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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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;

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			<td height="42" style="height:42px;width:216px;">0.01 M sodium citrate</td>
			<td style="width:121px;">SIGMA - ALDRICH</td>
			<td style="width:161px;"></td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">2.5% normal horse serum</td>
			<td style="width:121px;">SIGMA - ALDRICH</td>
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			<td>Liquid</td>
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			<td height="42" style="height:42px;width:216px;">4 % buffered formaldehyde solution</td>
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			<td height="63" style="height:63px;width:216px;">Anti-Amyloid Precursor Protein, C - terminal antibodyproduced in rabbit</td>
			<td style="width:121px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">Lot 056M4867V</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">biotinylated secondary antibody</td>
			<td style="width:121px;">Vector</td>
			<td style="width:161px;">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>
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			<td height="21" style="height:21px;width:216px;">bone-cutting forceps</td>
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			<td height="63" style="height:63px;width:216px;">DAB Peroxidase (HRP) Substrate Kit (with Nickel), 3,3&#8217;-diaminobenzidine</td>
			<td style="width:121px;">vector laboratory</td>
			<td style="width:161px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">embedding cassettes</td>
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			<td height="21" style="height:21px;width:216px;">ethanol 99.9 %</td>
			<td style="width:121px;">ROMICAL</td>
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			<td>Flammable Liquid</td>
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			<td height="21" style="height:21px;width:216px;">guillotine</td>
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			<td height="42" style="height:42px;width:216px;">Hematoxylin</td>
			<td style="width:121px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">H3136-25G</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Hydrogen peroxide solution</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:161px;">88597-100ML-F</td>
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			<td height="42" style="height:42px;width:216px;">Isofluran, USP 100%</td>
			<td style="width:121px;">Piramamal Critical Care, Inc</td>
			<td style="width:161px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Olympus BX 40 microscope</td>
			<td style="width:121px;">Olympus</td>
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			<td height="42" style="height:42px;width:216px;">paraffine</td>
			<td style="width:121px;">paraplast plus leica biosystem</td>
			<td style="width:161px;"></td>
			<td>Tissue embedding medium</td>
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		<tr height="42">
			<td height="42" style="height:42px;">phosphate-buffered saline (PBS)</td>
			<td style="width:121px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">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 &#176;C.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptavidin HRP</td>
			<td style="width:121px;">ABCAM</td>
			<td style="width:161px;">ab64269</td>
			<td>Streptavidin-HRP for use with biotinylated secondary antibodies during IHC / immunohistochemistry.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">xylene</td>
			<td style="width:121px;"></td>
			<td style="width:161px;"></td>
			<td></td>
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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 sec...

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

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8.3K Views.  Ben-Gurion University of the Negev. 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.

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

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

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 ...