Preliminary studies on differential expression of auditory functional genes in the brain after repeated blast exposures.
Article Type: Report
Subject: Enzymes
Brain
Antioxidants
Genes
Anopheles
Gene expression
Explosions
Histochemistry
Authors: Valiyaveettil, Manojkumar
Alamneh, Yonas
Miller, Stacy-Ann
Hammamieh, Rasha
Wang, Ying
Arun, Peethambaran
Wei, Yanling
Oguntayo, Samuel
Nambiar, Madhusoodana P.
Pub Date: 07/01/2012
Publication: Name: Journal of Rehabilitation Research & Development Publisher: Department of Veterans Affairs Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 Department of Veterans Affairs ISSN: 0748-7711
Issue: Date: July, 2012 Source Volume: 49 Source Issue: 7
Product: Product Code: 2831600 Enzymes; 2831630 Enzymes for Food Processing NAICS Code: 32519 Other Basic Organic Chemical Manufacturing; 325199 All Other Basic Organic Chemical Manufacturing
Accession Number: 309979817
Full Text: INTRODUCTION

Battlefield blast exposure is reported to cause auditory impairment in a large population of military personnel deployed to Iraq and Afghanistan [1-2]. Auditory/vestibular injuries from blast traumatic brain injury (TBI) can cause increased incidence of tinnitus and hearing loss, which worsens over time if not treated [1-4]. Shock waves generated from explosive blasts are reported to be destructive to both gas- and fluid-filled structures of the body, including the lungs, intestines, brain, eyes, nose, and middle ear [5-9]. Blast-induced damage to the auditory system can be the consequence of either direct exposure of the auditory canal to blast shock waves or TBI and impairment in the central auditory processing involving different brain regions after blast exposure. The literature on the neurobiological mechanisms of hearing impairment and development of tinnitus from blast TBI is limited.

A number of genes and their protein products have been reported to be involved in both age- and noise-related hearing loss [10-15]. Cadherin and protocadherin mutations were linked to digenic inheritance of deafness and have specific functional roles in noise-induced hearing loss [1314,16-17]. Other groups of proteins involved in deafness are otoferlin and otoancorin, which are also reported to have major roles in auditory functions, including central auditory processing [18-21]. Another large class of molecules involved in auditory signaling is centered on the calcium regulating proteins, which are known to have broad functions in age- and noise-related hearing loss or protection [18,22-26]. The significance of reactive oxygen species and heat shock proteins in age- and noise-related auditory impairments are also reviewed in detail [10,27-34].

Recent research on age- or noise-related hearing loss preferred mice as a suitable animal model because of the vulnerability of mice to sound compared to other rodents [15,35]. We have developed a preclinical mouse model of repeated blast exposures using an air-blast shock tube that closely mimics the repeated exposure to improvised explosive devices, grenades, or firing weapons used in the battlefield or breacher's studies [36-37]. The newly developed repetitive blast animal TBI model showed significant levels of neuropathology and neurobehavioral deficits after repeated blast exposures at 20.6 psi [36]. Using this mouse model of repeated blast exposures, we sought to determine differential expression of auditory-related genes in various regions of the brain by complementary DNA (cDNA) microarray analysis.

METHODS

Animal Blast Exposure Model

Experiments were performed in male mice (C57BL/ 6J, age 8-10 weeks, Jackson Laboratory; Bar Harbor, Maine). Groups of isoflurane (4%) anesthetized animals (n = 6 for sham and n = 6 for blast) were exposed to repeated blast exposures (20.6 psi), as reported previously, using a shock tube [9,36,38-39]. At a 6 h time point after the last blast exposure, three animals each from sham and blast groups were euthanized, and the brain tissue was collected after necropsy and separated into various regions as described earlier [39]. Different regions of the brain samples were immediately snap frozen and stored at -80[degrees]C until use. Remaining animals (n = 3) in each group were sacrificed at 24 h after the last blast exposure and used for histopathology.

