Mechanisms of Vestibular Deficits Induced by Blast Overpressure via the Ear Canal in Rats
by
David S. Sandlin, Ph.D.
A dissertation submitted to the School of Graduate Studies in the Health Sciences of the University of Mississippi Medical Center in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience
University of Mississippi Medical Center Jackson, MS March, 2018
© David S. Sandlin, Ph.D. March 2018
I certify that I have read this dissertation and that in my opinion it is fully adequate as a dissertation for the degree of Doctor of Philosophy. The Advisory Committee:
__________________________________ Wu Zhou, Ph.D., Chairperson Professor, Otolaryngology and Communicative Sciences
__________________________________ Hong Zhu, M.D., Ph.D. Professor, Otolaryngology and Communicative Sciences
__________________________________ Douglas E. Vetter, Ph.D. Associate Professor, Neuroscience
__________________________________ Yi Pang, Ph.D. Associate Professor, Pediatrics
__________________________________ John M. Schweinfurth, M.D. Professor, Otolaryngology and Communicative Sciences
Approved:
___________________________________ Joey P. Granger, Ph.D. Dean, School of Graduate Studies in the Health Sciences iii
MECHANISMS OF VESTIBULAR DEFICITS INDUCED BY BLAST OVERPRESSURE VIA THE EAR CANAL IN RATS David S. Sandlin, Ph.D. School of Graduate Studies in the Health Sciences University of Mississippi Medical Center March 2018 Blast overpressure has become an increasing cause of injury in both military and civilian populations. Dizziness and/or disequilibrium are frequent complaints of persons impacted by primary blast injury, i.e., that due to the overpressure of the blast. We hypothesize that blast overpressure can traverse the air- and fluid-filled tissues of the inner ear and cause injury to the peripheral vestibular system. To study the ear’s role in the spectrum of blast injury and blast’s impact on the peripheral vestibular system, we designed a blast generator that delivers controlled overpressure waves into the ear canal without impacting surrounding tissues, preventing systemic factors from whole-body exposure paradigms from confounding results. To validate our model, we exposed anesthetized adult male rats’ left ears to a single blast wave ranging from 0 to 110 PSI (0 to 758 kPa). Blast exposed rats exhibited tachypnea, bradycardia, and hypotension that worsened with increased blast intensity, similar to whole-body exposure results in the literature. Some rats exposed to blasts higher than 50 PSI (345 kPa) stopped breathing immediately and died. These autonomic responses were significantly reduced in vagally denervated rats, again similar to whole-body exposure literature. These results support our hypothesis that the ear provides a conduit for blast overpressure energy to traverse the skull and even cause brain injury. To test for peripheral vestibular injury in primary blast, we exposed rats in a similar manner to 40 PSI blasts. We studied behavioral changes using video-oculography to monitor the vestibulo-ocular reflex, functional changes using single-unit recording of vestibular afferent fibers, and end-organ damage by analyzing utricular and horizontal canal stereocilia bundle density changes over time after blast. We found that bundle density decreases progressively for 2 weeks after blast, but utricular (but not horizontal canal) stereocilia bundle density recovers by 8 weeks. Vestibular afferents exhibited decreased firing rates and decreased responses to linear and rotational stimuli. VOR gain decreased 24 hours after blast, gain from 2.0 Hz linear stimulation remained decreased, and VOR phase was more variable than baseline. These results support our hypothesis that blast induces vestibular deficits through damage in the peripheral vestibular system.
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DEDICATION
This dissertation is written in dedication to those who have been injured by blast, and with the hope that it contributes to the prevention, diagnosis, or treatment of those injuries.
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ACKNOWLEDGEMENTS First and foremost, I wish to acknowledge my mentors, Dr. Wu Zhou and Dr. Hong Zhu. Their guidance and support, in the good times and especially in the hard, helped to fortify the grit required for me to complete this endeavor and the many more that will come after. Thank you for those lessons, I will carry them with me for the rest of my life. I would also like to acknowledge the faculty and administration at the University of Mississippi Medical Center, both in the School of Medicine and the School of Graduate Studies in the Health Sciences. Whether as a member of my Advisory Committee, a professor in a course, a preceptor in the clinic, a collaborator in the laboratory, or a guiding hand in the office, I am grateful for the knowledge and direction you have imparted to me. Next, I would like to thank my peers in the School of Medicine, the School of Graduate Studies in the Health Sciences, and those few who shared my path in both. Having you there to compare notes, to cram for exams, to take care of patients, to run experiments, and to celebrate and commiserate afterwards has helped me keep a level head through this process. Finally, I must thank the love and support I have received from my family and friends through these years. The work that has gone into this has not been easy for me, and I know I have likewise not always been easy to be around while working. Thank you for being there for me when I’ve needed you most, especially when I didn’t deserve it. I love you all.
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TABLE OF CONTENTS
SIGNATURE PAGE ............................................................................................ iii ABSTRACT......................................................................................................... iv DEDICATION....................................................................................................... v ACKNOWLEDGEMENTS ................................................................................... vi LIST OF TALBES................................................................................................ ix LIST OF FIGURES .............................................................................................. x LIST OF ABBREVIATIONS ................................................................................. xi
I.
CHAPTER 1........................................................................................................ 1 INTRODUCTION AND REVIEW OF LITERATURE ............................................ 2 Literature Cited ..................................................................................................16 Tables ................................................................................................................28 Figure Legends ..................................................................................................29 Figures...............................................................................................................31
II.
CHAPTER 2.......................................................................................................36 Title Page ..........................................................................................................37 Statement of Permission and Contributions .......................................................38 Abstract .............................................................................................................39 Introduction ........................................................................................................40 Materials and Methods .......................................................................................42 Results...............................................................................................................46 Discussion .........................................................................................................48 Acknowledgements ............................................................................................53 Literature Cited ..................................................................................................54 Figure Legends ..................................................................................................65 Figures...............................................................................................................67
III.
CHAPTER 3.......................................................................................................74 Title Page ..........................................................................................................75 Statement of Permission and Contributions .......................................................76 Abstract .............................................................................................................77 Introduction ........................................................................................................78 Materials and Methods .......................................................................................80 vii
Results...............................................................................................................87 Discussion .........................................................................................................91 Acknowledgements ............................................................................................95 Literature Cited ..................................................................................................96 Figure Legends ................................................................................................102 Figures.............................................................................................................105
IV.
