Research paper
Antioxidant treatment reduces blast-induced cochlear damage and hearing loss

https://doi.org/10.1016/j.heares.2012.01.013Get rights and content

Abstract

Exposure to blast overpressure has become one of the hazards of both military and civilian life in many parts of the world due to war and terrorist activity. Auditory damage is one of the primary sequela of blast trauma, affecting immediate situational awareness and causing permanent hearing loss. Protecting against blast exposure is limited by the inability to anticipate the timing of these exposures, particularly those caused by terrorists. Therefore a therapeutic regimen is desirable that is able to ameliorate auditory damage when administered after a blast exposure has occurred. The purpose of this study was to determine if administration of a combination of antioxidants 2,4-disulfonyl α-phenyl tertiary butyl nitrone (HPN-07) and N-acetylcysteine (NAC) beginning 1 h after blast exposure could reduce both temporary and permanent hearing loss. To this end, a blast simulator was developed and the operational conditions established for exposing rats to blast overpressures comparable to those encountered in an open-field blast of 14 pounds per square inch (psi). This blast model produced reproducible blast overpressures that resulted in physiological and physical damage to the auditory system that was proportional to the number and amplitude of the blasts. After exposure to 3 consecutive 14 psi blasts 100% of anesthetized rats had permanent hearing loss as determined at 21 days post exposure by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) testing. Animals treated with HPN-07 and NAC after blast exposure showed a significant reduction in ABR threshold shifts and DPOAE level shifts at 2–16 kHz with significant reduction in inner hair cell (IHC) and outer hair cell (OHC) loss across the 5–36 kHz region of the cochlea compared with control animals.

The time course of changes in the auditory system was documented at 3 h, 24 h, 7 day and 21 day after blast exposure. At 3 h after blast exposure the auditory brainstem response (ABR) threshold shifts were elevated by 60 dB in both treated and control groups. A partial recovery of to 35 dB was observed at 24 h in the controls, indicative of a temporary threshold shift (TTS) and there was essentially no further recovery by 21 days representing a permanent threshold shift (PTS) of about 30 dB. Antioxidant treatment increased the amount of both TTS and PTS recovery relative to controls by 10 and 20 dB respectively. Distortion product otoacoustic emission (DPOAE) reached a maximum level shift of 25–30 dB measured in both control and treated groups at 3 h after blast exposure. These levels did not change by day 21 in the control group but in the treatment group the level shifts began to decline at 24 h until by day 21 they were 10–20 dB below that of the controls. Loss of cochlear hair cells measured at 21 day after blast exposure was mostly in the outer hair cells (OHC) and broadly distributed across the basilar membrane, consistent with the distribution of loss of frequency responses as measured by ABR and DPOAE analysis and typical of blast-induced damage. OHC loss progressively increased after blast exposure reaching an average loss of 32% in the control group and 10% in the treated group at 21 days. These findings provide the first evidence that a combination of antioxidants, HPN-07 and NAC, can both enhance TTS recovery and prevent PTS by reducing damage to the mechanical and neural components of the auditory system when administered shortly after blast exposure.

Highlights

► Exposure to blast overpressure was analyzed in a rodent model. ► The blast model gave reproducible blast overpressures that correlated with damage to the auditory system. ► Antioxidant treatment shortly after blast exposure significantly reduced blast-induced damage to the auditory system. ► Both temporary and permanent hearing loss was significantly improved.

Introduction

Experimental studies of blast injury in animals have indicated that auditory system is one of the most vulnerable systems to blast overpressure. The extreme physical force of the blast can rupture the tympanic membrane (TM), disarticulate the ossicular chain, fracture the ossicles, tear the IHCs and OHCs away from the basilar membrane and distort and break the ciliary projections of the hair cells (Patterson and Hamernik, 1997, Roberto et al., 1989, Hamernik, 1987). The incidence of TM rupture in studies of blast-exposed patients ranged from 17 to 29% (Garth, 1994, Jensen and Bonding, 1993, Patterson and Hamernik, 1997, Xydakis et al., 2007, Gondusky and Reiter, 2005). However, despite the potential for extensive cochlear damage, blast-induced hearing loss resulting from exposure to terrorist or military explosions resulted in moderate to severe sensorineural hearing loss (SNHL) not anacusis (Nageris et al., 2008). Permanent SNHL is the most prevalent type of auditory impairment associated with blast trauma of military personnel, with rates ranging from 33% to 78% (Hoffer and Balaban, 2010, Gondusky and Reiter, 2005, Cave et al., 2007, Fausti et al., 2009, Nageris et al., 2008).

The mechanisms of blast-induced hearing loss are not well understood. In addition to the mechanical damage to components of the auditory system, molecular and cellular processes are triggered by the blast that initiate molecular cascades that are responsible for much of the long-term damage (Patterson and Hamernik, 1997). Activation of cell death pathways and molecular mediators of inflammation cause secondary damage to the hair cells and supporting cells of the cochlea, as evidenced by cellular degeneration in the cochlea and accumulation of lymphocytes and macrophages in the scala tympani and perilymph (Patterson and Hamernik, 1997, Hoffer and Balaban, 2010). Increased oxidative stress is associated with both steady state noise-induced and impulse noise-induced hearing impairment (Ohlemiller et al., 1999, Henderson et al., 2006).