Preparation of RNA

Total RNA was isolated using Trizol reagent (Invitrogen Life Technology; Carlsbad, California) following the manufacturer's protocol. RNA quality and quantity were determined by using an Agilent 2100 Bioanalyzer (Agilent Technologies; Santa Clara, California).

cDNA Microarray Analysis

Microarray analysis was performed using Agilent 60-mer mouse genome 44K oligo microarrays (Agilent Technologies). We labeled 5 [micro]g of purified RNA with a commercially available kit (Agilent Low Input Quick Amp) by polymerase chain reaction amplification (Bio-Rad Laboratories; Hercules, California). Samples were fragmented and hybridized against universal mouse reference RNA (Stratagene; La Jolla, California) with a kit from Agilent. A 2-Color Microarray-Based Gene Expression Analysis (version 6.5) protocol was used for labeling and microarray processing. An Agilent G2565CA fluorescence scanner was used to quantitate the slides, and the resultant data were extracted using software (Agilent Feature Extraction, version 10.7.1). For filtering and normalization of the data, GeneSpring 10.1 software (Agilent) was used.

Statistical Analysis

The statistical analysis of the microarray data was performed with GeneSpring 10.1 software. Changes in the level of expression of various genes after blast exposure in comparison to sham controls were identified by Welsh's t-test statistical method (p-values < 0.05) in conjunction with multiple correction test (Benjamini-Hochberg) with 5 percent false discovery rate. To account for the small sample size, we used the reference design and filtered for genes with signal intensities that are twice the standard deviation of the background intensity levels. We determined that by performing gene-by-gene t-tests, for a samples size of 3 and 5 percent false discovery rate and a standard deviation of 0.5, the power is 75 percent. We also applied pathway and gene ontology analyses that offer extra power because it is statistically unlikely that a larger fraction of false positive genes end up in one specific pathway.

Histopathology of Auditory Cortex

Histopathology was performed in blast-exposed and sham control mice (n = 3 in each group), as described previously [36]. Brain sections were silver stained and microscopically examined for neurodegeneration exclusively in the auditory cortex region, and the severity of injury was scored as mild (+), moderate (++) and severe (+++).

RESULTS

Expression of Auditory-Related Genes in Hippocampus After Repeated Blast Exposures

The hippocampus of mice exposed to repeated blasts showed significant changes in the expression of multiple genes that are reported to be involved in age- or noise-induced hearing loss (Table 1). Otoancorin, a gene defective in autosomal recessive deafness, showed a significant increase (3.4-fold), while otoferlin, which is essential for glutamate exocytosis at the auditory ribbon synapse, showed a 1.8-fold decrease in the expression after repeated blast exposures. The expression of calcium binding protein 2 showed a 1.6-fold increase, whereas calcitonin-related polypeptide expression showed a 1.9-fold decrease after blast exposures. The expression of antioxidant enzyme superoxide dismutase 3 showed a 2.0-fold increase in the hippocampus of mice exposed to repeated blasts. The expression of heat shock protein 8 and heat shock transcription factor 5 showed significant increase in the hippocampus after repeated blast exposures. Protocadherin alpha 4 expression showed a 1.3-fold decrease after blast exposures.

Expression of Auditory-Related Genes in Cerebellum After Repeated Blast Exposures

The cerebellum of mice exposed to repeated blasts showed a 1.2-fold increase in protocadherins alpha 4 and beta 20 expression (Table 2). The expression of S100 calcium binding protein A7A showed a 1.4-fold increase, while multiple calcium channel proteins and calcium binding protein 2 expression showed a 1.1 to 1.2-fold decrease in the cerebellum after repeated blast exposures. Heat shock protein 8 expression also showed a 1.1-fold decrease after blast exposures.