CHAPTER 4.....................................................................................................117 SUMMARY AND DISCUSSION .......................................................................118 Literature Cited ................................................................................................129
APPENDIX.......................................................................................................136
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LIST OF TALBES
Table 1.1 Summary of studies examining the frequency of vestibular dysfunction in individuals with dizziness after blast exposure ............................................................... 28
ix
LIST OF FIGURES
Figure 1.1 Blast Injury Categories..................................................................................31 Figure 1.2 The Friedlander Curve ..................................................................................32 Figure 1.3 Anatomy of the Peripheral Vestibular System ...............................................33 Figure 1.4 Vestibular Hair Cell and Afferent Morphology ...............................................34 Figure 1.5 Three-neuron arc of the Vestibulo-Ocular Reflex (VOR) ...............................35 Figure 2.1 The Targeted Blast Generator ......................................................................67 Figure 2.2 Autonomic Recording Timeline .....................................................................68 Figure 2.3 Respiratory Rate Responses to Blast via the Ear .........................................69 Figure 2.4 Heart Rate Responses to Blast via the Ear ...................................................70 Figure 2.5 Blood Pressure Responses to Blast via the Ear ............................................71 Figure 2.6 ECG Responses to Blast via the Ear ............................................................72 Figure 2.7 Vagal Mediation of Autonomic Responses to Blast Via the Ear ....................73 Figure 3.1 VOR Recording Timeline ............................................................................105 Figure 3.2 Vestibular Afferent Recording Timeline .......................................................106 Figure 3.3 Histology Timeline ......................................................................................107 Figure 3.4 Steady-State Rotational VOR .....................................................................108 Figure 3.5 Transient Rotational VOR ...........................................................................109 Figure 3.6 Steady-State Linear VOR ...........................................................................110 Figure 3.7 Control Recording Example ........................................................................111 Figure 3.8 Baseline Firing Rates..................................................................................112 Figure 3.9 Vestibular Afferent Responses ...................................................................113 Figure 3.10 Utricular Stereocilia Images, Heatmaps, and Histograms .........................114 Figure 3.11 Horizontal Canal Images, Heatmaps, and Histograms ..............................115 Figure 3.12 Mean Stereocilia Bundle Density ..............................................................116
x
LIST OF ABBREVIATIONS
ABVN, Auricular Branch of the Vestibular Nerve AC, Anterior Semicircular Canal ANOVA, Analysis of Variance BINT, Blast-Induced Neurotrauma BPM, Beats Per Minute CR, Corneal Reflection cVEMP, Cervical Vestibular-Evoked Myogenic Potential d, Degree ECG, Electrocardiogram EDTA, Ethylenediaminetetraacetic Acid FFT, Fast Fourier Transform HC, Horizontal Semicircular Canal HIT, Head Impulse Test HPA, Hypothalamic-Pituitary-Adrenal Hz, Hertz ICP, Integrated Circuit Piezoelectric IED, Improvised Explosive Device i.p., Intra-Peritoneal IR, Infrared kHz, Kilohertz kPa, Kilopascal L, Liter
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LE, Long Evans LED, Light Emitting Diode M, Molar MAP, Mean Arterial Pressure min, Minute mL, Milliliter mm, Millimeter mmHg, Millimeter of Mercury ms, Millisecond mV, Millivolt MΩ, Megaohm NIH, National Institutes of Health NTS, Nucleus Tractus Solitarius OEF, Operation Enduring Freedom OIF, Operation Iraqi Freedom PBS, Phosphate Buffered Saline PC, Posterior Semicircular Canal PFA, Paraformaldehyde PPE, Personal Protective Equipment PSI, Pounds per Square Inch PTFE, Polytetrafluoroethylene PTSD, Post-Traumatic Stress Disorder ROS, Reactive Oxygen Species s, Second
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SEM, Standard Error of the Mean SD, Sprague Dawley TBI, Traumatic Brain Injury TM, Tympanic Membrane TNT, Trinitrotoluene um, micrometer US, United States VEMP, Vestibular-Evoked Myogenic Potential VOR, Vestibulo-Ocular Reflex
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CHAPTER 1
INTRODUCTION AND REVIEW OF LITERATURE
2
1.1 BLAST INJURY Explosive ordinance, be it in the form of artillery, claymore, landmine, or improvised explosive devices (IEDs), produce blasts which have become an increasing cause of injury not only in military personnel, but also in civilian victims of terrorist attacks, bystanders in warfare, or even workplace accidents (Ibolja Cernak & Noble-Haeusslein, 2010; Taber, Warden, & Hurley, 2006). Blasts have been deemed responsible for about two-thirds of the combat injuries in Operation Iraqi Freedom (OIF) and Operation Enduring Freedom (OEF), two of the United States’ most recent and highly impactful conflicts (Kocsis & Tessler, 2009). Additionally, civilian blast injuries from IEDs, suicide bombings, and other terror attacks as recent as the attempted New York subway bombing in Manhattan, New York on December 11, 2017 are becoming an all-too-normal injury. Blasts can cause a wide spectrum of injuries ranging in form and severity from a ruptured tympanic membrane (eardrum) to dismemberment, ruptured colon and lungs, and traumatic brain injury (TBI), and are frequently fatal. Permanent disability is a common result from blast and presents in many forms including chronic and/or recurrent dizziness and disequilibrium, loss of limbs or their function, cognitive disability resulting from brain injury, or emotional disabilities in the form of Post-Traumatic Stress Disorder (PTSD) and other emotional disorders. The significance, severity, increasing frequency, and chronic ramifications of blast injury have made its prevention, diagnosis, and treatment a highly important area of study both in the public eye and in scientific research, and as a result more clinical diagnoses have been attributed to the effects of blast exposure than in the past (Burgess et al., 2010; Svetlov et al., 2009).
1.1.1 Categories of blast injury As mentioned above, blast injuries are heterogeneous in both form and severity. Blast injuries have been classified into 4 distinct categories: Primary - injuries due to the overpressure of the blast wave; Secondary – injuries due to objects propelled by the blast wave striking the victim, Tertiary – injuries due to the victim themselves being propelled by the blast wave, and Quaternary – injuries due to any other factor of the blast (I Cernak, Savic, Ignjatovic, & Jevtic, 1999) (Figure 1.1). A single blast can cause multiple injuries in all four categories, or have just a single injury in one category. Primary blast injury is that due to the sudden changes in atmospheric pressure created by the blast wave itself. These changes in pressure have the most obvious effect 3
on organs that contain air-to-tissue interfaces, including multiple spaces in the ears, the lungs, and the intestines. As the blast waves impact the victim, the pressure of the air those organs are exposed to also suddenly and drastically change, frequently leading to rupture of those organs. The tympanic membrane (TM, also known as the eardrum) is one such organ that is exposed directly to the outside air. A blast can cause the pressure in the external ear canal to increase much faster than the pressure in the middle ear can compensate through the Eustachian tube, leading to rupture of the TM and potentially other windows of the ear discussed later. As such, it is the most frequently reported injury resulting from primary blast exposure. Likewise, the blast pressure can travel relatively unimpeded through the nose, pharynx, trachea, and bronchi to rapidly hyperinflate the alveoli of the lungs, leading to their rupture. Pulmonary blast injury can be very rapidly fatal, and was one of the first injuries recognized to be a direct result of the blast overpressure wave. However, current military field reports actually report fewer pulmonary injuries from blast (Bass et al., 2012). This may be due to improved chest protection worn during patrols or on the battlefield. Secondary blast injury is that due to an object being propelled by the blast striking the victim. Many explosive devices capitalize on this injury modality by incorporating outer shells that will fragment into smaller pieces as the blast expands, sending these pieces known as “shrapnel” as projectiles. Additionally, IEDs are frequently made containing loose hard items such as nails, ball-bearings, rocks, or glass shards to achieve a similar effect. Shrapnel injuries often present as penetrative trauma, like that of a gunshot wound, and as such can also be fatal or immediately life threatening due to the risk of trauma to the brain or hemorrhage due to lesion of a major blood vessel. Tertiary blast injury is that due to the victim themselves being propelled by the blast. The victim may then strike a solid object, such as a wall, a vehicle or the ground. Concussion and coup-contrecoup TBIs are common to this injury mechanism, wherein the victim’s head strikes a solid object and their brain’s inertia causes it to “bounce” forward and backward in the skull. Concussion is an active area of study in and of itself, and are frequently seen outside of blast in motor vehicle accidents, full-contact athletic injuries, and in injuries from everyday life (e.g. falls). Quaternary blast injury is that due to any other factor of the blast. This can include burns from the combustion of the explosive, smoke and/or toxic gas inhalation, radiation exposure, and much more. As such, these injuries are highly dependent on the unique 4
characteristics of the blast and the environment in which it occurs. The heterogeneity of these injuries results in their frequent omittance from descriptions of blast injuries and inclusion in studies of blast. While secondary, tertiary, and quaternary blast injuries present similarly to injuries not caused by blast (e.g., penetrative trauma, blunt-force trauma, and exposure, respectively), primary blast injuries are relatively unique. They are frequently insidious in that their signs are hidden during initial first-aid, workup, and treatment, only presenting themselves long after the victim has been stabilized (and possibly after a critical treatment period) (Bass et al., 2012; Burgess et al., 2010; Lemonick, 2011; Phillips, 1986). For these reasons, much less is known about primary blast injury than the other modalities. Since blast injury is increasing both on and off the battlefield, strategies for prevention, detection, and treatment of primary blast injuries have become a vitally important area of study.
1.1.2 Physics of primary blast In addition to their injuries, the blasts themselves occur through multiple mechanisms. An explosion could be produced from combustion of a fuel, such as trinitrotoluene (TNT), plastic explosives, gunpowder, or even powdered organic matter, such as sawdust suspended in air (Abbasi & Abbasi, 2007; Crowl, 2003; Eckhoff, 2005). Explosions can also occur from the sudden release of a pressurized gas, such as the rapid depressurization of a gas cylinder or even an aerosol can. Finally, explosions can also be produced by nuclear fission or fusion, leading to their own unique, and often gargantuan, impact. Blast waves can be produced by all of these explosions. Blast waves, in the physical sense, are produced by the rapid expansion of gas as the explosive detonates, canister depressurizes, or otherwise, producing a supersonic front that ultimately drives a shock wave out from the source (Alay, Skotak, Misistia, & Chandra, 2017; Cullis, 2001; Goel, Vasant, & Gupta, 2012; Ngo, Mendis, Gupta, & Ramsay, 2007). In high chemical explosives such as TNT, the explosion is a detonation, meaning the propagation of the combustion moves faster than the speed of sound. In low chemical explosives such as gunpowder, the explosion is a deflagration, which burns very quickly but does not actually exceed the speed of sound. The gasses produced, however, and the direction they are focused can still form a blast wave.
