Name: a novel model of mild traumatic brain injury for juvenile rats
Uploaded: 2014-12-08T13:00:00
Duration: 7 min 36 s
Description: Watch this Scientific Journal Video about A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats at JoVE.com

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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+the+development+of+rodent+models+of+mild+traumatic+brain+injury.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+traumatic+brain+injury.">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., Yan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+biology+and+the+anostic+approach+to+drug+discovery+and+development+to+treat+traumatic+brain+injury.">Systems biology and the anostic approach to drug discovery and development to treat traumatic brain injury.</a> Systems Biology in Drug Discovery and Development: Methods and Protocols, Methods in Molecular Biology. , .</li><li>Viano, D., Casson, I., Pellmen, E. <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+profession+football:+Biomechanics+of+the+struck+player+-+Part+14.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+diffuse+brain+injury+in+rats:+Part+1+-+Pathophysiology+and+biomechanics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+an+experimental+rodent+model+of+pediatric+mild+traumatic+brain+injury.">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/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> Journal of Neuroscience Methods. 203, 41-49 (2012).</li><li>Schallert, T., Woodlee, M., Fleming, S., Krieglstein, J., Klumpp, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disentangling+multiple+types+of+recovery+from+brain+injury.">Disentangling multiple types of recovery from brain injury.</a> Pharmacology of Cerebral Ischemia. , (2002).</li><li>Mychasiuk, R., Hehar, H., Farran, A., Esser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mean+Girls:+Sex+differences+in+the+effects+of+mild+traumatic+brain+injury+on+the+social+dynamics+of+juvenile+rat+play+behaviour.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Report to congress on mild traumatic brain injury in the United States: Steps to prevent a serious public health problem. , 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 border="0" cellpadding="0" cellspacing="0" width="824">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Brass Weights</td>
			<td style="width:155px;">Ginsberg Scientific</td>
			<td style="width:187px;">7-2500-2</td>
			<td style="width:219px;">Need to have metal loop attached to base</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Alluminum Foil</td>
			<td style="width:155px;">Alcan</td>
			<td style="width:187px;"></td>
			<td>Available at most grocery stores</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Masking Tape</td>
			<td style="width:155px;"></td>
			<td style="width:187px;"></td>
			<td>Commercially available&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">U-Shaped Plastic Stand</td>
			<td style="width:155px;"></td>
			<td style="width:187px;"></td>
			<td>Constructed by Laboratory</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Clamp Stand</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:187px;">Z190357</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Plastic Guide Tube</td>
			<td style="width:155px;"></td>
			<td style="width:187px;"></td>
			<td>Could be constructed or purchased at a hardware store</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fishing Line</td>
			<td style="width:155px;">Angler 10lb&#160;</td>
			<td style="width:187px;"></td>
			<td>Purchased from a sporting goods retailer&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:264px;">Isoflurane</td>
			<td style="width:155px;">Pharmaceutical Partners of Canada</td>
			<td style="width:187px;">DIN 02237518</td>
			<td>Inhalation Anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Topical Lidocaine (30ml)</td>
			<td style="width:155px;">Astra Zeneca</td>
			<td style="width:187px;">DIN 0001694</td>
			<td>Xylocaine Jelly 2%&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cotton Swabs</td>
			<td style="width:155px;"></td>
			<td style="width:187px;"></td>
			<td>Commercially available&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Heating Pad - 3 heat setting</td>
			<td style="width:155px;"></td>
			<td style="width:187px;"></td>
			<td>Commercially available&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Stop Watch</td>
			<td style="width:155px;">Sportline</td>
			<td style="width:187px;">L303</td>
			<td>Purchased from a sporting goods retailer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Video Camera</td>
			<td style="width:155px;">Sony</td>
			<td style="width:187px;">HDR-CX260V</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:264px;">Sprague Dawley Rats</td>
			<td style="width:155px;">Charles River Laboratories</td>
			<td style="width:187px;">SAS SD 40</td>
			<td>Male and females ordered from Charles River Laboratories and pups bred in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">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

Research

JoVE Journal

Medicine

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

A Novel Vertebral Stabilization Method for Producing  Contusive Spinal Cord Injury

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing reliable cervical SCI is difficult due to lack of satisfactory methods of vertebral stabilization. The conventional method to stabilize the spine is to suspend the rostral and caudal cervical spine via clamps attached to cervical spinous processes. &#160;However, this method of stabilization fails to prevent tissue yielding during the contusion as the cervical spinal processes are too short to be effectively secured by the clamps (Figure 1). &#160;Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury. &#160;This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs. &#160;We provide visual description of the equipment (Figure 2-4), methods, and a step-by-step protocol for the stabilization of the cervical 5 vertebra (C5) of adult rats, to perform laminectomy (Figure 5) and produce a contusive SCI thereafter. &#160;Although we only demonstrate a cervical hemi-contusion using the NYU/MASCIS impactor device, this vertebral stabilization technique can be applied to other regions of the spinal cord, or be adapted to other SCI devices. &#160;Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord. &#160;This vertebral stabilization method can also be used for stereotactic injections of cells and tracers, and for imaging using two-photon microscopy in various neurobiological studies.

Vertebral stabilization is necessary for minimizing variability, and for producing consistent experimental spinal cord injuries.&#160; Using a customized stabilizing apparatus in conjunction with the NYU/MASCIS impactor device, we have demonstrated here the proper equipment and procedure for generating reproducible hemi-contusive cervical (C5) spinal cord injuries in adult rats.

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing ...

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 ...

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 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 ...

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 ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human Brown-S&#233;quard syndrome&#160;is a common type of incomplete SCI caused by a lesion to one half of the spinal cord which results in paralysis and loss of&#160;proprioception&#160;on the same (or ipsilesional) side as the injury, and loss of pain and temperature sensation on the opposite (or contralesional) side. Adequate methodologies for producing a spinal cord lateral hemisection (HX) and assessing neurological impairments are essential to establish a reliable animal model of Brown-S&#233;quard syndrome. Although lateral hemisection model plays a pivotal role in basic and translational research, standardized protocols for creating such a hemisection and assessing unilateralized function are lacking. The goal of this study is to describe step-by-step procedures to produce a rat spinal lateral HX at the 9th thoracic (T9) vertebral level. We, then, describe a combined behavior scale for HX (CBS-HX) that provides a simple and sensitive assessment of asymmetric neurological performance for unilateral SCI. The CBS-HX, ranging from 0 to 18, is composed of 4 individual assessments which include unilateral hindlimb stepping (UHS), coupling, contact placing, and grid walking. For CBS-HX, the ipsilateral and contralateral hindlimbs are assessed separately. We found that, after a T9 HX, the ipsilateral hindlimb showed impaired behavior function whereas the contralateral hindlimb showed substantial recovery. The CBS-HX effectively discriminated behavioral functions between ipsilateral and contralateral hindlimbs and detected temporal progression of recovery of the ipsilateral hindlimb. The CBS-HX components can be analyzed separately or in combination with other measures when needed. Although we only provided visual descriptions of the surgical procedures and behavioral assessments of a thoracic HX, the principle may be applied to other incomplete SCIs and at other levels of the injury.

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral injury.