Expression Profile of Auditory-Related Genes in Frontal Cortex After Repeated Blast Exposures

The expression of calcium signaling-related molecules showed significant increase in the frontal cortex of mice exposed to repeated blasts, including calpain 3 (1.5-fold), S100 calcium binding protein A3 (1.4-fold), calcium/ calmodulin-dependent protein kinase kinase 1 (1.2-fold), and calcium binding domain 4A alpha polypeptide 7 (1.4-fold) (Table 3). Protocadherin beta 11 and calreticulin expression showed significant decrease (2.2- and 1.2-fold, respectively) in the frontal cortex of mice exposed to repeated blasts.

Expression of Auditory-Related Genes in Midbrain After Repeated Blast Exposures

The changes in the expression of auditory-related genes in the midbrain of repeated blast-exposed mice are shown in Table 4. Expression of cadherin-like 24 showed a 1.8-fold increase, while expression of cadherin 12 and protocadherin 8 showed significant decrease (1.7- and 1.4-fold, respectively) after the blast exposures. Multiple calcium signaling molecules, including calpain 9 (2.1-fold), S100 calcium binding protein A3 (1.2-fold), and calcium activated potassium channel beta 3 (2.1-fold), showed significantly increased expression in the midbrain after repeated blast exposures. At the same time, the expression of calcium binding protein 7 (2.2-fold), calcium channel voltage dependent L type alpha 1D subunit (1.6-fold), and calcium/ calmodulin-dependent protein kinase 2 gamma (1.1-fold) showed significant decrease after repeated blast exposures. The midbrain of repeated blast-exposed mice also showed significant decrease in the expression of heat shock protein 2 (1.3-fold), nicotinic alpha polypeptide 7 cholinergic receptor (1.5-fold), and stanniocalcin 2 (1.3-fold).

Histopathology of Auditory Cortex After Repeated Blast Exposures

To investigate whether blast exposure induces pathology of the auditory cortex, neuropathology analysis of the brain of repeated blast-exposed mice was performed by silver staining. As shown in the Figure, a significant level of neurodegeneration occurred in the auditory cortex at 24 h after repeated blast exposures. The pathology index in the inner layer of auditory cortex (Figure(b2)) was scored as + to ++, while the pathology index of the outer layer (Figure(a2)) was - to + compared to the respective sham controls.

DISCUSSION

Previous studies showed a significant level of neuropathology and neurobehavioral changes, with ~20 percent mortality rate after repeated blast exposures in mice at 20.6 psi [36]. The pathology was more evident in the prefrontal cortex and cerebellum of repeated blast-exposed mice. More recent results showed regional-specific changes in acetylcholinesterase activity in various regions of the brain after repeated blast exposures, indicating that the effects of blast exposure is heterogeneous in the brain [39]. The majority of the neurobiological changes in the brain were significant at 6 h after the last blast exposure [36]. Based on these observations, we analyzed the changes in the gene expression profile in different regions of the brain at 6 h after blast exposures in the present study.

The expression of otoferlin, which is known to be present in the brain and is essential for glutamate exocytosis at the auditory ribbon synapse and reported to be defective in a recessive form of human deafness, showed significant decrease in the hippocampus of mice exposed to repeated blasts [19-20,40-42]. In contrast, otoancorin, another hearing-related gene defective in autosomal recessive deafness and known to mediate the contact between the apical surface of sensory epithelial cells and acellular gels of the inner ear and the tectorial and otoconial membranes for proper auditory processing, showed significant increase in the hippocampus after repeated blast exposures [21,43]. Significant increase in the expression of otoancorin in the hippocampus after repeated blast exposures seems to be a compensatory mechanism to increase the sensitivity of hearing following injury to the auditory system and needs to be investigated in detail as a potential mechanism involved in the development of tinnitus.

Cadherins and protocadherins are another set of genes that showed differential expression in various regions of the brain after repeated blast exposures. Cadherin and protocadherin mutations are reported to be involved in noise-induced hearing loss [13-14,16-17,44]. The altered expression profile of cadherins and protocadherins in different regions of the brain after repeated blast exposures may impair central auditory processing. The functional significance of these gene modifications in the blast-induced impairment of central auditory processing has to be studied in detail to exploit them for therapeutic applications.

