The authors gratefully acknowledge Dr. Nathan Kleeorin (Department of Mechanical Engineering, Ben-Gurion University of the Negev) for his assistance with the biomechanical measurements. Also, we thank Professor Olena Severynovska, Maryna Kuscheriava, Maksym Kryvonosov, Daryna Yakumenko and Evgenia Goncharyk of the Department of Physiology, Faculty of Biology, Ecology, and Medicine, Oles Honchar Dnipro University, Dnipro, Ukraine for her support and helpful contributions to our discussions.

The experiments were performed following the recommendations of the Declarations of Helsinki and Tokyo and to the Guidelines for the Use of Experimental Animals of the European Community. The experiments were approved by the Animal Care Committee of Ben-Gurion University of the Negev.
1. Preparing rats for the experimental procedure
NOTE: Select adult male Sprague-Dawley rats weighing 300-350 g.
<ol>
	<li>Obtain approval for performing these experiments from the Institutional Animal Care and Use Committee.</li>
	<li>Maintain rats at a room temperature of 22 &#177; 1 &#176;C, with 12 hour light and 12 hour dark cycles. Provide rat chow and water ad libitum.</li>
	<li>Perform all experiments between 6:00 a.m. and 12:00 p.m.</li>
	<li>Use a continuous isoflurane administration system to induce anesthesia. Make sure the vaporizer system is filled with isoflurane.
	<ol>
		<li>Anesthetize the rats with 2% isoflurane.</li>
		<li>Confirm that the rat is fully anesthetized by observing a lack of movement or pedal reflex in response to an external stimuli.</li>
	</ol>
	</li>
</ol>
2. Induction of diffuse axonal injury
NOTE: The device consists of the following components: 1) transparent plastic cylinder, 2) iron weight (1308 g), 3) rotation mechanism consisting of a cylindrical tube, two bearings upon which the axis rotate and a head fixation (for ear pins); 4) horizontal platform on which are fixed two bearings.
<ol>
	<li>Place the device on a heavy, stable laboratory table.</li>
	<li>Attach the weight to a string that is elevated to a height of 120 cm.</li>
	<li>Allow the freely falling weight to hit the bolt, activating the rotational mechanism. Using the lateral head rotation device, the rodent&#8217;s head is turned rapidly from 0 to 90&#176;.</li>
	<li>After induction of diffuse axonal brain injury, transfer the rat to a recovery room.</li>
</ol>
3. Measurement of rotational Kinematics/Biomechanical parameters.
<ol>
	<li>Measure rotational kinematics/biomechanical parameters as follows: 
	<img alt="Equation 1" src="/files/ftp_upload/61198/61198eq1.jpg" /> 
	<img alt="Equation 2" src="/files/ftp_upload/61198/61198eq2.jpg" /> 
	<img alt="Equation 3" src="/files/ftp_upload/61198/61198eq3.jpg" /> 
	where Fo&#160;- force applied to animal head (kg); M &#8211; moment of force; K &#8211; kinetic energy; m &#8211; mass of the falling weight; g - gravitational acceleration; h &#8211; height (cm); D &#8211; distance between the ear pins (cm). 
	NOTE: To calculate the force applied to the animal&#8217;s head (Fo), it is necessary to know the mass of the falling weight, the height at which the weight falls, and the distance between the ear pins. The other parameters remain unchanged.</li>
</ol>
4. Evaluation of Neurological Severity Score after 48 hours
NOTE: Neurological deficits were assessed and graded using a Neurological Severity Score, as previously described17,18,19. Alterations in motor function and behavior are assessed by a point-system such that a maximum score of 24 represents severe neurological dysfunction. A score of 0 indicates intact neurological status. The following behavioral functions are assessed.
<ol>
	<li>Assess the rat&#8217;s inability to exit from a circle (50 cm in diameter) when left in its center. Perform this for three individual sessions lasting 30 min, 60 min, and more than 60 min.</li>
	<li>Test the rat for a loss of righting reflex in three sessions lasting 20 min, 40 min, and over 60 min.</li>
	<li>Perform the test for hemiplegia, the inability of the rat to resist forced changes in position.</li>
	<li>Raise the rat by its tail to test the flexion of the hindlimb.</li>
	<li>Place the rat on the floor to test its ability to walk straight.</li>
