We thank R. Gettens, N. St. Johns, P. Bennet and J. Robson for their contributions to the development of the TBI model. NARSAD Young Investigator Grants from the Brain &#38; Behavior Research Foundation (F.P. and M.J.R.), a Research Grant from the Darrell K. Royal Research Fund for Alzheimer&#8217;s Disease (F.P.) and a PhRMA Foundation Award (M.J.R.) supported this research. This work was supported through pre-doctoral fellowships from the American Foundation for Pharmaceutical Education (A.F.L and B.P.L.).

The protocol follows the animal care guidelines of the University of Cincinnati and West Virginia University. All procedures involving animals were approved by the Institutional Animal Care and Use Committees (IACUC), and were performed according to the principles of the Guide for the Care and Use of Laboratory Animals.
1. Installation of the blast TBI setup
<ol>
	<li>Acquire all the working parts that are required for the setup, including: shock tube consisting of steel driven- and driver section, polyester membrane, securing bolts, pressure sensors, polyvinyl chloride (PVC) pipe shield to protect peripheral organs, 9.53 mm high pressure hydraulic line and quick connect male and female attachments, high flow gas regulator and a gas cylinder with wall-mount bracket (see Figure 1A,B and Table of Materials). 
	NOTE: The specifications of the driven and driver section used here (see Figure 2 and Table of Materials) have been established to produce a consistent short-duration scaled blast wave (see Figure 3C,D) to induce mild to moderate TBI in mice. For this purpose, a taper-designed (6&#176; taper) short driver section was selected. The length and diameter of the driven and driver sections can be modified to specifically research blast wave29,30,31,32, compression wave18, or shock wave dynamics33. For experiments with rats, the dimensions of the shock tube need to be adapted to yield comparable forces according to retain pertinent body scaling parameters17 (see Table of Materials).</li>
	<li>Install the individual working parts of the setup on machine slide tables that are fixed on a stable, easy to clean surface (preferably stainless steel for use in rodents) in laboratory space approved for animal experiments. 
	NOTE: The blast wave experiments produce a considerable level of noise; therefore choose a location within sound absorbing laboratory space, where noise will not interfere with other experiments/laboratory groups.
	<ol>
		<li>Fix the PVC pipe shield perpendicular to the shock tube setup so that the body of the rodent will be fully covered and only the head protrudes. 
		NOTE: For the standard procedure to induce mild to moderate TBI described here, the center of the head is located 5 cm from the end of the driven section for mice.</li>
		<li>Wall mount gas cylinder in close proximity to setup in accordance with OSHA and all other pertinent safety regulations. 
		NOTE: Compressed air, helium or nitrogen gas are commonly used to generate the blast waves in rodent shock tube models. All data presented here has been generated using helium, as this gas produces higher overpressure over a shorter duration34, allowing for appropriate scaling for murine subjects.</li>
	</ol>
	</li>
</ol>
2. Evaluation of the setup and blast wave properties using pressure sensor recordings.
<ol>
	<li>Prepare the shock tube.
	<ol>
		<li>Carefully cut the polyester membrane without bending and producing fissures, in order to ensure consistent rupture.</li>
		<li>Insert the membrane between the driven and driver sections. Secure the sections by tightening connecting bolts.</li>
		<li>Check that the system is airtight and the membrane is tightly fixed between driver and driven sections.</li>
		<li>Connect the gas tank via a 9.53 mm high pressure hydraulic hose and quick connect attachments to the shock tube 
		NOTE: Driver and driven sections are machined to precise tolerances in order to afford a complete seal of membrane between sections. This allows for no gas leakage and precludes the use of any form of gasket/o-ring material and allows for greater consistency in generated waveform.</li>
	</ol>
	</li>
	<li>Install the pressure sensors for monitoring the blast waves (see Figure 1C).
	<ol>
		<li>Position one pressure sensor in the head placement area, and three sensors at the exit of the shock tube (see Figure 1C and 2).</li>
		<li>Initiate recording from pressure sensors, just prior to blast wave execution. Record the pressure wave data at 500,000 frames per second using a sensor signal conditioner and data acquisition board (see Table of Materials). 
		NOTE: Wear OHSA-approved earmuffs to ensure adequate hearing protection.</li>
		<li>Open the main valve of the compressed gas tank fully to allow the gas flow to produce a sudden, rapid pressure spike. 