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Primary blast can be modeled using what is referred to as the modified Friedlander equation (Friedlander, 1946; Goel et al., 2012). 𝑃(𝑡) = 𝑃0 + 𝑃𝑝𝑜𝑠 (1 −
𝑡 𝑡𝑝𝑜𝑠
)𝑒
−𝑏
𝑡 𝑡𝑝𝑜𝑠
Where P(t) is the pressure at any given time, P0 is atmospheric pressure, Ppos is the maximum pressure, t is the time at which pressure is measured, tpos is the time of maximum pressure, and b is a parametric measure of curve decay. Figure 1.2 shows the modified Friedlander curve. The Friedlander curve is useful in modeling a blast occurring in an open space, such as one produced by in-air exploding ordinance. However, real-world blasts are seldom “perfect” and occur with many obstacles, including the ground, walls, and the victim itself, influencing the characteristics of the blast wave. However, these complex waves are difficult to predict or control, leading to the acceptance of the Friedlander wave as an ideal for blast research since the singular energy of the blast can be more confidently stated to be the cause of any responses seen.
1.1.3 Mechanisms of primary blast injury The rapid pressure changes induced by blast exposure cause primary blast injury. Primary blast injury itself is heterogenic in form and severity, and is influenced by many factors including the intensity of the blast, the distance between the blast and the victim, the size, shape, and location of obstacles between the blast and the victim including the victim’s clothing and personal protective equipment (PPE), and even the direction the victim is facing. Injuries can be as minor as transient disorientation to as major as ruptured lungs and bowels and severe diffuse axonal injury, and may even cause death. However, for a blast to have that strong an effect, the victim will likely also endure other forms of blast injury, particularly tertiary and quaternary. For a survivor to have experienced strictly primary blast, they likely would have to have been far enough away that the effect of fire, heat, and toxic gasses would be negligible (Alay et al., 2017). Primary blast injury causes the most immediately noticeable injuries at air-tissue interfaces. The most common tissues affected include the ear (where the external ear canal communicates the blast overpressure to the TM, which may rupture and allow pressure to impact the round window and oval window), the lungs (where the airway 6
communicates the blast overpressure to the alveoli, which may rupture and allow pressure to force air into the pleural space), and the intestines (where although there is not a direct communication to the outside air, the shockwave may cause cavitation at the air-liquid interfaces of the lumen and result in perforations into the peritoneal space). For these immediately noticeable injuries, those to the lungs are immediately life-threating, those to the bowel is acutely life-threatening, but the injuries to the ear are appropriately triaged based on other injuries. However, many studies have drawn a correlation between a ruptured TM and blast-induced neurotrauma (BINT), suggesting that the presence of a ruptured TM is a useful biomarker in the diagnosis of BINT(DePalma, Burris, Champion, & Hodgson, 2005; Gan, Nakmali, Ji, Leckness, & Yokell, 2016; Hicks, Fertig, Desrocher, Koroshetz, & Pancrazio, 2010; Kronenberg, Ben-Shoshan, & Wolf, 1993). This is especially notable as BINT is not as immediately noticeable but can be significantly debilitating in the chronic blast condition.
1.1.4 Blast-induced neurotrauma (BINT) Traumatic brain injury, including BINT, is the “signature injury” of U.S. soldiers involved in recent conflicts in Iraq and Afghanistan (Huber et al., 2013; Mac Donald et al., 2014; Moss, King, & Blackman, 2009; Pham et al., 2015). Primary blast exposure may cause BINT through multiple pathways such as direct passage of the blast wave through the skull, compression of the torso resulting in transfer of the blast wave’s kinetic energy to the brain via hydraulic oscillations within the vasculature (also known as hydrostatic shock), and hypoxia through over-activation of the parasympathetic nervous system (Bolander, Mathie, Bir, Ritzel, & VandeVord, 2011; Ibolja Cernak, 2010; Ibolja Cernak, Wang, Jiang, Bian, & Savic, 2001; Chavko et al., 2011; Courtney & Courtney, 2009; Long et al., 2009; Moss et al., 2009; Säljö, Arrhén, Bolouri, Mayorga, & Hamberger, 2008; Simard et al., 2014). The parasympathetic response, specifically, is hypothesized to be a result of hyperinflation of the lungs stimulating alveolar juxtacapillary J-receptors innervated by vagal fibers, leading to apnea then tachypnea, bradycardia, and hypotension, which in turn lead to a Bezold-Jarish reflex further deepening bradycardia and hypotension (Ibolja Cernak, 2010; Krohn, Whitteridge, & Zuckerman, 1942; Zucker, 1986; Zuckerman, 1940). Another proposed mechanism suggests that the foramina of the skull (e.g., the acoustic meatus, optic canal, nasal cavity, or foramen magnum) can provide a conduit for the blast wave to enter the cranial vault in addition to the established 7
mechanisms mentioned above (Akula, Hua, & Gu, 2015; Hicks et al., 2010; Ropper, 2011). The advanced combat helmet, the current helmet of the U.S. Army, uses layers of Kevlar and a foam suspension to protect the skull from penetrating and blunt-force injuries, but leaves the ears, eyes, nose, and mouth all exposed to the surrounding air, permitting pressure from a blast to interact (Meaney, Morrison, & Dale Bass, 2014; Moore et al., 2009). Some studies have found that the presence of a helmet can actually amplify blast energy transmission to the ear (Gan et al., 2016; Moss et al., 2009). Nonetheless, soldiers frequently wear little to no ear protection, citing a necessity for situational awareness through unhindered sound localization (Abel, 2008; Brown et al., 2015; Clasing & Casali, 2014; Jones & Pearson, 2016; Killion, Monroe, & Drambarean, 2011). As a result of the lack of ear protection and blast’s great effects on air/tissue interfaces, a perforated eardrum is the most frequently reported blast injury (Ibolja Cernak & Noble-Haeusslein, 2010; Choi, 2012; Darley & Kellman, 2010; DePalma et al., 2005; Gan et al., 2016; Garth, 1994; Helling, 2004; Katz, Ofek, Adler, Abramowitz, & Krausz, 1989; Kronenberg et al., 1993; Mayorga, 1997; Patterson & Hamernik, 1997; Phillips, 1986)
1.2 PRIMARY BLAST EFFECTS ON BALANCE Dizziness and imbalance are frequent complaints among active duty service members and veterans who have experienced blast exposure (Akin & Murnane, 2011; Akin, Murnane, Hall, & Riska, 2017; Cohen et al., 2002; Fausti et al., 2009; Franke, Walker, Cifu, Ochs, & Lew, 2012; Hoffer et al., 2010; M. Scherer, Burrows, Pinto, & Somrack, 2007; Van Campen, Dennis, King, Hanlin, & Velderman, 1999). For example, a clinical study of 148 U.S. military personnel showed that 76% of the individuals evaluated 4-30 days after blast exposure complained of dizziness, and 47% complained of vertigo (Hoffer et al., 2010). Though most cases of dizziness after BINT are self-limiting, blastinduced dizziness have been documented to last for six months or even longer (Akin et al., 2017; Cohen et al., 2002; Dikmen, Machamer, Fann, & Temkin, 2010). The causes of chronic post-blast dizziness are unknown, and it has been shown that if dizziness is present at three months, the likeliness of it worsening over time is greater than other postconcussion symptoms (Chamelian & Feinstein, 2004; Yang, Tu, Hua, & Huang, 2007). Blast-induced vestibular deficits not only impair service members’ performance during deployment, but also have been correlated with delayed return to duty, impaired performance and daily living, diminished quality of life, increased self-perception of 8
disability, and increased anxiety and depression (Franke et al., 2012). Vestibular and cochlear injury have also been associated with a three-fold rise in incidence of PTSD among those with TBIs compared to those without vestibulocochlear damage (Haber, Chandler, & Serrador, 2016; Tigno, Armonda, Bell, & Severson, 2017). Thus, the development of programs for the prevention, assessment, and treatment of blast-induced vestibular deficits is critical to relieve this burden in military and civilian populations. Although there is considerable clinical evidence of blast-induced vestibular injuries in humans, little is known about the underlying mechanisms (Akin et al., 2017).
1.2.1 Potential mechanisms of blast-induced dizziness Post-blast dizziness is a challenging problem in that blast can cause multiple injuries that could lead to dizziness. The visual system, the vestibular system, and proprioception all contribute to balance. It is well known that blast exposure can induce widespread brain injuries (I Cernak et al., 1999; Ibolja Cernak, Savic, Malicevic, Zunic, Radosevic, Ivanovic, et al., 1996; Ibolja Cernak et al., 2001; Du et al., 2013). Pathological changes in any of these pathways would contribute to the dizziness symptoms reported by TBI patients. Dizziness and imbalance, therefore, may not be specific indicators for vestibular injury (Di Fabio, 1995). Thus, empirical studies on the effects of blast on the vestibular system are important in elucidating the specific injuries caused by blast. The peripheral vestibular system, i.e., the organs which detect changes in head position and contribute to balance, are housed in the inner ear. They consist of three ampullae of semicircular canals, which detect rotation, and two maculae of otolith organs, which detect linear translation (Figure 1.3). These end organs consist of two types of hair cells, type I and type II, which synapse with afferents via calyxes and boutons, respectively (Figure 1.4) (Eatock & Songer, 2011). Those afferents can be calyx-only, which synapse with type 1 hair cells, are irregular in firing frequency, are highly sensitive to fast (high frequency) stimulation but also extinguish quickly; bouton-only, which synapse with type II hair cells, are regular in firing frequency, are not as sensitive to fast stimulation as irregulars, but have much slower extinguishing rates thus contributing to the sensation of the body’s static position; and dimorphic, which synapse with both type I and type II hair cells, have irregular firing frequencies, and have sensitivities and extinguishing characteristics in the spectrum between calyx- and bouton-only afferents (Cullen, 2012).
