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

Summary

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.

Abstract

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.

Introduction

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 ‘silent disease’ 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 dysfunction^3,4,5,6. Due to the rising incidence of blast-related mild TBI^7,8, 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 effects^12,13. 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 spectrum^2,14,15. 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 investigations^16,17,18. In addition, this model allows for investigation of coup and contrecoup injury due to rapid rotational forces of the animal’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 studies^{17,19,20,21,22,23}. 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 models^10,24. Previous research^19,21,22,23 has shown that this preclinical model can be used to characterize neurobiological and pathophysiological processes underlying the etiology of mild and moderate TBI^{17,19,20,21,22,23}. 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 interventions^19,21,22,23.

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 waves^{2,14,17,25,26,27,28}. 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.

Protocol

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

1. 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° taper) short driver section was selected. The length and diameter of the driven and driver sections can be modified to specifically research blast wave^29,30,31,32, 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).
2. 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.
   1. 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.
   2. 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.

2. Evaluation of the setup and blast wave properties using pressure sensor recordings.

1. Prepare the shock tube.
   1. Carefully cut the polyester membrane without bending and producing fissures, in order to ensure consistent rupture.
   2. Insert the membrane between the driven and driver sections. Secure the sections by tightening connecting bolts.
   3. Check that the system is airtight and the membrane is tightly fixed between driver and driven sections.
   4. 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.
2. Install the pressure sensors for monitoring the blast waves (see Figure 1C).
   1. Position one pressure sensor in the head placement area, and three sensors at the exit of the shock tube (see Figure 1C and 2).
   2. 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.
   3. 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.
   4. 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.
   5. 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.
3. 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.
   1. 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).
   2. 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.
   3. Repeat steps 2.1.1 to 2.2.4 to establish desired blast wave properties (if needed) and control for reproducibility.
   4. 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).

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.

1. Prepare all materials required for the experiment and check setup for proper installation (e.g., adjust parameters according to aim of study) (~5 – 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 μm is utilized for mild to moderate TBI in mice³⁵. We have previously utilized membranes with a thickness of 76.2 to 127 μm to produce mild to moderate TBI in rats¹⁹.
   1. Carefully cut the polyester membrane, insert it between the driven and driver sections and secure by tightening the connecting bolts.
   2. 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.
   3. Place three pressure sensors at the exit of the shock tube, 120° apart, to monitor the blast wave properties during the TBI induction as described in step 2.2.2 and 2.2.5.
   4. Ensure distance from end of shock tube apparatus is correct for each respective subject using installed micrometer. Keep the positioning of the rodent’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.
   5. Initiate the recording from the pressure sensors using the graphical user interface (GUI) of the software.
2. Anesthesia and positioning of rodents in setup
   1. 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.
   2. 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.
   3. 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’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 TBI^36,37,38.
3. Exposure of rodents to blast wave.
   1. 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.
   2. Turn off the gas flow immediately after hearing the explosion.
4. Recovery from blast wave exposure
   1. 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.
   2. 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).
   3. 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.
   4. After the initial monitoring period, subjects can be analyzed using various biochemical, neuropathological, neurophysiological, and behavioral assays of researcher’s choosing (see below).
5. Prepare setup and room for next experiment.
   1. Clean setup with detergent to remove odor.

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.

1. Biochemical analysis
   1. 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.
   2. Use tissue for biochemical analysis (i.e., immunoblotting, ELISA, etc.) to assess the effect of mild TBI on neurobiological and pathophysiological processes.
2. Neuropathological analysis
   1. 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).
   2. 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.
3. Neurophysiological analysis in brain slices
   1. At defined experimental time points (hours to days post-mild TBI), sacrifice rodents by decapitation, remove brain and prepare brain slices as described¹⁹.
   2. Perform electrophysiological recordings as described¹⁹ to assess the effect of mild TBI on basal synaptic properties and synaptic plasticity.
4. Behavioral analysis
   1. 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 & memory (e.g., fear conditioning, Barnes maze, Morris water maze).

Results

The scalability of the blast wave setup was tested using three different membrane thicknesses, 25.4, 50.8 and 76.2 μm. 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 & Figure 2). Peak pressures increase in concordance with membrane thickness at both sensor locations (Figure 3A,B), demonstrating that th...

Discussion

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 organs^2,39,

Materials

Name	Company	Catalog Number	Comments
3/8 SAE High Pressure Hydraulic Hose	Eaton Aeroquip	R2-6-6-36M	Available from Grainger
3/8'' Quick Connect Female Plugs	Karcher	KAR 86410440
3/8'' Quick Connect Male Plugs	Karcher	KAR 86410440
ANY-maze video tracking software	Stoelting Co.	ANY-maze software
Clear Mylar membrane	ePlastics.com	POLYCLR0.003	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.
Compound Slide Table (X2)	Grizzly Industrial	G5757
Deadman Gas Control Ball Valve	Coneraco Inc.	71-502-01	"Apollo", Available from Grainger
Driver and driven section (murine)	own design/production	n/a	For further information please contact the authors
Driver and driven section (rat)	own design/production	n/a	For further information please contact the authors
Ear Muffs	3M	37274	Available from Grainger
Gas Regulator - Hi Flow 3500-600-580	Harris	3003539
Helium Gas	AirGas	HE 300	Tanks are available in various sizes
Inhalation Anesthesia System	VetEquip	901806
Input Module	National Instruments	NI 9223
Isoflurane	Baxter	NDC 10019-360-40	Ordered by veterinarian
Laboratory Timer/Stopwatch	Fisher Scientific	50-550-352
Labview version 12.0	National Instruments		Data Acquistion Software
Magnetic Dial Indicator/Micrometer	Grizzly Industrial	G9849
MATLAB	MathWorks		Software for pressure recording analysis
Oxygen Regulator	Medline	HCS8725M
PC for Data Processing	Dell
Polyvinylchloride Tubing - 25.4 mm	FORMUFIT	P001FGP-WH-40x3
Pressure sensors	PCB Piezotronics	102A05
Receiver USB Chassis	National Instruments	DAQ-9171
Sensor Signal Conditioner	PCB Piezotronics	482C series
Stainless NSF-Rated Mounting Table	Gridmann	GR06-WT2448
T Handle Allen Wrench - 3/16''	S&K	73310