	<li>Test for three separate reflexes: the pinna reflex, the corneal reflex, and the startle reflex.</li>
	<li>Rate the rat with a clinical grade based on loss of seeking behavior and prostration.</li>
	<li>Test limb reflexes for placement. Perform the test on the left and right forelimbs, and then the left and right hindlimbs.</li>
	<li>Perform a functional test via the beam balancing task. Beam should measure 1.5 cm wide. Run the test for sessions of 20 s, 40 s, and more than 60 s.</li>
	<li>Perform beam walking test on the rat with beams of three different widths: 8.5 cm wide, 5 cm wide, and 2.5 cm wide.</li>
</ol>
5. Brain collection for histological examination after 48 hours
<ol>
	<li>At 48 hours post injury, euthanize the rats by replacing their inspired gas mixture with 20% O2/80% CO2. Ensure that CO2 is delivered at a predetermined rate in accordance with Institutional Animal Care and Use Committee guidelines.
	<ol>
		<li>Ensure death confirmation in accordance with Institutional Animal Care and Use Committee guidelines.</li>
	</ol>
	</li>
	<li>Transcardiacally perfuse the rat with 0.9% heparinized saline at temperature 4 &#176;C, followed by 500 mL of 4% paraformaldehyde in 0.1 M phosphate buffer saline (pH 7.4).</li>
	<li>After perfusion, perform decapitation with a guillotine.</li>
	<li>Perform brain collection by removing the calvarias with bone-cutting forceps to avoid damaging brain tissue.</li>
	<li>Remove the brain immediately and fix in a 4% buffered formaldehyde solution for 48 h at 4 &#176;C.</li>
	<li>Block brains into 5 mm coronal sections from the olfactory bulb face to the visual cortex and bisect cerebellums and brain stems.</li>
	<li>Following paraffin embedding, cut coronal and sagittal sections (5 &#181;m) away from the thalamus by microtome sectioning.</li>
</ol>
6. Immunochemical staining and examination
<ol>
	<li>Gently place the slices on glass slides with a soft brush, 1 slice per slide.</li>
	<li>Produce immunochemical staining of &#946;APP.
	<ol>
		<li>Deparaffinize slices with xylene (3 times for 5 min each) and rehydrate with gradually-reduced concentrations of ethanol at room temperature: 3 min in 100% ethanol twice, 3 min in 95% ethanol twice, 3 min in 90% ethanol, 3 min in 70% ethanol, and 3 min in DDW.</li>
		<li>Treat deparaffinized and rehydrated brain sections with 3% H2O2 for 15 min at room temperature to block endogenous peroxidase activity.</li>
		<li>Incubate sections with 0.01 M sodium citrate (pH 6.0) at 98 &#176;C for 5 min for antigen retrieval.</li>
		<li>Keep the slides in the buffer for 20 min at room temperature to cool.</li>
		<li>Wash sections with phosphate-buffered saline (PBS) solution twice for 5 min.</li>
		<li>Block the sections with 2.5% normal horse serum for 1 h at room temperature and incubate overnight at 4 &#176;C in primary rabbit anti-APP (1:4000) diluted in the blocking serum.</li>
		<li>After incubation in primary antibody, wash sections in PBS at room temperature.</li>
		<li>Incubate sections in appropriately diluted biotinylated secondary antibody for 15 min and wash with PBS for 3 min twice at room temperature.</li>
		<li>Incubate in streptavidin&#8211;peroxidase for 15 min and wash again in PBS for 3 min twice at room temperature.</li>
		<li>Incubate sections with buffered substrate solution (pH 7.5) containing hydrogen peroxide and 3,3-diaminobenzidine chromogen solution and protect from light until the color is developed.</li>
		<li>Incubate the slides with DDW at room temperature for 5 min in order to stop the reaction.</li>
		<li>Counterstain sections with Hematoxylin for 3 min at room temperature and wash for 5 min with flowing tap water.</li>
		<li>Dehydrate the slides with gradually increasing concentrations of ethanol at room temperature: 2 min in DDW, 2 min in 70% ethanol, 2 min in 90% ethanol, 2 min in ethanol 95%, 2 min in 100% ethanol, and 3 min in xylene three times.</li>
		<li>Dry and mount with mounting&#160;medium.</li>
	</ol>
	</li>
	<li>Examine the slices under microscope magnification of 200x with a 20 mm objective lens using a microscope.</li>
</ol>

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</ol>

The authors have nothing to disclose.

