Name: Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration (Video) | JoVE Journal
Uploaded: 2020-05-09T15:00:00.000+00:00
Duration: 6 min 14 s
Description: Learn to induce diffuse axonal brain injury in rats using a rotational acceleration device. This model enables reproducible TBI research without skull fractures, facilitating neurological deficit assessment and therapeutic development.

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5 minutes

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 ± 1 °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’s head is turned rapidly from 0 to 90°.</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="Moment calculation, M=2*(K+mgl1)/π equation, physics problem-solving." src="/files/ftp_upload/61198/61198eq1.jpg" data-altupdatedat="1788250015000" data-alt="Equation 1"> 
	<img alt="Static equilibrium equation diagram; M=0.225m*(1+14.2mh/(1.664+1.037m)); educational use." src="/files/ftp_upload/61198/61198eq2.jpg" data-altupdatedat="1788250015000" data-alt="Equation 2"> 
	<img alt="static equilibrium formula diagram with force equation Fo=(2.5/D)m*(1+(14.2mh)/(1.664+1.037m))" src="/files/ftp_upload/61198/61198eq3.jpg" data-altupdatedat="1788250015000" data-alt="Equation 3"> 
	where Fo - force applied to animal head (kg); M – moment of force; K – kinetic energy; m – mass of the falling weight; g - gravitational acceleration; h – height (cm); D – distance between the ear pins (cm). 
	NOTE: To calculate the force applied to the animal’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’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 °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 °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 µm) 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 βAPP.
	<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 °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 °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–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 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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<li>Meaney, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modification+of+the+cortical+impact+model+to+produce+axonal+injury+in+the+rat+cerebral+cortex%5BTitle%5D)+AND+%22Journal+of+Neurotrauma%22%5BJournal%5D)">Modification of the cortical impact model to produce axonal injury in the rat cerebral cortex.</a> Journal of Neurotrauma. 11, 599-612 (1994).</li>
</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 lo...

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

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 &lt; 0.001, r = 0.78 (see Table 2). The data are measured in counts and are presented as median and 25–75 percentile range.
Representative photomicrographs of thalamic sections o...

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

Research

JoVE Journal

Neuroscience

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.

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

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...

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

Source: Macheda, T., et. al. Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice. J. Vis. Exp. (2022)This video demonstrates the procedure to induce a closed-head injury in a mouse model using a stereotaxic frame. The controlled impact replicates mild traumatic brain injury, allowing researchers to study neurological effects and recovery by measuring post-injury reflexes and behavior.

Induction of Mild Traumatic Brain Injury in a Mouse Model

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.

A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats

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.

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

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.

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

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

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

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.

Assessing Changes in Synaptic Plasticity Using an Awake Closed-Head Injury Model of 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.

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

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.

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

Learn to induce diffuse axonal brain injury in rats using a rotational acceleration device. This model enables reproducible TBI research without skull fractures, facilitating neurological deficit assessment and therapeutic development.

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

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