is used as marker of mTBI, the researcher should try to ensure that animals receiving a mTBI and animals receiving a sham injury are exposed to similar levels of anesthetic. A major advantage to this technique over many other TBI procedures is the low level and duration of anesthesiology. However, the juvenile rat needs to be non-responsive to a toe or tail pinch to ensure they do not wake-up on the stage before the injury is induced. Finally, in order to maintain a consistent injury etiology, the positioning of the rat&#8217;s head is particularly important. Ideally the weight should impact the center of the dorsal surface of the head. Caution should be taken to avoid positioning the path of the weight too near the caudal/posterior portion of the head, as impacting the brainstem and cerebellum is associated with increased mortality and seizure activity. 
Based upon the biomechanical pathophysiology of injury induction and the behavioural outcomes examined, the modified weight-drop technique appears to be a reliable model for the investigation of paediatric mTBI and concussion. Although preliminary studies of this novel model have assessed some basic molecular and structural changes 7 future studies will be needed to ascertain how the brain responds to a mTBI with this injury etiology. An in-depth analysis of the neuroanatomical and biological changes that occur at the cellular and epigenetic level would increase model validity and translational applicability. In addition to stimulating the generation of targeted pharmacological therapies, understanding the pathophysiological changes that occur in the brain in response to mTBI and concussion would also direct the research related to clinical biomarkers that have the ability to predict outcomes following injury.

Although there are many widely used methods for the generation of moderate-to-severe traumatic brain injury (TBI), very few techniques have been developed to induce mild, closed head injuries in rodents. Owing to the fact that mild traumatic brain injury (mTBI) is three times more common than moderate and severe brain injury combined 1, a reliable model of mTBI is needed to facilitate research regarding pathophysiology, neurobiological and behavioural outcomes, and therapeutic strategies. For example, partially due to the limitations of current animal models 2, in the past decade there have been over 200 failed clinical drug trials for the treatment of TBI 3. When modelling systems are generated for translational research studies, the applicability of the findings are dependent upon the validity of the model implemented. For the study of mTBI/concussion, a reliable animal model would not only mimic the biomechanical forces responsible for injury etiology, but would also induce symptoms consistent with those reported by the clinically relevant population. Furthermore, because children are at particularly high risk for mTBI, optimal modelling systems would be applicable to young and juvenile rodents, in addition to their adult equivalents.
Biomechanical analyses of circumstances in which athletes have sustained mTBIs or concussive brain injuries indicate that the most critical predictive factors for injury are rapid head acceleration and high velocity impacts 4. The majority of rodent models currently employed for the induction of TBI permit little or no movement of the head 5 (for review see 2). The model outlined here, delivers a high-velocity impact to the head of a physically&#160;unrestrained juvenile rat that is accompanied by an 180&#176; rotation and free-fall that applies acceleration/deceleration forces to the subject&#8217;s head and body. There are two main advantages associated with this modified weight drop technique for the induction of mTBI. First, the model produces clinically relevant concussive like symptomology without causing any overt damage to the brain (for complete description of behavioural outcomes see 6). Also consistent with clinical reports of post-concussive syndrome, this modified weight-drop technique produces heterogeneous outcomes. Although the effects from the mTBI are significant, there is substantial variation between rodents that experienced a mTBI when examined on multiple outcome measures. Second, the method permits the study of repetitive mTBI 7. As the majority of existing TBI models inflict such severe injuries, it is often difficult to induce a second injury, and nearly impossible to study repetitive TBI without extensive damage to the entire cortex.
Therefore, the primary rationale for using the modified weight drop technique for the induction of mTBI is to produce an injury that more closely represents the pathophysiology and symptomology of concussion and repetitive TBI in juvenile populations. With the increasing incidence of mTBI related to sports, falls, and automobile accidents, especially during childhood, this unique rodent model of mTBI provides researchers with a valuable tool for the study of concussive-like brain injury that can be easily applied to multiple-hit paradigms.