This protocol describes a rodent model of DAI. In DAI, rotational acceleration on the brain causes a shear effect that triggers axonal and biochemical changes that lead to loss of axonal function in a progressive process. Secondary axonal changes are produced by a rapid axonal stretch injury and are variable in their extent and severity4,5,10. Within hours to days after the primary injury, biochemical changes will lead to the loss of axonal function4,5,10. Following the injury, the permeability of the axon membrane changes, allowing a massive calcium influx. The intake of calcium causes the mitochondria to swell and break, releasing caspases and triggering caspase mediated progressive cell death4,5,10,11,20. Secondary axonal injury can present in the form of the axonal bulbs at the ruptured end or in the form of varicosities along the length of the axon4,21,22. The loss of nerve impulse transition is expressed by the aggregation of the &#946;-amyloid precursor protein (&#946;APP), a single transmembrane protein present in most cells and tissues4,23,24,25,26. Immunohistochemical analysis of &#946;APP accumulation is currently the gold standard clinical and experimental technique for assessment of DAI4,9,10,20,27. Studies have reported &#946;APP immunoreactivity starting approximately 2 hours after injury, but there is evidence that ongoing changes continue for one or more years post-injury23,28,29. The most vulnerable areas are the brainstem, parasagittal white matter of the cerebral cortex, and corpus callosum11.
Common in vivo animal models of DAI are the lateral fluid percussion model30,31, the impact acceleration injury32,33 and the controlled cortical impact model34,35,36. These models provide some useful results but with significant limitations.
Fluid percussion models in animal models induce brain injury by injecting varying volumes of saline into the closed cranial cavity at the midline, especially in cat and rabbit models, or laterally in rodent models30,31. Injury severity can be varied from mild to severe by adjusting the fluid pressure. Although this model is reliable and reproducible, it is not an ideal model of human DAI, because percussion injury produces contusion and/or subarachnoid hemorrhage and the type of primary impact is distinct from real life injuries37,38. Furthermore, the effects of brain geometry and intracranial structure on direction, displacement and velocity make it very difficult to perform a precise biomechanical analysis of the injury39.
The impact acceleration injury model32,33 uses segmented brass weights free-falling from a specified height through a Plexiglas guide tube onto a metallic helmet fixed by dental acrylic to the skull vertex of the rat. This model is inexpensive, easy to perform, and can produce graded DAI, but there is also a possibility of contusions and skull fractures, compromising the reproducibility of the model. In addition, the induced injury involves a disproportionately smaller volume of the brain than in humans39.
The model of controlled cortical impact employs a pneumatic or electromagnetic impact device to drive a rigid impactor onto the exposed, whole dura through a unilateral craniotomy, which leads to deformation of the underlying cortex16,17. Air pressure is responsible for the impact velocity, and the depth of cortical deformation is regulated by vertical adjustment of the crossbar where the cylinder is attached. Like the fluid percussion model, it causes mainly focal injury.
Regarding these disadvantages, a new modified rodent model has been developed with opening of the dura mater over the contralateral hemisphere to produce more widespread axonal injury40. However, most previous models require craniotomy, and results of axonal pathogenesis may be affected by contusion and hemorrhage that usually appear in previous models. Moreover, the mechanism of injury in these models is different from the human DAI caused by the acceleration&#8211;deceleration movements of the brain.
There are several steps in the protocol that are critical and merit careful consideration. One should consider that head of the rat should be tightly fixed to the ear pins, or the rat may fall from device. When falling, other forces may play a role that will affect the accuracy of any calculations. Also, the iron weight must be the specific weight and dropped at the specific height noted in this protocol. These measurements have been determined empirically and are mandatory conditions for the reproduction of the model. The installation of the plastic cylinder should be at an angle of 90&#176; relative to the rotational mechanism, namely the bolt. This is because it is the hit to the bolt that drives the rotational mechanism. Otherwise, the friction of the iron weight relative to the plastic cylinder is introduced, which will lead to a decrease in the force applied to the rat&#8217;s head.
There are some limitations to this model. The development of DAI in humans is mainly secondary to an impact from another object. In this case, either the person moves towards the object, the object moves towards the person, or they both move toward each other. In such a collision, a patient develops a combined head injury, where diffuse axonal damage is only part of the TBI. Here, the applied rotational acceleration is the main mechanism that leads to the development of DAI without others elements of head injury.
The model proposed here appears to alleviate the complications of skull fractures and contusions that caused widespread white matter damage without limited additional injury. Similar to other recent rodent models, this model is effective and provides a low (0%) mortality rate. It is a reproducible and affordable technique that could serve as a valuable resource for better understanding the pathophysiology of DAI to develop more effective treatments.