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These afferents synapse with the central vestibular nuclei in the brainstem, where the vestibular inputs are processed. A number of reflexes are mediated through the vestibular system, including the vestibulo-ocular reflex (VOR, Figure 1.5) (Purves et al., 2001). The VOR is important in keeping the eyes’ gaze steady as the head moves, and acts through a three-neuron arc (hair cell to vestibular afferent, afferent to central vestibular nuclei, nucleus to ocular motor neuron, ocular motor neuron to extraocular muscle). It is one of the fastest known reflexes with a latency of about 10 milliseconds. Deficits in the VOR are detrimental to an individual’s ability to ambulate and frequently result in symptoms of vertigo and disequilibrium, especially when moving. Similar vestibular reflexes are the vestibulo-collic reflex and the vestibulo-spinal reflex, which help keep head position steady through activation of neck and spine muscles, respectively. Another vestibular reflex is the vestibulo-cardiac reflex, in which activation of the vestibular system induces an increase in blood pressure, which may help prevent orthostatic hypotension (Radtke, Popov, Bronstein, & Gresty, 2000). As an air- and fluid-filled organ directly exposed to the surrounding air, the ear is particularly vulnerable to injury by primary blast. Though blasts’ effects on the auditory system are an active area of study (S.-I. Cho et al., 2013; Dougherty et al., 2013; Garth, 1994; Patterson & Hamernik, 1997), much less is known about the impact of blast on the vestibular organs. All data on vestibular injuries to blast are prospective/retrospective studies in humans; to date, no animal studies exist on PubMed, CINAHL, or the Mendeley catalog that experimentally test the effects of blast on the peripheral vestibular system (search terms: Vestibular and Blast). This may be in part due to the difficulty in isolating the blasts’ effects on and through the ear from the systemic response of the rest of the body to the blast, as described below. Thus, a model of blast exposure that can exclusively expose a small area of tissue to blast while having negligible effects on areas of known systemic activation is necessary for study of the vestibular system’s response to blast.
1.2.2 Semicircular canal vs. otolith injury in blast In the studies in humans, some histological studies have been performed postmortem, and the limited analyses have suggested that the maculae of the otolith organs are more vulnerable to the strain/shear effects of blast than the semicircular canals (Kerr, 1980; Kerr & Byrne, 1975). Clinicians assessing patients after blast exposure typically use tests of the vestibulo-ocular reflex (VOR) including the caloric test, and, less frequently, 10
vestibular-evoked myogenic potentials (VEMPs) as a measure of vestibular function. Of the clinical studies that performed specific vestibular tests to study blast-induced dizziness, caloric testing (a marker for horizontal semicircular canal injury) is the only test performed in a vast majority of cases, and rates of horizontal canal involvement range from 0 to 40% and are dependent on the intensity and closeness of the blast (Akin & Murnane, 2011; Akin et al., 2017; Cohen et al., 2002; M. R. Scherer, Burrows, et al., 2011; Shupak, Doweck, Nachtigal, Spitzer, & Gordon, 1993; Van Campen et al., 1999). However, two of these studies did include cervical vestibular evoked myogenic potentials (cVEMPs), which showed that otolith organ involvement was greater than that of the horizontal semicircular canals (Akin & Murnane, 2011; M. R. Scherer, Burrows, et al., 2011). These studies are summarized in Table 1.1, adapted from Akin et al., 2017. Serrador and others used unilateral centrifugation and found that approximately 30% of blast-exposed veterans exhibited unilateral otolith dysfunction without horizontal canal impairment (Serrador, Blatt, Acosta, Ghobreal, & Wood, 2012). These findings are supported by studies supporting that the otolith organs are more susceptible to noise injury than the semicircular canals, particularly due to their location in a relatively less protected portion of the inner ear, and their proximity to the oval window (Akin et al., 2012; Hsu, Wang, Lue, Day, & Young, 2008; Perez, Freeman, Cohen, & Sohmer, 2002; Y. P. Wang, Hsu, & Young, 2006; Wu & Young, 2009). Thus, testing for vestibular injury in victims of blast exposure should not be limited to nonspecific (e.g. oculomotor, orthostatic) or canalspecific (caloric, rotary VOR) tests.
1.3 MODELS OF BLAST INJURY Blast was likely first noted to cause injury with the development of gunpowder by Chinese alchemists in the 9th century. However, injury due to primary blast is not described in empirical medical detail until written in notes written by doctors treating bombing victims in the first world war (Mott, 1916). By the second world war, researchers in both the Allied and Axis powers were performing experiments investigating primary blast injury and mortality in animals and, in the case of Nazi scientists, on human concentration camp victims (German aviation medicine, World War II., 1950; Zuckerman, 1940). Those early experiments were performed using what is currently referred to as the open field model of blast, which simply put is the use of actual explosives in an open field with sensors and/or restrained animal. As time progressed and experiments moved from fields into 11
laboratories, the shock tube began to be employed for animal studies of blast injury. To this day, the shock tube is currently the most popular laboratory model of blast (Alay et al., 2017; Fomin, 2010). There are a few other, more exotic methods for producing blast waves in experimental setups including high-energy laser activation.
1.3.1 Open field model of blast injury The open field model of blast injury is just that: exposure to a blast from a conventional explosive in an open field. Its use has waned in laboratory research in favor of the more precisely controllable shock tube, but its accurate representation of blast waves, especially as they would occur in a real-world explosion, result in its continued use in blast injury research (Pun et al., 2011; Rubovitch et al., 2011; Song et al., 2016). In the open field model of blast, subjects are placed a specific distance from an explosive device. The device itself may be suspended or located directly on the ground. The device is loaded with a specific amount of fuel to produce a blast of relatively predictable intensity. The device is then activated, and the resultant shockwave strikes the subjects. Additionally, the fireball, debris, and other aspects of a conventional explosive will contribute to the injury. The open field model is considered to be the most accurate representation of the human condition in blast injury (Kovacs, Leonessa, & Ling, 2014; Song et al., 2016; Xiong, Mahmood, & Chopp, 2013). However, it is also one of the least controllable modalities of blast exposure. The ground, for example, will provide a point of reflection for the blast wave to reflect and cause a more complex injury than intended. Additionally, since an actual detonation is produced, the fireball, debris, blast wind, and more will produce primary, secondary, tertiary, and quaternary blast injuries. Pun et al, 2011 attempted to prevent secondary injury by placing a concrete block directly between the animals and the explosive charge, but this resulted in the creation of a more complex blast wave impacting the animals (Nakagawa et al., 2011; Pun et al., 2011; Song et al., 2016). Finally, due to the nature of explosive materials, the permitting required for their acquisition, and the toxic byproducts they produce, these experiments are highly dangerous to the investigators, can be very expensive to undertake, and are very time consuming in their set-up and resultant clean-up.
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1.3.2 Shock tube model of blast injury Shock tubes were used to produce shockwaves as early as 1899 for the study of the physical processes of combustion and detonation (Fomin, 2010; Vieille, 1899). The shock tube continued to be used primarily for the physical study of combustion and of the resultant shock waves themselves until around World War II, when shock tubes were employed to study the effects of shockwaves on various material properties. At this point, the open field model of blast was employed for studying blast injury. In 1955, a compressed-gas driven shock tube was developed for the study of blast waves’ effects on mice (Celander, Clemedson, Ericsson, & Hultman, 1955). Shock tubes can employ either actual combustion or compressed gas to generate a blast wave. The wave then propagates down a tube to a specific point where the experimental subject is loaded. This tube allows the properties of the shock wave to be more precisely controlled than those in open field models, especially regarding producing primary blast injuries with little to negligible secondary or quaternary injuries. The blast wind still can have a significant tertiary effect, however, especially if the subject is not secured (Alay et al., 2017). Thanks to this exclusivity, the effects of primary blast injury can now be studied without confounding factors of the other mechanisms of blast injury. Secondly, shock tubes are more economical and safe compared to open-field blasts. However, the shock tube has its own caveats. The nature of the tube itself, a relatively narrow cylinder, can cause internal reflection of the blast wave. This is amplified in closed-end shock tubes, in which the blast pressures can be higher with less fuel and/or compressed air, but the closed tube causes internal reflections through the length of the tube, exposing the animal to repeated complex shock waveforms (Y. Chen & Constantini, 2013). This is reduced in open-ended shock tubes, but the animals themselves still produce a reflection of the wave that causes issues related to high pressure gas confinement. Thus, shock tubes tend to cause complex shock waves following the original shock front, reducing their accuracy in approximating real-world blasts (Y. Chen & Huang, 2011).