		NOTE: The gas overpressure ruptures the polyester membrane to release a shock wave that transitions into a compression wave within the driven section and exits the tube in the direction of the head placement area.</li>
		<li>Turn off the gas flow immediately after the procedure. 
		NOTE: The setup can be equipped with a spring return valve to automatically and rapidly stop the gas flow.</li>
		<li>Analyze the pressure wave recordings using custom written computer program to determine peak overpressure and graph data. Data can be graphed with each sensor individually or overlaid on one another to demonstrate planarity of the wave generated (see Figure 3C,D). 
		NOTE: The analysis can technically be done using more readily available software, but due to the large data sets, these programs have long delays in generating plots.</li>
	</ol>
	</li>
	<li>Establish experimental conditions that are adequate for the aim of the designated TBI study, and confirm that the model produces a consistent blast wave with a peak over-pressure, duration, and impulse measurement comparable to a Friedlander wave (see Figure 3). Verify these parameters using the aforementioned computer software.
	<ol>
		<li>Calibrate the setup by repeating steps 2.1.1. to 2.2.5. and use the pressure wave recordings to determine whether the setup needs adjustment (for representative data see Figure 3).</li>
		<li>Modify the setup (if needed). 
		NOTE: The blast wave properties can be adjusted by minor modifications of the setup. For example, the distance of the head to the end of the driven section impacts the blast wave force at the level of the head. The thickness of the polyester membrane determines the level of peak overpressure, with thicker membranes increasing peak levels (see Figure 3A,B). Additionally, the setup allows selecting the direction by which the blast wave strikes/penetrates the head (i.e., head-on, side, top or underneath) and therefore different aspects can be investigated, such as blast wave injury alone or in combination with coup and contrecoup injury due to rapid rotational forces.</li>
		<li>Repeat steps 2.1.1 to 2.2.4 to establish desired blast wave properties (if needed) and control for reproducibility.</li>
		<li>Repeat steps 2.1.1 to 2.2.4 with polyester membranes of different thickness to evaluate scalability of the setup (for representative data see Figure 3A,B).</li>
	</ol>
	</li>
</ol>
3. Preparation of experimental setup and induction of mild TBI in rodents
NOTE: Transfer rodents to holding area 30 min to 1 h prior to start of TBI experiments to acclimatize. Select holding area that is minimally affected by noise of the procedure.
<ol>
	<li>Prepare all materials required for the experiment and check setup for proper installation (e.g., adjust parameters according to aim of study) (~5 &#8211; 10 minutes). 
	NOTE: Injury severity can be adjusted by selecting the thickness of the polyester membrane. Based on our studies, a membrane thickness of 25.4 to 102 &#956;m is utilized for mild to moderate TBI in mice35. We have previously utilized membranes with a thickness of 76.2 to 127 &#956;m to produce mild to moderate TBI in rats19.
	<ol>
		<li>Carefully cut the polyester membrane, insert it between the driven and driver sections and secure by tightening the connecting bolts.</li>
		<li>Connect the gas tank to the shock tube through the use of quick-release fittings. Ensure that the membrane is tightly fixed between driver and driven sections.</li>
		<li>Place three pressure sensors at the exit of the shock tube, 120&#176; apart, to monitor the blast wave properties during the TBI induction as described in step 2.2.2 and 2.2.5.</li>
		<li>Ensure distance from end of shock tube apparatus is correct for each respective subject using installed micrometer. Keep the positioning of the rodent&#8217;s head (i.e., position, distance) constant within studies to allow for consistent injury evaluation. 
		NOTE: As stated in 1.2.1., different types of injury can be induced by selecting the direction, in which the blast wave impacts the head. For the procedure to induce mild to moderate TBI described here, the body is placed perpendicular to the shock tube that the blast wave impacts the side of the head. In this setting, the head is allowed free mobility and hence is exposed to the blast wave and rapid rotational forces allowing the generation of coup and contrecoup effects.</li>
		<li>Initiate the recording from the pressure sensors using the graphical user interface (GUI) of the software.</li>
	</ol>
	</li>
	<li>Anesthesia and positioning of rodents in setup
	<ol>
		<li>Transfer rodents from holding room and induce anesthesia with 4% isoflurane in oxygen and maintain with 2% isoflurane in oxygen to reduce distress and pain. 