[FIGURE OMITTED]

Molecules involved in calcium influx and calcium-dependent proteins/enzymes are predominant signal transducers in auditory neurons [22-23,45-48]. The frontal cortex and midbrain of blast-exposed mice showed significant increase in the expression of calcium-dependent cysteine proteases and calpain 3 and 9, respectively (Tables 3 and 4). Calpains are essential for initiation and promotion of cell death, and treatment with calpain inhibitors are known to prevent the hearing loss induced by aminoglycoside ototoxicity [45]. The hippocampus of blast-exposed mice showed a significant decrease in the expression of calcitonin-related peptide, a suggested peptide therapeutic treatment for hearing loss (Table 1) [22,48]. The cerebellum and midbrain regions showed significant decrease in the expression of voltage-dependent calcium channel genes after repeated blast exposures, while multiple calcium binding proteins showed differential expression in the hippocampus, cerebellum, frontal cortex, and midbrain after repeated blast exposures (Tables 1-4). It is known that L-type voltagegated calcium channels are involved in the pathogenesis of acoustic injury in the cochlea, and treatment with calcium channel blockers can reduce the damage to the auditory neurons [26].

Other types of molecules involved in calcium regulation, such as calreticulin and calmodulin-dependent protein kinase expression, showed significant decrease in the frontal cortex and midbrain of blast-exposed mice, respectively (Tables 3 and 4) [23,47]. Interestingly, two calcium binding proteins, calretinin and parvalbumin, that were upregulated in the cerebellum at 24 and 48 h after blast exposures by proteomic analysis were not found to be altered in cDNA microarray analysis at 6 h after blast exposures. ** One possible reason for this difference is that calretinin and parvalbumin expression might be regulated at the translational level after blast exposures, which needs to be investigated further. Second, cDNA microarray analysis at 24 and 48 h after blast exposures needs to be done to find any significant changes in calretinin and parvalbumin gene expression. Western-blotting of the hippocampal region at 6 h after blast exposures showed significant increase in calretinin, further supporting the idea that blast exposure possibly modulates the protein expression at the translational level. The differential expression of calcium-dependent proteins/receptors in the brain after repeated blast exposures could be the consequence of increased/decreased calcium buffering in the auditory neurons. Thus, these results suggest that repeated blast exposures lead to an imbalance in the regulation of calcium homeostasis in different regions of the brain that can directly influence the central auditory processing and lead to auditory impairment.

Heat shock proteins or factors are one of the best-characterized families of protective proteins that are usually upregulated after stress, offering cellular protection and survival [10,27-28]. Repeated blast exposures in mice showed significant increase in the expression of heat shock protein 8 and factor 5 in the hippocampus, while cerebellum and midbrain showed significant decrease in heat shock protein 8 and heat shock protein 2, respectively (Tables 1, 2, and 4). The functional significance of heat shock proteins in hyper thermia and noise overstimulation is well documented [10,28]. The differential expression of heat shock proteins in the brains of repeated blast-exposed mice needs to be investigated further. Additionally, repeated blast exposure in mice showed significant reduction in the expression of the cholinergic receptor nicotinic alpha polypeptide 7 in the midbrain, suggesting a possible role of these receptors in aberrant central auditory processing (Table 4). The nicotinic receptor of cochlear hair cells has been proposed by others as a potential therapeutic target in acoustic trauma [11,49].

The functional role of reactive oxygen species and the protective efficacy of antioxidants in noise-induced hearing loss are well documented [32,50-51]. Repeated blast exposure in mice showed significant increase in the expression of antioxidant enzymes, superoxide dismutase 3, and glutathione peroxidase 4 in the hippocampus and midbrain, suggesting a protective mechanism in central auditory processing (Tables 1 and 4). The influence of glutathione peroxidase and superoxide dismutase in noiseinduced hearing loss has also been reported [12,31,33-34]. Reactive oxygen species showed an increase in the brain following repeated blast exposures [36].