1.3.3 Alternative models of blast injury The inherent caveats of open-field and shock tube models of blast injury have led to the development of unique blast injury paradigms that vary greatly from laboratory to
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laboratory. One such paradigm uses a laser to cause a shockwave at a particular point in the ear (Niwa et al., 2016). While these approaches aim to reduce one or more of the caveats of open-field and shock-tube models, they introduce new, unforeseen challenges that reduce repeatability (Y. Chen & Huang, 2011). The design and use of models of blast exposure is a topic of debate between physicists, who are frequently strongly in favor of precisely controlled primary blast overpressure waves (section 1.1.2), and biomedical scientists, who tend to posit that the energy transfer is more important in the study of blast injury than the mechanisms of the blast wave itself (Mediavilla Varas et al., 2011; Moore et al., 2009; Needham, Ritzel, Rule, Wiri, & Young, 2015; Risling et al., 2011; Sundaramurthy et al., 2012; Turner et al., 2013; Wood et al., 2013; Xiong et al., 2013)
1.4 A LOCAL EXPOSURE MODEL FOR STUDYING THE EAR’S ROLE IN BINT AND THE EFFECTS OF PRIMARY BLAST ON THE VESTIBULAR SYSTEM As mentioned above, dizziness and disequilibrium are common complains among those exposed to primary blast overpressure. Studies regarding the vestibular system’s response to blast have all been observational in humans. To date, no studies of the mechanisms of vestibular deficit following blast exposure have been performed in animals. This may be in part due to the difficulty in isolating the blasts’ effects on and through the ear from the systemic response of the rest of the body to the blast. Thus, a model of blast exposure that can exclusively expose a small area of tissue to blast while having negligible effects on areas of known systemic activation is necessary for study of the ear’s role in blast injury and the vestibular system’s response to blast.
To achieve such a study, a model that specifically and exclusively impacts the ear would be an ideal method. The goals of this dissertation were twofold:
(1) To develop a model of blast overpressure injury that exclusively impacts the ear. This was to reduce the confounding effects that systemic injury will have on the organs of the peripheral vestibular system. It must be predictable, reliable, and overcome the caveats mentioned in section 1.2. In validating this model, it was found that the autonomic responses to blast, which previously were reported to be
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due to lung injury, could also be elicited by ear-only blast exposure. As described in section 1.2.3, any blast model will have its own unique caveats, so for this model the caveats must also be recognized and reconciled against the alternative methods. And (2) To use the new model of blast overpressure to initiate studies on the effects that blast overpressure has on the peripheral vestibular system. Hair cell stereocilia injury was quantified at varying durations out to 2 months after blast exposure. The responses of vestibular afferent fibers were also assessed to determine the functional deficits of the peripheral vestibular system. Finally, the vestibulo-ocular reflex was tested before and at varying timepoints after blast to determine the timecourse of vestibular functional deficits following blast exposure.
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1.5 LITERATURE CITED Abbasi, T., & Abbasi, S. A. (2007). Dust explosions-Cases, causes, consequences, and control. Journal of Hazardous Materials. https://doi.org/10.1016/j.jhazmat.2006.11.007 Abel, S. M. (2008). Barriers to Hearing Conservation Programs in Combat Arms Occupations. Aviation, Space, and Environmental Medicine, 79(6), 591–598. https://doi.org/10.3357/ASEM.2262.2008 Akin, F. W., & Murnane, O. D. (2011). Head injury and blast exposure: Vestibular consequences. Otolaryngologic Clinics of North America. https://doi.org/10.1016/j.otc.2011.01.005 Akin, F. W., Murnane, O. D., Hall, C. D., & Riska, K. M. (2017). Vestibular consequences of mild traumatic brain injury and blast exposure: a review. Brain Injury, 31(9), 1188– 1194. https://doi.org/10.1080/02699052.2017.1288928 Akin, F. W., Murnane, O. D., Tampas, J. W., Clinard, C., Byrd, S., & Kelly, J. K. (2012). The Effect of Noise Exposure on the Cervical Vestibular Evoked Myogenic Potential. Ear and Hearing, 1. https://doi.org/10.1097/AUD.0b013e3182498C5f Akula, P., Hua, Y., & Gu, L. (2015). Blast-induced mild traumatic brain injury through ear canal: A finite element study. Biomedical Engineering Letters, 5(4), 281–288. https://doi.org/10.1007/s13534-015-0204-0 Alay, E., Skotak, M., Misistia, A., & Chandra, N. (2017). Dynamic loads on human and animal surrogates at different test locations in compressed-gas-driven shock tubes. Shock Waves, 1–12. https://doi.org/10.1007/s00193-017-0762-4 Bass, C. R., Panzer, M. B., Rafaels, K. A., Wood, G., Shridharani, J., & Capehart, B. (2012). Brain Injuries from Blast. Annals of Biomedical Engineering, 40(1), 185–202. https://doi.org/10.1007/s10439-011-0424-0 Bolander, R., Mathie, B., Bir, C., Ritzel, D., & VandeVord, P. (2011). Skull Flexure as a Contributing Factor in the Mechanism of Injury in the Rat when Exposed to a Shock Wave. Annals of Biomedical Engineering, 39(10), 2550–2559. https://doi.org/10.1007/s10439-011-0343-0
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Risling, M., Plantman, S., Angeria, M., Rostami, E., Bellander, B.-M. B.-M., Kirkegaard, M., … Davidsson, J. (2011). Mechanisms of blast induced brain injuries, experimental studies in rats. NeuroImage. https://doi.org/10.1016/j.neuroimage.2010.05.031 Ropper, A. (2011). Brain injuries from blasts. The New England Journal of Medicine, 364(22), 2156–2157. https://doi.org/10.1056/NEJMe1102187 Rubovitch, V., Ten-Bosch, M., Zohar, O., Harrison, C. R., Tempel-Brami, C., Stein, E., … Pick, C. G. (2011). A mouse model of blast-induced mild traumatic brain injury. Experimental Neurology, 232(2), 280–289. https://doi.org/10.1016/j.expneurol.2011.09.018 Säljö, A., Arrhén, F., Bolouri, H., Mayorga, M., & Hamberger, A. (2008). Neuropathology and pressure in the pig brain resulting from low-impulse noise exposure. Journal of Neurotrauma, 25(12), 1397–1406. https://doi.org/10.1089/neu.2008.0602 Scherer, M., Burrows, H., Pinto, R., & Somrack, E. (2007). Characterizing self-reported dizziness and otovestibular impairment among blast-injured traumatic amputees: a pilot study. Military Medicine, 172(7), 731–737. https://doi.org/Article Scherer, M. R., Burrows, H., Pinto, R., Littlefield, P., French, L. M., Tarbett, A. K., & Schubert, M. C. (2011). Evidence of central and peripheral vestibular pathology in blast-related traumatic brain injury. Otology & Neurotology : Official Publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology, 32(4), 571–580. https://doi.org/10.1097/MAO.0b013e318210b8fa Serrador, J. M., Blatt, M., Acosta, A., Ghobreal, B., & Wood, S. (2012). Blast exposure is associated with unilateral vestibular damage in US veterans. In Proceedings of the 35th annual association for research in otolaryngology midwinter meeting. Baltimore, MD. Shupak, A., Doweck, I., Nachtigal, D., Spitzer, O., & Gordon, C. R. (1993). Vestibular and Audiometric Consequences of Blast Injury to the Ear. Archives of Otolaryngology-Head & Neck Surgery, 119(12), 1362–1367. https://doi.org/10.1001/archotol.1993.01880240100013
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Simard, J. M., Pampori, A., Keledjian, K., Tosun, C., Schwartzbauer, G., Ivanova, S., & Gerzanich, V. (2014). Exposure of the thorax to a sublethal blast wave causes a hydrodynamic pulse that leads to perivenular inflammation in the brain. Journal of Neurotrauma, 31(14), 1292–1304. https://doi.org/10.1089/neu.2013.3016 Song, H., Cui, J., Simonyi, A., Johnson, C. E., Hubler, G. K., DePalma, R. G., & Gu, Z. (2016, August 21). Linking blast physics to biological outcomes in mild traumatic brain injury: Narrative review and preliminary report of an open-field blast model. Behavioural Brain Research. https://doi.org/10.1016/j.bbr.2016.08.037 Sundaramurthy, A., Alai, A., Ganpule, S., Holmberg, A., Plougonven, E., & Chandra, N. (2012). Blast-Induced Biomechanical Loading of the Rat: An Experimental and Anatomically Accurate Computational Blast Injury Model. Journal of Neurotrauma, 29, 2352–2364. https://doi.org/10.1089/neu.2012.2413 Svetlov, S. I., Larner, S. F., Kirk, D. R., Atkinson, J., Hayes, R. L., & Wang, K. K. W. W. (2009). Biomarkers of blast-induced neurotrauma: profiling molecular and cellular mechanisms of blast brain injury. Journal of Neurotrauma, 26(6), 913–921. https://doi.org/10.1089/neu.2008.0609 Taber, K. H., Warden, D. L., & Hurley, R. A. (2006). Blast-related traumatic brain injury: what is known? The Journal of Neuropsychiatry and Clinical Neurosciences, 18(2), 141–145. https://doi.org/10.1176/jnp.2006.18.2.141 Tigno, T. A., Armonda, R. A., Bell, R. S., & Severson, M. A. (2017). The vestibulocochlear bases for wartime posttraumatic stress disorder manifestations. Medical Hypotheses, 106, 44–56. https://doi.org/10.1016/j.mehy.2017.06.027 Turner, R. C., Naser, Z. J., Logsdon, A. F., DiPasquale, K. H., Jackson, G. J., Robson, M. J., … Rosen, C. L. (2013). Modeling clinically relevant blast parameters based on scaling principles produces functional & histological deficits in rats. Experimental Neurology, 248, 520–529. https://doi.org/10.1016/j.expneurol.2013.07.008 Van Campen, L. E., Dennis, J. M., King, S. B., Hanlin, R. C., & Velderman, A. M. (1999). One-year vestibular and balance outcomes of Oklahoma City bombing survivors. Journal of the American Academy of Audiology, 10(9), 467–483.