Traumatic brain injury (TBI) is a major cause of death and disability in the United States. TBIs contribute to about 30% of all injury-related deaths1,2. The leading causes of TBI differ among age groups and include falls, high-speed collisions during sports, intentional self-harm, motor vehicle crashes and assaults1,2,3.
Brain diffuse axonal injury (DAI) is a specific type of TBI induced by rotational acceleration, shaking or blast injury of the brain resulting from unrestricted head movement in the instant after injury4,5,6,7,8. DAI involves widespread axonal damage leading to long-lasting neurological impairment that is associated with poor outcome, burdensome health-care costs, and a 33-64% mortality rate1,2,4,5,9,10,11. Despite significant recent research into the pathogenesis of DAI, there has not been a consensus on best treatment options11,12,13,14.
Over the last decades, numerous experimental models have attempted to accurately replicate different aspects of DAI11,12,15,16. However, these models have limitations given the unique presentation of DAI compared to other focal injuries. These prior models not only cause axonal injury in white matter regions but also result in focal cerebral injuries. Clinically, DAI is accompanied by micro hemorrhages, which may constitute a major cause of damage to white matter.
Only two animal models have been shown to replicate the key clinical features of DAI. Gennarelli and colleagues produced the first lateral head rotation device in 1982, using nonimpact head rotational acceleration to induce coma with DAI in a nonhuman primate model15. This primate model employed controlled single rotation for acceleration and deceleration to displace the head through 60&#176; within 10-20 ms. This technique was able to emulate impaired consciousness and widespread axonal damage that resembled the effects of severe TBI observed in human brains. However, primate models are very expensive4,11,16. Based in part on the previous model, a pig model of rotational acceleration brain injury was designed in 1994 (Ross et al.) with similar results14.
These two animal models, though they produced different presentations of typical pathology, have added greatly to the concepts of DAI pathogenesis. Rapid head rotation is generally accepted as the best method for inducing DAI, and rodents provide a less expensive model for the rapid head rotation studies11,16. Here, we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions. This current model will enable better understanding of the pathophysiology of DAI and development of more effective treatments.&#8195;

<table><tbody><tr><td>0.01 M sodium citrate</td><td>SIGMA - ALDRICH</td><td></td><td></td></tr><tr><td>2.5% normal horse serum</td><td>SIGMA - ALDRICH</td><td>H0146</td><td>Liquid</td></tr><tr><td>4 % buffered formaldehyde solution</td><td></td><td></td><td></td></tr><tr><td>Anti-Amyloid Precursor Protein, C - terminal antibodyproduced in rabbit</td><td>SIGMA - ALDRICH</td><td>Lot 056M4867V</td><td></td></tr><tr><td>biotinylated secondary antibody</td><td>Vector</td><td>BA-1000-1.5</td><td>10 mM sodium phosphate, pH 7.8, 0.15 M NaCl, 0.08% sodium azide, 3 mg/ml bovine serum albumin</td></tr><tr><td>bone-cutting forceps</td><td></td><td></td><td></td></tr><tr><td>DAB Peroxidase (HRP) Substrate Kit (with Nickel), 3,3&amp;rsquo;-diaminobenzidine</td><td>vector laboratory</td><td></td><td></td></tr><tr><td>embedding cassettes</td><td></td><td></td><td></td></tr><tr><td>ethanol 99.9 %</td><td>ROMICAL</td><td></td><td>Flammable Liquid</td></tr><tr><td>guillotine</td><td></td><td></td><td></td></tr><tr><td>Hematoxylin</td><td>SIGMA - ALDRICH</td><td>H3136-25G</td><td></td></tr><tr><td>Hydrogen peroxide solution</td><td>Millipore</td><td>88597-100ML-F</td><td></td></tr><tr><td>Isofluran, USP 100%</td><td>Piramamal Critical Care, Inc</td><td></td><td></td></tr><tr><td>Olympus BX 40 microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>paraffine</td><td>paraplast plus leica biosystem</td><td></td><td>Tissue embedding medium</td></tr><tr><td>phosphate-buffered saline (PBS)</td><td>SIGMA - ALDRICH</td><td>P5368-10PAK</td><td>Contents of one pouch, when dissolved in one liter of distilled or deionized water, will yield 0.01 M phosphate buffered saline (NaCl 0.138 M; KCl - 0.0027 M); pH 7.4, at 25 &amp;deg;C.</td></tr><tr><td>Streptavidin HRP</td><td>ABCAM</td><td>ab64269</td><td>Streptavidin-HRP for use with biotinylated secondary antibodies during IHC / immunohistochemistry.</td></tr><tr><td>xylene</td><td></td><td></td><td></td></tr></tbody></table>