Neuropathology analysis of the auditory cortex of repeated blast-exposed mice showed significant injury (Figure). The injury level was more on the medial contralateral side of the brain than the ipsilateral side. The neuropathology of the auditory cortex is in line with the significant level of auditory-related gene expression changes in the brain of blast-exposed mice. It is not clear whether the neuropathology is responsible for the changes in gene expression or vice versa. It has been reported that blast-induced mild to moderate TBI leads to neurobiological and behavioral changes with multifocal axonal injury [52-53]. In these reports, neuropathological changes were observed at 7 and 14 d after blast exposure, although gene expression changes were observed at day 1, indicating that molecular changes contributes to the neuropathology. In our studies, neuropathology was prominent at 24 h after repeated blast exposures, but changes in gene expression were observed much earlier, suggesting that molecular changes can occur earlier as a direct effect of blast exposures. Preliminary data on brain DNA damage after blast exposure using comet assay showed breakage of DNA after repeated blast exposures. Studies with rats exposed to low-levels of explosive blast showed terminal dUTP nick end labeling-positive cells in the white matter in day 1 without any changes in day 7 [54].

Changes in many hearing-related genes after blast exposure in the brain indicate that these genes play specific roles in central auditory processing. The gene expression changes may be the consequence of initial protection against blast-induced central auditory processing and later as injury mechanism of central auditory processing. Gene expression changes can vary with respect to blast overpressure or number of blasts and may also depend on the severity of injury. The contribution of the shock waves transmitting through the auditory canal or directly through the skull in central auditory processing impairment is currently being investigated in the laboratory by using ear protection. Unraveling the functional role of these genes in central auditory processing and how they cross-talk with each of the brain regions to perform sound perception, hearing, speech recognition, and long-term memory will help us to understand how exactly their modulation plays a role in central auditory processing impairments. The linkage of these gene modulations to concurrent neuropsychiatric changes after blast exposure is also important to understand the complex neurobiological mechanisms of blast affecting central auditory processing and aid in rehabilitation.

CONCLUSIONS

In summary, preliminary results indicate that repeated blast exposures in mice showed significant alterations in multiple genes that are reported to be involved in age- or noise-related hearing loss at 6 h after blast exposure. The repeated blast exposure also showed significant neuropathology at 24 h in the auditory cortex, suggesting that blast exposure damages central auditory processing systems. Gene expression changes occur at early time points after blast exposure and may not be the consequence of apoptotic or necrotic changes in the brain. The gene expression profile showed differential pattern in various regions of the brain of mice exposed to repeated blasts. Otoferlin and otoancorin, which are involved in deafness, showed significant alteration in the hippocampus after repeated blast exposure. Similarly, cadherins and protocadherins, which are involved in noise-induced hearing loss, showed significant changes in all the brain regions tested. The expression profile of calcium-regulating proteins/receptors in various brain regions also showed differential expression, indicating an imbalance in calcium homeostasis after repeated blast exposures. The heat shock proteins and antioxidant enzyme expressions also showed significant changes in various regions of the brain after repeated blast exposure, indicating possible protective effects. The differential expression of multiple auditory-related genes in various regions of the brain after repeated blast exposures in mice needs to be investigated further to draw specific biochemical pathways involved in the functional significance of central auditory processing in blast-induced auditory dysfunction and tinnitus.

at a Glance

[ILLUSTRATION OMITTED]

Repeated blast exposures of mice showed significant alteration in multiple genes involved in age- or noise-related hearing loss. Blast exposure also showed significant neuropathology in the auditory cortex, suggesting that blast exposure damages central auditory processing systems. Differentially expressed genes include otoferlin, otoancorin, cadherins, and calcium regulating proteins/receptors, which are known to play various roles in auditory processing and hearing impairment. Changes in hearing-related gene expression after blast exposures need to be investigated further to draw specific biochemical pathways involved in defective central auditory processing leading to auditory dysfunction and tinnitus.

Abbreviations: cDNA = complementary DNA, TBI = traumatic brain injury.

ACKNOWLEDGMENTS

Author contributions:

Study concept and design: M. Valiyaveettil, M. P. Nambiar.