		NOTE: Make sure the animal is non-responsive to toe or tail pinch prior to proceeding. Make sure the induction of anesthesia is consistent for all experimental animals, including sham controls. This procedure requires a low-level and short duration of anesthesia.</li>
		<li>Place the fully anesthetized rodent in the PVC pipe shield with cushioning to protect peripheral organs from the blast wave. 
		NOTE: Control subjects are anesthetized and placed in proximity to the setup, but are not directly subjected to the blast wave. Ensure that the controls are subjected to the noise generated by the shock tube.</li>
		<li>Place the head of the rodent within the head placement area and support it from below, either by a support built directly into the shielding apparatus or a gauze pad. Determine the head alignment according to each individual rodent&#8217;s anatomy, with the occipital condyle aligned with the edge of the protective shielding. 
		NOTE: Avoid directing the pressure wave directly towards the brainstem to decrease mortality. Injury to the respiratory center of the brainstem and the cervical spinal cord is known to contribute to breathing abnormalities and even death in rodent models of TBI36,37,38.</li>
	</ol>
	</li>
	<li>Exposure of rodents to blast wave.
	<ol>
		<li>Rapidly open the main valve of the compressed gas tank to produce a pressure spike that ruptures the membrane and produce a loud explosion that confirms the generation of a pressure wave. The membrane will be visually ruptured when removed after the experiment. 
		NOTE: A high-speed camera can be used to capture the coup and contrecoup effects of the rotational acceleration experienced by the rodent for further analysis.</li>
		<li>Turn off the gas flow immediately after hearing the explosion.</li>
	</ol>
	</li>
	<li>Recovery from blast wave exposure
	<ol>
		<li>After blast wave exposure, remove the rodent from the apparatus and place on a flat surface directly adjacent to the shock tube on their side.</li>
		<li>Monitor subjects to determine righting reflex time (RRT). Use a stopwatch to record time from blast wave exposure until they regain inherent righting reflex. (see Figure 4A).</li>
		<li>As soon as subjects regain their righting reflex, place them in their respective home cage where they are monitored for adverse reactions (i.e., seizures, difficulty breathing, bleeding from a bodily orifice) for the next 24 h.</li>
		<li>After the initial monitoring period, subjects can be analyzed using various biochemical, neuropathological, neurophysiological, and behavioral assays of researcher&#8217;s choosing (see below).</li>
	</ol>
	</li>
	<li>Prepare setup and room for next experiment.
	<ol>
		<li>Clean setup with detergent to remove odor.</li>
	</ol>
	</li>
</ol>
4. Downstream applications for rodents exposed to blast wave/rotational forces and controls
NOTE: In previous studies, the effects of mild to moderate TBI at various time points after exposure to a blast wave and rotational forces were assessed in rodents using downstream applications, including biochemical, neuropathological, neurophysiological, and behavioral analyses19.
<ol>
	<li>Biochemical analysis
	<ol>
		<li>At defined experimental time points (hours to days post-mild TBI), harvest tissue (e.g., brain, blood) for biochemical analysis using standard protocols as described19.</li>
		<li>Use tissue for biochemical analysis (i.e., immunoblotting, ELISA, etc.) to assess the effect of mild TBI on neurobiological and pathophysiological processes.</li>
	</ol>
	</li>
	<li>Neuropathological analysis
	<ol>
		<li>At defined experimental time points (hours to days post-mild TBI), perfuse rodents transcardially with saline solution followed by 4% paraformaldehyde solution to fix tissue as described19. 
		NOTE: Some applications are not compatible with paraformaldehyde fixation (e.g., silver staining, some antibodies for immunohistochemistry).</li>
		<li>Use perfused, fixed tissue for anatomical, histological and molecular analyses to assess neuropathological changes associated with mild TBI, including neuroinflammation, neurodegeneration, and neurochemical changes as described19.</li>
	</ol>
	</li>
	<li>Neurophysiological analysis in brain slices
	<ol>
		<li>At defined experimental time points (hours to days post-mild TBI), sacrifice rodents by decapitation, remove brain and prepare brain slices as described19.</li>
		<li>Perform electrophysiological recordings as described19 to assess the effect of mild TBI on basal synaptic properties and synaptic plasticity.</li>
	</ol>
	</li>
	<li>Behavioral analysis
	<ol>
		<li>At defined experimental time points (hours to days post-mild TBI), evaluate behavioral performance, including motor function (e.g., open field, rotarod, locomotor activity; see Figure 4D) and learning &#38; memory (e.g., fear conditioning, Barnes maze, Morris water maze).</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing interests.