induction of diffuse axonal brain injury in rats based on rotational acceleration

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

Table 1 illustrates the protocol timeline. The mortality rate in this model of DAI was 0%. A Mann-Whitney test indicated that neurological deficit was significantly greater for the 15 DAI rats compared to the 15 sham rats at 48 hours following intervention (Mdn = 1 vs. 0), U = 22.5, p &#60; 0.001, r = 0.78 (see Table 2). The data are measured in counts and are presented as median and 25&#8211;75 percentile range.
Representative photomicrographs of thalamic sections of brain tissue are shown in Figure 1. Photomicrographs revealed axonal and neuronal &#946;APP immunoreactivities following isolated DAI in rats 48 hours post injury compared to the control group (67.46 &#177; 30 vs. 0 &#177; 0), U = 0, p &#60; 1.1E-06, r = 0.92. The data are measured as counts and presented as mean &#177; SD.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Groups</td>
			<td>Time</td>
			<td>Procedures</td>
		</tr>
		<tr>
			<td>DAI (15 rats)</td>
			<td>0 h</td>
			<td>Induction Diffuse Axonal Injury</td>
		</tr>
		<tr>
			<td>Sham (15 rats)</td>
			<td>48 h</td>
			<td>Neurological Severity Score Assessment,</td>
		</tr>
		<tr>
			<td>DAI (15 rats)</td>
			<td></td>
			<td>Immunochemical staining of BAPP.</td>
		</tr>
	</tbody>
</table>
Table 1: Demonstration of the protocol timeline. The various groups of rats at different times are shown: DAI = Diffuse axonal brain injury at the beginning of the experiment; At 48 hours, a Neurological Severity Score was determined and immunochemical staining of &#946;APP was performed in both groups.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td colspan="3">NSS values of the various groups at 48 hours</td>
		</tr>
		<tr>
			<td>Animal Group</td>
			<td>N</td>
			<td>NSS 48 hours after DAI</td>
		</tr>
		<tr>
			<td>Sham</td>
			<td>15</td>
			<td>0 (0-0)</td>
		</tr>
		<tr>
			<td>DAI</td>
			<td>15</td>
			<td>1 (1-1)*</td>
		</tr>
	</tbody>
</table>
Table 2: Neurological severity score. Neurological deficit 48 hours following DAI for 2 study groups. A Mann-Whitney test indicated that neurological deficit was significantly greater for the 15 DAI rats compared to the 15 sham rats at 48 hours following intervention (Mdn = 1 vs. 0), U = 22.5, p &#60; 0.001, r = 0.78. The data are measured in counts and are presented as median and 25&#8211;75 percentile range.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61198/61198fig1.jpg" /> 
Figure 1: Immunochemical examination. Representative photomicrographs of thalamic sections of brain tissue revealed axonal and neuronal immunoreactivities following isolated DAI in rats (B) 48 hours post injury compared to the control group (A). &#946;APP immunoreactivity was detected in the region of interest in all 15 DAI rats, and not at all in any of the sham-operated rats. Mann-Whitney test indicated that number of &#946;APP -positive axons was significantly greater for 15 DAI rats than for sham-injured animals at 48h following DAI (67.46 &#177; 30 vs. 0 &#177; 0), U = 0, p &#60; 1.1E-06, r = 0.92. Images are at the original magnification * 200. The data are measured as counts and presented as mean &#177; SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61198/61198fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration at JoVE.com

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

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

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

induction-diffuse-axonal-brain-injury-rats-based-on-rotational

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.4K Views.  Ben-Gurion University of the Negev. Traumatisch hersenletsel is een van de belangrijkste oorzaken dood en invaliditeit. Diffuus axonaal hersenletsel is een wijdverspreide axonale schade te ontwikkelen na TBI. In dit onderzoek wil ik ons model presenteren over inductie diffuse axale hersenletsel op basis van rotatieversnellingsmechanisme resultaat TBI.Selecteer volwassen mannelijke Sprague-Dawley ratten met een gewicht van 300 tot 350 gram. Zorg voor rat chow en water ad libitum. Voer alle experimenten uit tussen 06:00 en...

