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 Acquire all the working parts that are required for the setup, including: shock tube consisting of steel dr…

Representative 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,<sup clas…

Materials

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-40×3
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