We present here a preclinical mild TBI model that is cost-effective, easy to set up and execute, and allows for high-throughput, reliable, and reproducible experimental outcomes. This model provides protective shielding to peripheral organs to allow for focused investigation into mild TBI mechanisms while limiting the confounding variables of systemic injury. In contrast, other blast models are known to inflict damage to peripheral organs2,39,<sup clas...

Traumatic brain injury (TBI) accounts for more than two million hospital visits each year in the United States alone. Mild TBI commonly resulting from car accidents, sporting events, or falls represent approximately 80% of all TBI cases1. Mild TBI is considered the &#8216;silent disease&#8217; as patients often experience no overt symptoms in the days and months following the initial insult, but can develop serious TBI-related complications later in life2. Moreover, blast-induced mild TBI is prevalent among military service-members, and has been associated with chronic CNS dysfunction3,4,5,6. Due to the rising incidence of blast-related mild TBI7,8, preclinical modeling of neurobiological and pathophysiological processes associated with mild TBI has thus become a focus in the development of novel therapeutic interventions for TBI.
Historically, TBI research has primarily focused on severe forms of neurotrauma, despite the relatively lower number of severe human TBI cases. Preclinical rodent models for severe human TBI have been developed, including the controlled cortical impact (CCI)9,10 and fluid percussion injury (FPI)11 models, which are both well established to produce reliable pathophysiological effects12,13. These models have laid the groundwork for what is known today about neuroinflammation, neurodegeneration, and neuronal repair in TBI. Although considerable knowledge of the pathophysiology of TBI has been developed, there are currently no effective, FDA-approved treatments available for TBI.
More recently, the focus of TBI research has been broadened to include a wider spectrum of TBI-related pathologies with the ultimate goal of developing effective therapeutic interventions. Nevertheless, few preclinical models for mild TBI have been established that have shown measurable effects, and only a small number of studies have investigated the mild TBI spectrum2,14,15. As mild TBI accounts for the large majority of all TBI cases, reliable models of mild TBI are urgently needed to facilitate research into the etiology and neuropathophysiology of the human condition, in order to develop novel therapeutic strategies.
In conjunction with biomedical engineers and aerospace physicists, we have established a scalable, closed-head blast wave model for mild to moderate TBI. This preclinical rodent model has been specifically developed to investigate the effects of force dynamics, including blast waves and acceleration/deceleration movement, that are associated with human mild TBI obtained in military combat, sporting events, car accidents, and falls. As blast waves correlate with the force dynamics that cause mild TBI in humans, this model was designed to produce a consistent Friedlander waveform with an impulse, which is measured as pounds per square inch (psi)*millisecond (ms). The impulse level is scaled to fall below defined lung lethality curves for mice and rats in order to conduct preclinical investigations16,17,18. In addition, this model allows for investigation of coup and contrecoup injury due to rapid rotational forces of the animal&#8217;s head. This kind of injury is inherent to several types of clinical TBI presentations, including those observed in both military and civilian populations. Therefore, this versatile model fits a need that encompasses multiple clinical presentations of TBI.