Acquisition of data: Y. Alamneh, S. Miller, Y. Wang, P. Arun, Y. Wei, S. Oguntayo.

Analysis and interpretation of data: Y. Alamneh, S. Miller, R. Hammamieh, Y. Wang, P. Aran, Y. Wei, S. Oguntayo, M. P. Nambiar.

Drafting of manuscript: M. Valiyaveettil, M. P. Nambiar.

Study supervision: M. Valiyaveettil, R. Hammamieh, Y. Wang, P. Arun, M. P. Nambiar.

Financial Disclosures: The authors have declared that no competing interests exist.

Funding/Support: This material was unfunded at the time of manuscript preparation.

Additional Contributions: Collaborative help from COL Paul Bliese, Dr. Joseph Long, and Blast-Induced Neurotrauma Branch members is greatly acknowledged.

Institutional Review: Experiments were performed in compliance with the Animal Welfare Act and other Federal statutes and regulations relating to animals and experiments involving animals and adhered to principles stated in the Guide for the Care and Use of Laboratory Animals with an approved Institutional Animal Care and Use Committee protocol.

Disclaimer: The contents, opinions, and assertions contained herein are private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense.

REFERENCES

[1.] Lew HL, Jerger JF, Guillory SB, Henry JA. Auditory dysfunction in traumatic brain injury. J Rehabil Res Dev. 2007; 44(7):921-28. [PMID:18075949] http://dx.doi.org/10.1682/JRRD.2007.09.0140

[2.] Fausti SA, Wilmington DJ, Gallun FJ, Myers PJ, Henry JA. Auditory and vestibular dysfunction associated with blast-related traumatic brain injury. J Rehabil Res Dev. 2009; 46(6):797-810. [PMID:20104403] http://dx.doi.org/10.1682/JRRD.2008.09.0118

[3.] Mao JC, Pace E, Pierozynski P, Kou Z, Shen Y, Vande-Vord PJ, Haacke EM, Zhang X, Zhang J. Blast-induced tinnitus and hearing loss in rats: behavioral and imaging assays. J Neurotrauma. 2012;29(2):430-44. [PMID:21933015] http://dx.doi.org/10.1089/neu.2011.1934

[4.] Mrena R, Savolainen S, Kuokkanen JT, Ylikoski J. Characteristics of tinnitus induced by acute acoustic trauma: a long-term follow-up. Audiol Neurootol. 2002;7(2):122-30. [PMID:12006740] http://dx.doi.org/10.1159/000057660

[5.] Bauman RA, Ling G, Tong L, Januszkiewicz A, Agoston D, Delanerolle N, Kim Y, Ritzel D, Bell R, Ecklund J, Armonda R, Bandak F, Parks S. An introductory characterization of a combat-casualty-care relevant swine model of closed head injury resulting from exposure to explosive blast. J Neurotrauma. 2009;26(6):841-60. [PMID:19215189] http://dx.doi.org/10.1089/neu.2008.0898

[6.] Lew HL, Garvert DW, Pogoda TK, Hsu PT, Devine JM, White DK, Myers PJ, Goodrich GL. Auditory and visual impairments in patients with blast-related traumatic brain injury: Effect of dual sensory impairment on Functional Independence Measure. J Rehabil Res Dev. 2009;46(6): 819-26. [PMID:20104405] http://dx.doi.org/10.1682/JRRD.2008.09.0129

[7.] Dougherty AL, MacGregor AJ, Han PP, Heltemes KJ, Galarneau MR. Visual dysfunction following blast-related traumatic brain injury from the battlefield. Brain Inj. 2011; 25(1):8-13. [PMID:21117919] http://dx.doi.org/10.3109/02699052.2010.536195