<table><tbody><tr><td>Brass Weights</td><td>Ginsberg Scientific</td><td>7-2500-2</td><td>Need to have metal loop attached to base</td></tr><tr><td>Alluminum Foil</td><td>Alcan</td><td></td><td>Available at most grocery stores</td></tr><tr><td>Masking Tape</td><td></td><td></td><td>Commercially available&amp;nbsp;</td></tr><tr><td>U-Shaped Plastic Stand</td><td></td><td></td><td>Constructed by Laboratory</td></tr><tr><td>Clamp Stand</td><td>Sigma-Aldrich</td><td>Z190357</td><td></td></tr><tr><td>Plastic Guide Tube</td><td></td><td></td><td>Could be constructed or purchased at a hardware store</td></tr><tr><td>Fishing Line</td><td>Angler 10lb&amp;nbsp;</td><td></td><td>Purchased from a sporting goods retailer&amp;nbsp;</td></tr><tr><td>Isoflurane</td><td>Pharmaceutical Partners of Canada</td><td>DIN 02237518</td><td>Inhalation Anesthetic</td></tr><tr><td>Topical Lidocaine (30ml)</td><td>Astra Zeneca</td><td>DIN 0001694</td><td>Xylocaine Jelly 2%&amp;nbsp;</td></tr><tr><td>Cotton Swabs</td><td></td><td></td><td>Commercially available&amp;nbsp;</td></tr><tr><td>Heating Pad - 3 heat setting</td><td></td><td></td><td>Commercially available&amp;nbsp;</td></tr><tr><td>Stop Watch</td><td>Sportline</td><td>L303</td><td>Purchased from a sporting goods retailer&amp;nbsp;</td></tr><tr><td>Video Camera</td><td>Sony</td><td>HDR-CX260V</td><td></td></tr><tr><td>Sprague Dawley Rats</td><td>Charles River Laboratories</td><td>SAS SD 40</td><td>Male and females ordered from Charles River Laboratories and pups bred in-house</td></tr><tr><td>Balance Beam</td><td></td><td></td><td>Constructed by Laboratory</td></tr></tbody></table>

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 described above is a reliable method for the induction of mild traumatic brain injury (mTBI) in juvenile rats. Utilizing an impact weight of 150 g, this technique has been successfully applied to juvenile rats that range from 50&#8211;120 g. In addition, the procedure can be easily repeated in the same animals for the study of repetitive mTBI. Although animals that experience a single mTBI exhibit an increase in the time-to-right (Figure 3) and appear stunned upon waking, they rapidly resume normal activities and are visually indistinguishable from sham-injured animals. Given that the injury is mild, topical lidocaine which eliminates any pain associated with the glancing impact, is the only analgesic required. This is important for research as pain medications are known to interfere with typical inflammatory and recovery processes. Owing to the lack of overt symptomology, the beam walking task is a reliable tool that can be used to validate the induction of the mTBI. It is important to note, that not all animals that experience a mTBI will exhibit deficits on the beam walking task, but as a group, juvenile rats with a mTBI demonstrate significantly more hind leg foot-slips when compared to juvenile rats with a sham injury (Figure 4).
Another key characteristic of this modified weight-drop technique is the lack of restraint applied to the juvenile rat during injury induction. By delivering a glancing blow to the head followed by rapid rotational acceleration and deceleration, this model more closely represents the biomechanical forces attributed to mTBI and concussion. When this procedure is applied to juvenile rats or adult mice, mortality rates are extremely low (7/202 juvenile animals ~3.4% mortality rate), and skull fracture and intracranial bleeding are exceptionally rare 6,7. Additionally, the model produces clinically relevant symptomology. Juvenile rodents that experienced a single mTBI demonstrated deficits in balance and motor behaviours, along with deficits in executive function, increased depressive-like behaviours, and altered social interactions 6,9. Similarly, adult mice also display mild balance and coordination deficits that recover with time 7. Finally, induction of mTBI using this model requires minimal anesthetic and does not involve surgical preparation or burrowing into the skull. Results are therefore not biased by confounding inflammatory or immunological effects triggered by the surgery or anesthetic. Furthermore, the rapid recovery time and lack of open wounds enables the commencement of additional testing paradigms to occur shortly after rodents experience the mTBI.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51820/51820fig1highres.jpg" width="500" /> 
Figure 1: Cartoon representation of the U-shaped plastic stage and collection sponge with all pertinent dimensions. A distance of 10 cm must be maintained between the collection sponge and the top of the plastic stage to ensure the juvenile rat has enough time to complete the 180&#176; rotation.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51820/51820fig2highres.jpg" width="500" /> 
Figure 2: (A) Photographic representation of the injury induction platform. The juvenile rat is placed chest-down on the scored tin foil so that the head is directly below the falling weight. (B) Side view of the injury induction platform. (C) Photographic demonstration of the weight used in the induction of the mTBI.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51820/51820fig3highres.jpg" width="500" /> 
Figure 3: Graphical representation of the average differences in time-to-right between juvenile rats that experienced a single mTBI and juvenile rats that experienced a sham injury (* p &#60;0.01). Rats that have received a mTBI exhibit a significant increase in the duration of time needed to right themselves from the supine position.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51820/51820fig4highres.jpg" width="500" /> 
Figure 4: Graphical representation of the average number of hind leg foot-slips exhibited on the Beam Walking task by juvenile rats that experienced a single mTBI and juvenile rats that experienced a sham injury (* p &#60;0.05).