The preclinical model presented here produces reliable and reproducible pathophysiological changes associated with clinical mild TBI as demonstrated by a number of prior studies17,19,20,21,22,23. Studies with this model showed that rats subjected to a low-intensity blast wave exhibited neuroinflammation, axonal injury, microvascular damage, biochemical changes related to neuronal injury and deficits in short-term plasticity and synaptic excitability19. However, this mild TBI model did not induce any macroscopic neuropathological changes, including tissue damage, hemorrhage, hematoma and contusion19 that have been commonly observed in studies using moderate to severe invasive TBI models10,24. Previous research19,21,22,23 has shown that this preclinical model can be used to characterize neurobiological and pathophysiological processes underlying the etiology of mild and moderate TBI17,19,20,21,22,23. This model also permits for testing of new therapeutic compounds and strategies, as well as the identification of novel, suitable targets for the development of effective TBI interventions19,21,22,23.
This model was developed to investigate effects induced by blast waves as well as rapid rotational forces on molecular, cellular and behavioral outcomes in rodents. Analogous to the blast wave model presented here, a number of preclinical models has been developed that attempt to recapitulate mild to moderate TBI using gas-driven overpressure waves2,14,17,25,26,27,28. Some of the limitations of other models include: the animal is fixed to a wire-mesh gurney and the head is immobilized upon impact; the peripheral organs are exposed to the wave in addition to the brain, which creates the confounding variables of polytrauma; and the models are large and stationary, which limits changing and adapting critical parameters to better model conditions reminiscent of human TBI.
The benefits of this bench-top, gas-driven shock tube setup are its relative low-cost for acquisition and running expenses, as well as ease of installation and use. Furthermore, the setup allows for high-throughput operation and generation of controlled reproducible blast waves and in vivo outcomes in both mice and rats. In order to control for consistent test conditions (i.e., constant blast wave and overpressure) the setup is equipped with pressure sensors. The advantages of this model for TBI include scalability of the injury severity and that mild TBI is induced using a non-invasive, closed-head procedure. Peak overpressure and subsequent brain injury increase with thicker polyester membranes in a consistent scalable manner17. The ability to scale TBI severity through membrane thickness is a useful tool to determine the level, at which specific outcome measures (e.g., neuroinflammation) become evident. Providing protective shielding for the peripheral organs, also allows focused investigation into mild TBI mechanisms by avoiding or reducing confounding variables of systemic injury, such as lung- or thoracic injury. Moreover, this setup allows selecting the direction, by which the blast wave strikes/penetrates the head (i.e., head-on, side, top or underneath) and therefore different types of TBI-inducing insults can be investigated. The standard procedure to induce mild to moderate TBI described here employs side exposure to evaluate the effects of blast wave injury in combination with coup and contrecoup injury due to rapid rotational forces. Furthermore, in order to investigate exclusively blast-induced injury, top down blast wave exposure can be employed in this model.