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Vieille, P. (Labatoire C. des P. et S. (1899). Sur les discontinuites produites par la detente bruque de gaz comprimes. Comptes Rendus, 129(1228), 68. Wang, Y. P., Hsu, W. C., & Young, Y. H. (2006). Vestibular evoked myogenic potentials in acute acoustic trauma. Otol Neurotol, 27(7), 956–961. https://doi.org/10.1097/01.mao.0000231590.57348.4b\r00129492-200610000-00012 [pii] Wood, G. W., Panzer, M. B., Yu, A. W., Rafaels, K. A., Matthews, K. A., Bass, C. R., … et al. (2013). Scaling in blast neurotrauma. In International Research Council on the Biomechanics of Injury Conference, IRCOBI 2013 (pp. 549–558). Retrieved from http://www.scopus.com/inward/record.url?eid=2-s2.084896648583&partnerID=40&md5=2e7b921b9abc0213a389eb1d252ff787 Wu, C. C., & Young, Y. H. (2009). Ten-year longitudinal study of the effect of impulse noise exposure from gunshot on inner ear function. International Journal of Audiology, 48(9), 655–660. https://doi.org/10.1080/14992020903012481 Xiong, Y., Mahmood, A., & Chopp, M. (2013). Animal models of traumatic brain injury. Nature Reviews. Neuroscience, 14(2), 128–142. https://doi.org/10.1038/nrn3407 Yang, C.-C., Tu, Y.-K., Hua, M.-S., & Huang, S.-J. (2007). The association between the postconcussion symptoms and clinical outcomes for patients with mild traumatic brain injury. The Journal of Trauma, 62(3), 657–663. https://doi.org/10.1097/01.ta.0000203577.68764.b8 Zucker, I. H. (1986). Left ventricular receptors: physiological controllers or pathological curiosities? Basic Research in Cardiology, 81(6), 539–557. https://doi.org/10.1007/BF02005179 Zuckerman, S. (1940). EXPERIMENTAL STUDY OF BLAST INJURIES TO THE LUNGS. The Lancet, 236(6104), 219–224. https://doi.org/10.1016/S01406736(01)08726-8
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1.6 TABLES Table 1.1 Summary of studies examining the frequency of vestibular dysfunction in individuals with dizziness after blast exposure N
TBI
Abnormal Caloric
Abnormal
Presence
Test
cVEMP
Not Specified
2/5 (40.0%)
Not Tested
(Shupak et al., 1993)
5
(Van Campen et al.,
27 16/27
2/27 (7.4%)
Not Tested
(Cohen et al., 2002)
17 Not Specified
0/17 (0.0%)
Not Tested
(Akin & Murnane, 2011)
31 15/31
7/31 (22.5%)
16/31 (51.6%)
(M. R. Scherer,
11 5/11
1/11 (9.1%)
3/11 (27.3%)
1999)
Burrows, et al., 2011)
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1.7 FIGURE LEGENDS Figure 1.1 Blast Injury Categories Blast injuries can be divided into four categories. Primary injury is that due to the overpressure wave of the blast and is unique to blast. Secondary injury is from a solid object propelled by the blast striking the victim, frequently resulting in a “shrapnel” penetrative trauma. Tertiary injury is from the victim themselves being propelled by the blast and striking a solid object, frequently resulting in a “coup-contracoup” concussive injury. Quaternary injury refers to any other blast-induced injury that does not fall in any of the previous categories, e.g., burns, toxic smoke inhalation, and radiation exposure.
Figure 1.2 The Friedlander Curve The Friedlander curve illustrates the waveform from the Friedlander equation, which describes the overpressure waveform produced by an “ideal” blast in an open space. It consists of a sharp overpressure followed by exponential decay into negative pressure (i.e., vacuum), followed by slow return to ambient pressure. (adapted from Goel, 2014)
Figure 1.3 Anatomy of the Peripheral Vestibular System The peripheral vestibular system is housed in the inner ear and consists of three ampullae of semicircular canals (Superior, Horizontal, and Posterior Canals) which detect rotation in pitch (forward-backward nodding movement), roll (side-to-side tilt movement), and yaw (left-right head-shaking movement), and two maculae of otolith organs (Utricle and Saccule) with detect linear translation with respect to lateral movement (forward, backward, left, right) or vertical movement (up, down, gravity). (adapted from “Neuroscientifically Challenged. Know your brain: Vestibular System,” 2015)
Figure 1.4 Vestibular Hair Cell and Afferent Morphology The vestibular end organs consist of two types of hair cells, type I and type II, which synapse with afferents via calyxes and boutons, respectively. Those afferents can be calyx-only, which synapse with type 1 hair cells, are irregular in firing frequency, are highly sensitive to fast (high frequency) stimulation but also extinguish quickly; bouton-only, which synapse with type II hair cells, are regular in firing frequency, are not as sensitive to fast stimulation as irregulars, but have much slower extinguishing rates thus contributing to the sensation of the body’s static position; and dimorphic, which synapse with both type 29
I and type II hair cells, have irregular firing frequencies, and have sensitivities and extinguishing characteristics in the spectrum between calyx- and bouton-only afferents. (adapted from “Eatock Lab: Signaling in the inner ear • Current Projects,”) Figure 1.5 Three-neuron arc of the Vestibulo-Ocular Reflex (VOR) The stereocilia of the vestibular hair cells are deflected by motion, which changes the release of glutamate from the hair cell to the afferent synapse. The afferent carries that signal from the hair cells in the inner ear to the central vestibular nuclei in the brainstem, which process the signal. They then synapse with ocular motor neurons, which in turn induce contraction or relaxation to keep the eyes’ gaze steady during head movement.