Administration of antioxidants has been shown to block some of the non-auditory pathological outcomes of exposure to blast overpressure or other impact injury in animal models: 1) N-acetylcysteine amide was found to reduce the inflammatory response and infiltration of neutrophils in the lungs of chinchillas (Chavko et al., 2009); 2) N-acetylcysteine (NAC) was shown to restore mitochondrial electron transfer, energy coupling capacity, calcium uptake activity and reduced calcium content absorbed to brain mitochondrial membranes of rats following cortical impact (Xiong et al., 1999); 3) NAC was shown to attenuate the inflammatory response in the injured rat brain (Chen et al., 2008); and 4) 2,4-disulfonyl α-phenyl tertiary butyl nitrone (HXY-059, now called HPN-O7) was found to reduce loss of injured brain tissue and improve cognitive function when administered to rats after percussion induced brain injury (Clausen et al., 2008).

Oxidative stress has been associated with pathological processes involved in auditory trauma including: mitochondrial injury, activation of cell death pathways, activation of mediators of inflammation, glutamate excitotoxicity, and increased levels of lipid peroxidase (Abi-Hacehm et al., 2010, Clausen et al., 2008, Haase et al., 2011, Elsayed et al., 2000, Elsayed and Gorbunov, 2003, Kopke et al., 2007, Chavko et al., 2009, Wu et al., 2006). These findings suggest that antioxidants have the potential to block the molecular cascades that are triggered by the auditory trauma which induces oxidative stress and results in permanent threshold shifts (PTS) and hearing loss.

Studies of auditory physiology have demonstrated a protective effect of antioxidants against hearing loss resulting from auditory trauma. Administering three antioxidant compounds, 4-hydroxy phenyl N-tert-butyl nitrone (4-OHPBN) and NAC, and acetyl-L-carnitine (ALCAR) 4 h following acute steady state noise [105 dB, sound pressure level (SPL)], prevented noise-induced hearing loss (NIHL) in 100% of the test animals (Choi et al., 2008). Similarly, treatment with two antioxidants, either ALCAR or NAC, significantly reduced PTS in chinchillas exposed to impulse noise, 155 dB SPL (Kopke et al., 2005). The level of exposure in these studies was comparable to repeated exposure to rifle fire.

The question addressed here is whether a similar combination of antioxidants could decrease damage to the auditory system caused by blast overpressure levels (>14 psi, corresponding to 194 dB SPL) which exceed that of steady state noise or impulse noise in previous experiments. We chose a combination of two antioxidants for this study, NAC and HPN-O7. NAC, which functions to increase the intra-cellular pool of the antioxidant glutathione, has been approved by the FDA for more than two decades to treat liver necrosis from acetaminophen overdose (Kopke et al., 2007). HPN-07 is a free radical spin-trapping agent that has shown activity as a neuroprotectant, and inhibits iNOS, decreases glutamate excitotoxicity, and may decrease cell death (Floyd et al., 2008). HPN-07 has been tested in multiple clinical stroke trials and found to be very safe (Chen et al., 2008, Lyden et al., 2007, Nilsson et al., 2007).

Section snippets

Blast simulator

To study the effects of antioxidants on blast-induced hearing loss a blast simulator was developed to simulate the expanding blast wave of an open-field blast. The blast wave generator consists of a steel pressure reservoir with a film inserted between it and a one inch blast nozzle. A pneumatic piston pushes the chamber against the film, sealing the chamber. Compressed nitrogen is delivered to the pressure reservoir until the burst strength of the plastic film is exceeded, rupturing the film

Blast exposure conditions

Preliminary studies showed that a single exposure with high-level blast overpressures (>20 psi) consistently produced permanent hearing loss as well as rupture of the TM. At moderate blast levels (<15 psi) ABR threshold shifts at 21 d post exposure as well as the frequency of TM ruptures correlated with the number of exposures in a dose dependent manner. Rats were subjected to repeated (2, 3, and 4 times), blast overpressures of 14 psi with a 1.5 min interval between exposures. The incidence of

Blast conditions producing permanent hearing loss

A variety of methods have been used to generate blast overpressure ranging from the use of actual weaponry (Ylikosi, 1987, Price et al., 1989), or explosive charges (Cheng et al., 2010), or pressure discharges from ruptured film (Jaffin et al., 1987) either in an open space (Long et al., 2008, Svetlov et al., 2010), or an enclosure (shock tube) (Hoffer and Balaban, 2010). Published reports on blast pressure-time history recordings vary in the suddenness of the onset of pressure rise, magnitude

Conclusion

The blast simulator developed for these studies produced reproducible blast overpressures and physiological and physical damage to the auditory system of rats, consistent with open-field blast exposures of humans. Results of auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) testing and outer hair cell analysis suggest that blast exposure causes damage to both the synchronous synaptic activity of the auditory nerve and brainstem and the mechanical activity of

Author disclosure statement

Dr. Kopke and Dr. Floyd have financial interests in Otological Pharmaceutics Inc. Drs Ewert, Lu, Du, and Li have no conflicts of interest.

Acknowledgments

The authors appreciate the efforts of Joel Young in the design and construction of the blast simulator and of Dr. Ning Hu and Dr. Charles Stewart and Weihua Cheng for their outstanding technical assistance. This research was supported by grant N00014-09-1-0999 from the US Department of Navy, Office of Naval Research.

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