<table>
	<tbody>
		<tr>
			<td>3/8 SAE High Pressure Hydraulic Hose</td>
			<td>Eaton Aeroquip</td>
			<td>R2-6-6-36M</td>
			<td>Available from Grainger</td>
		</tr>
		<tr>
			<td>3/8&#39;&#39; Quick Connect Female Plugs</td>
			<td>Karcher</td>
			<td>KAR 86410440</td>
			<td></td>
		</tr>
		<tr>
			<td>3/8&#39;&#39; Quick Connect Male Plugs</td>
			<td>Karcher</td>
			<td>KAR 86410440</td>
			<td></td>
		</tr>
		<tr>
			<td>ANY-maze video tracking software</td>
			<td>Stoelting Co.</td>
			<td>ANY-maze software</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear Mylar membrane</td>
			<td>ePlastics.com</td>
			<td>POLYCLR0.003</td>
			<td>http://www.eplastics.com/Plastic/Clear_Polyester_Film/POLYCLR0-003; Clear Mylar membrane is sold in various thicknesses. All are sold by vendor listed above.</td>
		</tr>
		<tr>
			<td>Compound Slide Table (X2)</td>
			<td>Grizzly Industrial</td>
			<td>G5757</td>
			<td></td>
		</tr>
		<tr>
			<td>Deadman Gas Control Ball Valve</td>
			<td>Coneraco Inc.</td>
			<td>71-502-01</td>
			<td>&#34;Apollo&#34;, Available from Grainger</td>
		</tr>
		<tr>
			<td>Driver and driven section (murine)</td>
			<td>own design/production</td>
			<td>n/a</td>
			<td>For further information please contact the authors</td>
		</tr>
		<tr>
			<td>Driver and driven section (rat)</td>
			<td>own design/production</td>
			<td>n/a</td>
			<td>For further information please contact the authors</td>
		</tr>
		<tr>
			<td>Ear Muffs</td>
			<td>3M</td>
			<td>37274</td>
			<td>Available from Grainger</td>
		</tr>
		<tr>
			<td>Gas Regulator - Hi Flow 3500-600-580</td>
			<td>Harris</td>
			<td>3003539</td>
			<td></td>
		</tr>
		<tr>
			<td>Helium Gas</td>
			<td>AirGas</td>
			<td>HE 300</td>
			<td>Tanks are available in various sizes</td>
		</tr>
		<tr>
			<td>Inhalation Anesthesia System</td>
			<td>VetEquip</td>
			<td>901806</td>
			<td></td>
		</tr>
		<tr>
			<td>Input Module</td>
			<td>National Instruments</td>
			<td>NI 9223</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>NDC 10019-360-40</td>
			<td>Ordered by veterinarian</td>
		</tr>
		<tr>
			<td>Laboratory Timer/Stopwatch</td>
			<td>Fisher Scientific</td>
			<td>50-550-352</td>
			<td></td>
		</tr>
		<tr>
			<td>Labview version 12.0</td>
			<td>National Instruments</td>
			<td></td>
			<td>Data Acquistion Software</td>
		</tr>
		<tr>
			<td>Magnetic Dial Indicator/Micrometer</td>
			<td>Grizzly Industrial</td>
			<td>G9849</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td></td>
			<td>Software for pressure recording analysis</td>
		</tr>
		<tr>
			<td>Oxygen Regulator</td>
			<td>Medline</td>
			<td>HCS8725M</td>
			<td></td>
		</tr>
		<tr>
			<td>PC for Data Processing</td>
			<td>Dell</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylchloride Tubing - 25.4 mm</td>
			<td>FORMUFIT</td>
			<td>P001FGP-WH-40x3</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure sensors</td>
			<td>PCB Piezotronics</td>
			<td>102A05</td>
			<td></td>
		</tr>
		<tr>
			<td>Receiver USB Chassis</td>
			<td>National Instruments</td>
			<td>DAQ-9171</td>
			<td></td>
		</tr>
		<tr>
			<td>Sensor Signal Conditioner</td>
			<td>PCB Piezotronics</td>
			<td>482C series</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless NSF-Rated Mounting Table</td>
			<td>Gridmann</td>
			<td>GR06-WT2448</td>
			<td></td>
		</tr>
		<tr>
			<td>T Handle Allen Wrench - 3/16&#39;&#39;</td>
			<td>S&#38;K</td>
			<td>73310</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The scalability of the blast wave setup was tested using three different membrane thicknesses, 25.4, 50.8 and 76.2 &#956;m. Peak pressure levels were assessed at the head placement area and the exit of the shock tube apparatus using piezoelectric pressure sensors (see Figure 1 &#38; Figure 2). Peak pressures increase in concordance with membrane thickness at both sensor locations (Figure 3A,B), demonstrating that th...

Watch this Scientific Journal Video about Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents at JoVE.com

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

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 ...

low-intensity-blast-wave-model-for-preclinical-assessment-closed-head

Research

JoVE Journal

Neuroscience

2.5K Views.  Veterans Affairs. 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.

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

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 ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

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 ...

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 ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...

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

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

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

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

Validating Injury Response and Behavioral Assessments in an mTBI Rat Model

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

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

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

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

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