Watch this Scientific Journal Video about A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats at JoVE.com

A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats

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-novel-model-of-mild-traumatic-brain-injury-for-juvenile-rats

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23.6K Views.  University of Calgary. 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. 

Video: A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, few studies have investigated the long-term effects of injury in rodent models of TBI. Shown here are behavioral measures that are well-established in TBI research for times early after injury, such as two weeks, until two months. Some of these methods have previously been used at later times after injury, up to one year, but by very few laboratories. The methods demonstrated here are a short neurological assessment to test reflexes, a Beam-Balance to test balance, a Beam-Walk to test balance and motor coordination, and a working memory version of the Morris water maze that can be sensitive to deficits in reference memory. Male rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid percussion injury (FPI) or sham injury. Rats can be tested on the short neurological assessment (neuroscore), the beam-balance, and the Beam-Walk multiple times, while testing on the water maze can only be done once. This difference is because rats can remember the task, thus confounding the results if repeated testing is attempted in the same animal. When testing from one to three days after injury, significant differences are detected in all three non-cognitive tasks. However, differences in the Beam-Walk task were not detectable at later time points (after 3 months). Deficits were detected at 3 months in the Beam-Balance and at 6 months in the neuroscore. Deficits in working memory were detected out to 12 months after injury, and a deficit in a reference memory first appeared at 12 months. Thus, standard behavioral tests can be useful measures of persistent behavioral deficits after FPI.

The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, ...

Behavior

Assessing Dominant-Submissive Behavior in Adult Rats Following Traumatic Brain Injury

Competition over resources such as food, territory, and mates significantly influences relationships within animal species and is mediated through social hierarchies that are often based on dominant-submissive relationships. The dominant-submissive relationship is a normal behavioral pattern among the individuals of a species. Traumatic brain injury is a frequent cause of social interaction impairment and the reorganization of dominant-submissive relationships in animal pairs. This protocol describes submissive behavior in adult male Sprague-Dawley rats after the induction of traumatic brain injury using a fluid-percussion model compared to naive rats through a series of dominant-submissive tests performed between 29 days and 33 days after induction. The dominant-submissive behavior test shows how brain injury can induce submissive behavior in animals competing for food. After traumatic brain injury, the rodents were more submissive, as indicated by them spending less time at the feeder and being less likely to arrive first at the trough compared to the control animals. According to this protocol, submissive behavior develops after traumatic brain injury in adult male rats.