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

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats

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

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

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

Medicine

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 ...

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 ...

Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the United States. In fact, TBI is a contributing factor to over a third of all injury-related mortality. Nonetheless, the cellular and molecular mechanisms underlying the pathophysiology of TBI are poorly understood. Thus, preclinical models of TBI capable of replicating the injury mechanisms pertinent to TBI in human patients are a critical research need. The controlled cortical impact (CCI) model of TBI utilizes a mechanical device to directly impact the exposed cortex. While no model can full recapitulate the disparate injury patterns and heterogeneous nature of TBI in human patients, CCI is capable of inducing a wide range of clinically applicable TBI. Furthermore, CCI is easily standardized allowing investigators to compare results across experiments as well as across investigative groups. The following protocol is a detailed description of applying a severe CCI with a commercially available impacting device in a murine model of TBI.

Here we describe a protocol for the induction of murine traumatic brain injury via an open-head controlled cortical impact.

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the ...

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

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

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

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

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

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

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

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

rmTBI Induction in Mice via a Closed-Head Injury Method

Modified Mouse Model of Repetitive Mild Traumatic Brain Injury Incorporating Thinned-Skull Window and Fluid Percussion

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models with well-defined pathologies are urgently needed for studying the mechanisms of neuropathology after mild TBI and testing therapeutics. Replicating the entire sequelae of TBI in animal models has proven to be a challenge. Therefore, the availability of multiple animal models of TBI is necessary to account for the diverse aspects and severities seen in TBI patients. CHI is one of the most common methods for fabricating rodent models of rmTBI. However, this method is susceptible to many factors, including the impact method used, the thickness and shape of the skull bone, animal apnea, and the type of head support and immobilization utilized. The aim of this protocol is to demonstrate a combination of the thinned-skull window and fluid percussion injury (FPI) methods to produce a precise mouse model of CHI-associated rmTBI. The primary objective of this protocol is to minimize factors that could impact the accuracy and consistency of CHI and FPI modeling, including skull bone thickness, shape, and head support. By utilizing a thinned-skull window method, potential inflammation due to craniotomy and FPI is minimized, resulting in an improved mouse model that replicates the clinical features observed in patients with mild TBI. Results from behavior and histological analysis using hematoxylin and eosin (HE) staining suggest that rmTBI can lead to a cumulative injury that produces changes in both behavior and gross morphology of the brain. Overall, the modified CHI-associated rmTBI presents a useful tool for researchers to explore the underlying mechanisms that contribute to focal and diffuse pathophysiological changes in rmTBI.

Author Spotlight: Optimizing Rodent Models for Investigating the Mechanisms and Rehabilitation in Repeated Mild Traumatic Brain Injury

This protocol presents a modified mouse model of repetitive mild traumatic brain injury (rmTBI) induced via a closed-head injury (CHI) method. The approach features a thinned-skull window and fluid percussion to reduce the inflammation commonly caused by meninges exposure, along with improved reproducibility and accuracy in modeling rmTBI in rodents.

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models ...

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 ...

Induction of Traumatic Brain Injury in a Rat Model Using a Lateral Fluid Percussion Device

Source: McGuire, M. J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59637">Use of a Wireless Video-EEG System to Monitor Epileptiform Discharges Following Lateral Fluid-Percussion Induced Traumatic Brain Injury.</a> J. Vis. Exp. (2019)
This video demonstrates the induction of traumatic brain injury (TBI) in a rat using a lateral fluid percussion injury (FPI) device. The procedure involves creating a hole in the skull of an anesthetized rat to expose the dura mater, onto which a cannula is attached. The rat is then connected to the device, which generates a pressure wave that reaches the brain, mimicking TBI by causing neural cell death and vascular damage

Source: McGuire, M. J., et al. Use of a Wireless Video-EEG System to Monitor Epileptiform Discharges Following Lateral Fluid-Percussion Induced Traumatic ...

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

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