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1.8 FIGURES
Figure 1.1 Blast Injury Categories
31
Adapted from Friedlander 1946, Goel 2014
Figure 1.2 The Friedlander Curve
32
(adapted from “Neuroscientifically Challenged. Know your brain: Vestibular System,” 2015)
Figure 1.3 Anatomy of the Peripheral Vestibular System
33
(adapted from “Eatock Lab: Signaling in the inner ear • Current Projects”)
Figure 1.4 Vestibular Hair Cell and Afferent Morphology
34
Figure 1.5 Three-neuron arc of the Vestibulo-Ocular Reflex (VOR)
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CHAPTER 2
AUTONOMIC RESPONSES TO BLAST OVERPRESSURE CAN BE ELICITED BY EXCLUSIVELY EXPOSING THE EAR IN RATS David S. Sandlin1,2, Yue Yu3, Jun Huang3, Chunming Zhang3,7, Alberto A. Arteaga3, John K. Lippincott2, Erin O.H. Peeden2, Ryan R. Guyton1, Lan Chen4, Laura L.S. Beneke2, Jerome C. Allison3, Hong Zhu3,5, Wu Zhou3,5,6* Journal of Otology https://doi.org/10.1016/j.joto.2018.01.001 Submitted December 7th, 2017, Revised January 28th, 2018, Accepted January 30th, 2018, Available online March 9th, 2018. 1
Graduate Program in Neuroscience, University of Mississippi Medical Center, Jackson, Mississippi, United States of America 2
School of Medicine, University of Mississippi Medical Center, Jackson, Mississippi, United States of America 3
Department of Otolaryngology and Communicative Sciences, University of Mississippi Medical Center, Jackson, Mississippi, United States of America 4
Summer Undergraduate Research Experience, University of Mississippi Medical Center, Jackson, Mississippi, United States of America 5
Department of Neurobiology and Anatomical Sciences, University of Mississippi Medical Center, Jackson, Mississippi, United States of America 6
Department of Neurology, University of Mississippi Medical Center, Jackson, Mississippi, United States of America 7
Department of Otolaryngology, First Affiliated Hospital, Shanxi Medical University, 85 Jiefang S Rd, Yingze Qu, Taiyuan Shi, Shanxi Sheng, China
* Corresponding author Wu Zhou, Ph.D. Department of Otolaryngology and Communicative Sciences University of Mississippi Medical Center 2500 North State St. Jackson, MS 39216 Email:
[email protected] Phone: (601) 815-4735 Fax: (601) 984-5085 HZ and WZ are Joint Senior Authors
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STATEMENT OF PERMISSION AND CONTRIBUTIONS
Autonomic responses to blast overpressure can be elicited by exclusively exposing the ear in rats
David S. Sandlin, Yue Yu, Jun Huang, Chunming Zhang, Alberto A. Arteaga, John K. Lippincott, Erin O.H. Peeden, Ryan R. Guyton, Lan Chen4, Laura L.S. Beneke, Jerome C. Allison, Hong Zhu, Wu Zhou
This work has been accepted for publication by a peer-reviewed journal. Permission for use of this manuscript has been obtained and is included in the appendix.
Contributions of authors and co-authors: David S. Sandlin contributed to the design of the study and the methods used, gathering and analyzing data, interpreting results, creating and editing figures, and writing and editing the manuscript. Hong Zhu and Wu Zhou are to be considered joint senior authors and contributed equally to the design of the study and the methods used, gathering and analyzing data, interpreting results, and editing the figures and the manuscript. Jerome C. Allison contributed to the design of the study and the methods used, gathering and analyzing data, and editing the manuscript. Yue Yu, Jun Huang, Chunming Zhang, Alberto A. Arteaga, John K. Lippincott, Erin O.H. Peeden, Ryan R. Guyton, Lan Chen, Laura L.S. Beneke contributed to gathering and analyzing data and editing the manuscript.
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ABSTRACT Blast overpressure has become an increasing cause of brain injuries in both military and civilian populations. Though blast’s direct effects on the cochlea and vestibular organs are active areas of study, little attention has been given to the ear’s contribution to the overall spectrum of blast injury. Acute autonomic responses to blast exposure, including bradycardia and hypotension, can cause hypoxia and contribute to blast-induced neurotrauma. Existing literature suggests that these autonomic responses are elicited through blast impacting the thorax and lungs. We hypothesize that the unprotected ear also provides a vulnerable locus for blast to cause autonomic responses. We designed a blast generator that delivers controlled overpressure waves into the ear canal without impacting surrounding tissues in order to study the ear’s specific contribution to blast injury. Anesthetized adult rats' left ears were exposed to a single blast wave ranging from 0 to 110 PSI (0 to 758 kPa). Blast exposed rats exhibited decreased heart rates and blood pressures with increased blast intensity, similar to results gathered using shock tubes and whole-body exposure in the literature. While rats exposed to blasts below 50 PSI (345 kPa) exhibited increased respiratory rate with increased blast intensity, some rats exposed to blasts higher than 50 PSI (345 kPa) stopped breathing immediately and ultimately died. These autonomic responses were significantly reduced in vagally denervated rats, again similar to whole-body exposure literature. These results support the hypothesis that the unprotected ear contributes to the autonomic responses to blast.
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2.1 INTRODUCTION Blast, such as that produced by explosive devices, has become a frequent cause of injury in both military and civilian populations (Ibolja Cernak & Noble-Haeusslein, 2010; Taber et al., 2006). Injuries from blast wave overpressure are a source of uncertainty in that they often lack clinically apparent signs and can be insidious, only presenting themselves after other injuries have been managed (Bass et al., 2012; Burgess et al., 2010; Lemonick, 2011; Phillips, 1986). Detection, management, and prevention of blast injury has become an active area of study in blast literature, and more clinical diagnoses can be attributed to the effect of blast overpressure than in the past (Burgess et al., 2010; Svetlov et al., 2009). Traumatic brain injury, including blast-induced neurotrauma (BINT), is the “signature injury” of U.S. soldiers involved in recent conflicts in Iraq and Afghanistan (Huber et al., 2013; Mac Donald et al., 2014; Moss et al., 2009; Pham et al., 2015). Blast exposure may cause BINT through multiple pathways such as direct passage of the blast wave through the skull, compression of the torso resulting in transfer of the blast wave’s kinetic energy to the brain via hydraulic oscillations within the vasculature, and hypoxia through over-activation of the parasympathetic nervous system (Ibolja Cernak, 2010; Ibolja Cernak et al., 2001; Courtney & Courtney, 2009; Long et al., 2009; Moss et al., 2009; Simard et al., 2014). The parasympathetic response, specifically, is hypothesized to be a result of hyperinflation of the lungs stimulating alveolar juxtacapillary J-receptors innervated by vagal fibers, leading to apnea then tachypnea, bradycardia, and hypotension, which in turn lead to a Bezold-Jarish reflex further deepening bradycardia and hypotension (Ibolja Cernak, 2010; Krohn et al., 1942; Zucker, 1986; Zuckerman, 1940). Another proposed mechanism suggests that the foramina of the skull (e.g., the acoustic meatus, optic canal, nasal cavity, or foramen magnum) can provide a conduit for the blast wave to enter the cranial vault in addition to the established mechanisms mentioned above (Hicks et al., 2010; Ropper, 2011; Sundaramurthy et al., 2012). The advanced combat helmet, the current helmet of the U.S. Army, uses layers of Kevlar and a foam suspension to protect the skull from penetrating and blunt-force injuries, but leaves the ears, eyes, nose, and mouth all exposed to the surrounding air, permitting pressure from a blast to interact (Meaney et al., 2014; Moore et al., 2009). Nonetheless, soldiers frequently wear little to no ear protection, citing a necessity for situational awareness through unhindered sound localization (Abel, 2008; Brown et al., 2015; Clasing & Casali, 2014; Jones & Pearson, 2016). A perforated eardrum is the most frequently reported blast 40
injury (Ibolja Cernak & Noble-Haeusslein, 2010; Choi, 2012; Darley & Kellman, 2010; DePalma et al., 2005; Gan et al., 2016; Garth, 1994; Helling, 2004; Katz et al., 1989; Kronenberg et al., 1993; Mayorga, 1997; Patterson & Hamernik, 1997; Phillips, 1986). We hypothesize that energy from blast overpressure could enter the unprotected ear canal, traverse these soft tissues into the cranial vault, and directly impact the brain. In this study, we tested the hypothesis that the ear provides a vulnerable locus for blast energy to impact the brain while causing acute autonomic responses typically observed in whole-body paradigms of blast exposure (Guy, Kirkman, Watkins, & Cooper, 1998; Krohn et al., 1942; Sawdon, Ohnishi, Watkins, & Kirkman, 2002). To do so, we developed a blast generator that delivers precisely controlled blast overpressure waves targeted to a small area of tissue, with minimal impact on the surrounding tissues (Figure 2.1). Though placing an animal in a “shock tube” is widely accepted to be the most accurate laboratory model of primary blast, i.e. the shockwave effect of blast, (Alay et al., 2017; Needham et al., 2015), there is not a feasible way to prevent the blast wave from causing flexion of the skull and compression of the torso that would introduce confounding factors in our study of the ear’s role. Instead, our blast generator produces an overpressure wave similar to that of shock tube literature but isolates the wave’s impact to the ear and minimizes exposure of the rest of the body, including the lungs. This approach allows us to specifically study the ear’s role in blast injury, minimizing confounding factors encountered when blast is delivered over the whole animal (Ibolja Cernak, 2005; Mediavilla Varas et al., 2011; Xiong et al., 2013; Yarnell et al., 2013). Blast-induced injuries to the organs of the inner ear are active areas of study using whole-body exposure paradigms (Akin & Murnane, 2011; Chandler & Edmondt, 1997; W. Chen, Wang, Chen, Chen, & Chen, 2013; S.-I. Cho et al., 2013; Choi, 2012; Cohen et al., 2002; Darley & Kellman, 2010; Dougherty et al., 2013; Fausti et al., 2009; Gan et al., 2016; Garth, 1994; Helling, 2004; Hoffer et al., 2010; Jagade et al., 2008; Kerr & Byrne, 1975; Niwa et al., 2016; M. R. Scherer, Shelhamer, & Schubert, 2011; Singh & Ahluwalia†, 1968; Teter, Newell, & Aspinall, 1970; Tungsinmunkong, Chongkolwatana, Piyawongvisal, Atipas, & Namchareonchaisuk, 2007). However, to the best of our knowledge, this is the first study of the ear’s specific contribution to the systemic autonomic responses induced by blast injury.