The present protocol describes a rat model of fluid percussion-induced traumatic brain injury followed by a series of behavioral tests to understand the development of dominant and submissive behavior. Using this model of traumatic brain injury in conjunction with specific behavioral tests enables the study of social impairments following brain injury.

Competition over resources such as food, territory, and mates significantly influences relationships within animal species and is mediated through social ...

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

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

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

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

A 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

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 ...

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 ...

Validating Injury Response and Behavioral Assessments in an mTBI Rat Model

Rat Model of Closed-Head Mild Traumatic Injury and its Validation

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful insights, developing a stable and reproducible animal model is essential. In this study, we report a detailed description of a closed-head mTBI model and a representative validation method using Sprague-Dawley rats to verify the modeling effect. The model involves dropping a 550 g mass weight from a height of 100 cm directly onto the head of a rat on a destructible surface, followed by a 180-degree turn. To assess the injury, rats underwent a series of neurobehavioral assessments 10 min post-injury, including time of loss of consciousness, first seeking-behavior time, escape ability, and beam balance ability test. During the acute and subacute stages following the injury, behavioral tests were conducted to assess motor coordination ability (Beam task), anxiety (Open Field test), and learning and memory abilities (Morris Water Maze test). The closed-head mTBI model produced a consistent injury response with minimal mortality and replicated real-life situations. The validation method effectively verified the model development and ensured the stability and consistency of the model.

Author Spotlight: Advancing Traumatic Brain Injury Research - A Closed-Head Model for Accurate Replication and Rapid Assessment 

Here, we present a closed-head mild traumatic brain injury (mTBI) rat model and its validation exhibiting remarkable similarity to human mTBI concerning behavioral manifestations during the acute and subacute stages.

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful ...

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 ...

CHILD Model &#8212; Recapitulating Juvenile Concussion and Long-Term Outcomes in Mice

A Pediatric Concussion Model in Mice: Closed Head Injury with Long-Term Disorders (CHILD)

Concussions and mild traumatic brain injuries (mTBI) during childhood represent a significant health endangerment, with many patients later in life exhibiting debilitating physiological, neurological, and psychosocial outcomes. The cellular and molecular pathophysiological mechanisms are relatively unknown, and this significant gap effectively precludes investigations into specific therapeutic strategies to mitigate chronic sequelae. No single animal concussion model recapitulates all the features reported in human subjects, and current models are designed to answer specific research questions. We set out to develop a juvenile concussion that recapitulates clinical early, and long-term symptomology encountered, termed Closed Head Injury with Long-term Disorder (CHILD). In this model, a concussive force via an electromagnetic impactor is delivered to the head of a lightly anesthetized unrestrained post-natal day 17 (P17) mouse, allowing free rotation of the head. Mice are placed on a tightly stretched tin foil across a stereotactic device, and the impactor is carefully aligned with the targeted cortical region. The electromagnetic impactor facilitates the selection of impact depth, dwell, and duration to determine injury severity. A strength of this model is that it allows head rotation, an important clinical feature. After impact, mice are immediately monitored for righting time and time to explore their environment, followed by their return to their dam. Increasing the severity of concussion results in intracranial bleeds in a subset of mice. Mice can be routinely monitored for behavior and neuroimaging over their lifespan, as desired. Various injury severities mimic the heterogeneous nature of juvenile concussions. The current standard CHILD model exhibits no skull fracture, no observable conventional neuroimaging change (similar to clinical), but leads to progressive and persistent neuronal death, altered diffusion MRI, modified neuronal activity and plasticity, increased gliosis, and progressive behavioral perturbations with age. In summary, this CHILD model mimics the early and long-term features observed in many clinical concussion patients.

Pediatric concussions can result in long-term sequelae with physiological and psychological perturbations. This pediatric concussion mouse model results in long-term neuroinflammation, white matter alterations, and neuronal modifications with behavioral dysfunction. The model can be adapted to differential impact locations or repeated concussions to assess lifetime changes.

Concussions and mild traumatic brain injuries (mTBI) during childhood represent a significant health endangerment, with many patients later in life ...

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

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

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

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