41
2.2 MATERIALS AND METHODS 2.2.1 Animals Adult male Sprague-Dawley (SD) rats (Harlan Sprague-Dawley, Indianapolis, IN) weighing 250-500 grams were used for this experiment. In total, 31 rats were used in this study; 22 Rats were used for the autonomic response measurements, and 9 rats were used for the subsequent vagal denervation study. Rats were assigned to exposure intensities randomly. Male rats were used due to the prevalence of males as active-duty soldiers. Sprague-Dawley rats were used due to our previous use in autonomic studies (Zhu et al., 2007) and their frequent use in studies of blast-induced neurotrauma (Budde et al., 2013; Chavko et al., 2011; Chavko, Koller, Prusaczyk, & McCarron, 2007; H. J. Cho, Sajja, VandeVord, & Lee, 2013; Kabu et al., 2015; Long et al., 2009; Newman et al., 2015; Niwa et al., 2016; Pham et al., 2015; Readnower et al., 2010; Reneer et al., 2012; Sawyer et al., 2016; Sundaramurthy et al., 2012; Xiong et al., 2013; Yeoh, Bell, & Monson, 2013). All procedures were performed in accordance with NIH guidelines and approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center.
2.2.2 Blast wave generator and calibration A modified high-power airgun was used as the basis for the blast generator (Figure 2.1A). All seals and connectors were replaced and/or reinforced with high-quality o-rings and poly-tetra-fluoro-ethylene (PTFE) tape to minimize unwanted pressure escape and increase repeatability. An M1-3KPSI digital pressure gauge (Crystal Engineering Corp., San Luis Obispo, CA) was connected to the air canister to measure input air pressure at a resolution of 0.1 PSI (0.7 kPa). The muzzle of the blast generator was threaded and an aircraft-grade aluminum t-fitting (Eaton, Dublin, Ireland) was affixed for use as a sensor bung. A high-frequency integrated-circuit-piezoelectric (ICP) pressure sensor connected to a signal conditioner (102B04, 480C02, PCB Piezotronics, Depew, NY) was installed in the sensor bung. The blast generator was installed on a stereotaxic frame (David Kopf Instruments, Tujunga, CA) using an adjustable multi-arm instrument holder. A 3.0 mm stainless steel speculum was attached to the instrument holder and held even with the level of the muzzle, leaving open space for excess blast wind to escape. For calibration, a second arm was attached holding another high-frequency ICP pressure sensor and adjusted such that the sensor was in the position of the rat’s tympanic membrane (2.5mm 42
from the tip of the speculum). The blast generator was charged to 500 PSI (3447 kPa) input pressure, allowed to come to equilibrium, and then activated with both the bung sensor and the output sensor recording at 500 kHz. After the blast, the generator was given time to return to equilibrium and then activated again. This was repeated until the air canister was depleted (about 45 blasts). This process was repeated twice to verify repeatability. Each of the waveforms produced by the blast generator were then analyzed for rise time, total positive pressure time, maximum pressure, and return to baseline pressure. The blast pressures were linearly related to the input pressure as well as the bung pressure. Thus, desired blast pressure was achieved by adjusting the input pressure and was verified by the bung pressure.
2.2.3 Blast exposure Figure 2.2 shows the timeline for the blast exposure and autonomic recording study. Rats were maintained under 2% inhaled isoflurane anesthesia. Before blast exposure, a photograph of the left and right tympanic membranes was captured using a digital macroview otoscope (Welch-Allyn, Skaneateles Falls, NY) and evaluated for intactness and lack of erythema or effusion. If either factor was present, the rat was not used for this study. A PE-50 polyethylene catheter (Intramedic by BD, Franklin Lakes, NJ) was implanted in the left femoral artery and connected to a disposable transducer (Argon Medical, Athens, TX) for blood pressure monitoring. If a pulsatile waveform was recorded, the catheterization was deemed successful. If such a waveform was not present or if the catheterization was incomplete, the catheter was left in place and the experiment was carried out normally, but the blood pressure data was not recorded. Stainless steel 27gauge subdermal needle electrodes (CareFusion, Middleton, WI) were placed at the ventral right shoulder, left shoulder, and left hip of the rat for electrocardiography using a D360 isolated patient amplifier (Digitimer, Ft. Lauderdale, FL). The rat was then laid in the right lateral decubitus position and a piezo transducer (Radio Shack, Ft. Worth, TX) was placed under the ribcage to detect inspiratory effort. The blast generator was lowered into the left ear canal, and the blast apparatus was rotated away to allow direct visualization of the speculum position within the ear canal using the digital macroview otoscope. The speculum was lowered further, and the rat’s head position adjusted until the head was held in place on the platform by the speculum, and the tympanic membrane was in the center of the speculum’s line of sight. The blast generator was then rotated back into 43
position and locked. Electrocardiography (ECG), pulse oximetry, piezo respiratory effort sensing, and blood pressure were continuously monitored prior to, during, and after blast exposure to evaluate the physiologic effects of the blast. The rats were monitored for stabilization of the heart rate and respiratory rate. One blast of varying pressure was delivered. At first, a wide range of blast pressures were experimented with (0-110 PSI). However, when it became evident that blasts of 50 PSI and above could be immediately lethal, the remaining blasts were performed below that threshold. Control rats underwent the same anesthesia, preparation, and monitoring, except the blast generator was not charged (0 PSI, or 0 kPa). After blast exposure and autonomic response recording, rats were sacrificed via intracardiac perfusion with 4% Paraformaldehyde in phosphatebuffered saline for tissue fixation for separate studies.
2.2.4 Vagus nerve cervical segment ligation and blast exposure Rats were maintained under isoflurane anesthesia and placed supine on a feedback-controlled heating pad (FHC Inc., Bowdoin, ME). A midline incision was made on the neck and the fat and submandibular glands were retracted. The left sternocleidomastoid muscle was rotated laterally to expose the carotid sheath. The sheath was opened at the level of the thyroid cartilage and the vagus nerve bundle separated from the carotid artery and jugular vein using blunt dissection with fine forceps. A 4-0 silk suture was loosely tied around the nerve, which was then allowed to rest back under the sternocleidomastoid muscle. The contralateral nerve was then isolated in the same fashion. Once the cervical portion of the vagus nerve had been isolated bilaterally, the nerves were lifted using the suture, the sutures were tied tightly, and the nerves were cut using micro-scissors. An increase in heart rate when tying and cutting the nerves was used to verify adequate separation. Rats receiving sham surgery underwent the midline incision, retraction of fat and glands, and reflection of both sternocleidomastoid muscles, but the carotid sheath was not opened. Rats then underwent blast exposure as outlined above.
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2.2.5 Data collection and analysis The blast sensor signal conditioners, blood pressure monitor, ECG amplifier, and respiratory rate monitor were connected to a CED Power1401 data acquisition system connected to a personal computer running CED Spike2 data acquisition and analysis software (Cambridge Electronic Design Limited, Cambridge, England). Respiratory rate and heart rate were computed as the inverse of the period between breaths and heartbeats, respectively. Baseline respiratory rate and heart rate were calculated as the mean rates for 1 minute prior to blast exposure. Peak/trough respiratory rate and heart rate were defined as the highest or lowest value after blast exposure that was not due to a double/absent breath or heartbeat. Blast pressure was calculated using the bung pressure. Relationships were analyzed using linear regression and t-tests using SigmaPlot software (Systat Software Inc., San Jose, CA). P-value of 0.05 was used to determine significance of the results, expressed as mean ± standard error.
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2.3 RESULTS 2.3.1 Blast waveforms and calibration The waveforms produced by the blast generator exhibited a rise time of 2.2±0.3 ms and a total positive phase duration of 7.3±0.5 ms (Figure 2.1B), resembling the waveforms reported in the literature using shock tubes based on the Friedlander waveform, i.e., the ideal waveform produced by a blast in an open space (Figure 2.1B insert) (Ibolja Cernak et al., 2011; Friedlander, 1946; Goel et al., 2012; Mediavilla Varas et al., 2011; Sundaramurthy et al., 2012). The output pressure was linearly related to the input tank pressure (POutput=0.12*PInput+1.76, R2=0.997, p