[8.] Garman RH, Jenkins LW, Switzer RC 3rd, Bauman RA, Tong LC, Swauger PV, Parks SA, Ritzel DV, Dixon CE, Clark RS, Bayir H, Kagan V, Jackson EK, Kochanek PM. Blast exposure in rats with body shielding is characterized primarily by diffuse axonal injury. J Neurotrauma. 2011; 28(6):947-59. [PMID:21449683] http://dx.doi.org/10.1089/neu.2010.1540

[9.] Long JB, Bentley TL, Wessner KA, Cerone C, Sweeney S, Bauman RA. Blast overpressure in rats: recreating a battlefield injury in the laboratory. J Neurotrauma. 2009;26(6): 827-40. [PMID:19397422] http://dx.doi.org/10.1089/neu.2008.0748

[10.] Fairfield DA, Lomax MI, Dootz GA, Chen S, Galecki AT, Benjamin IJ, Dolan DF, Altschuler RA. Heat shock factor 1-deficient mice exhibit decreased recovery of hearing following noise overstimulation. J Neurosci Res. 2005;81(4): 589-96. [PMID:15952177] http://dx.doi.org/10.1002/jnr.20417

[11.] Shen H, Lin Z, Lei D, Han J, Ohlemiller KK, Bao J. Old mice lacking high-affinity nicotine receptors resist acoustic trauma. Hear Res. 2011;277(1-2):184-91. [PMID:21272629] http://dx.doi.org/10.1016/j.heares.2011.01.009

[12.] Keithley EM, Canto C, Zheng QY, Wang X, Fischel-Ghodsian N, Johnson KR. Cu/Zn superoxide dismutase and age-related hearing loss. Hear Res. 2005;209(1-2):76-85. [PMID:16055286] http://dx.doi.org/10.1016/j.heares.2005.06.009

[13.] Zheng QY, Yan D, Ouyang XM, Du LL, Yu H, Chang B, Johnson KR, Liu XZ. Digenic inheritance of deafness caused by mutations in genes encoding cadherin 23 and protocadherin 15 in mice and humans. Hum Mol Genet. 2005;14(1):103-11. [PMID:15537665] http://dx.doi.org/10.1093/hmg/ddi010

[14.] Noben-Trauth K, Zheng QY, Johnson KR. Association of cadherin 23 with polygenic inheritance and genetic modification of sensorineural hearing loss. Nat Genet. 2003;35(1):21-23. [PMID:12910270] http://dx.doi.org/10.1038/ng1226

[15.] Ohlemiller KK, Wright JS, Heidbreder AF. Vulnerability to noise-induced hearing loss in 'middle-aged' and young adult mice: a dose-response approach in CBA, C57BL, and BALB inbred strains. Hear Res. 2000;149(1-2):239-47. [PMID:11033262] http://dx.doi.org/10.1016/S0378-5955(00)00191-X

[16.] Siemens J, Lillo C, Dumont RA, Reynolds A, Williams DS, Gillespie PG, Muller U. Cadherin 23 is a component of the tip link in hair-cell stereocilia. Nature. 2004;428(6986): 950-55. [PMID:15057245] http://dx.doi.org/10.1038/nature02483

[17.] Sollner C, Rauch GJ, Siemens J, Geisler R, Schuster SC, Muller U, Nicolson T; Tubingen 2000 Screen Consortium. Mutations in cadherin 23 affect tip links in zebrafish sensory hair cells. Nature. 2004;428(6986):955-59. [PMID:15057246] http://dx.doi.org/10.1038/nature02484

[18.] Beurg M, Michalski N, Safieddine S, Bouleau Y, Schneggenburger R, Chapman ER, Petit C, Dulon D. Control of exocytosis by synaptotagmins and otoferlin in auditory hair cells. JNeurosci. 2010;30(40):13281-90. [PMID:20926654] http://dx.doi.org/10.1523/JNEUROSCI.2528-10.2010

[19.] Schug N, Braig C, Zimmermann U, Engel J, Winter H, Ruth P, Blin N, Pfister M, Kalbacher H, Knipper M. Differential expression of otoferlin in brain, vestibular system, immature and mature cochlea of the rat. Eur J Neurosci. 2006;24(12):3372-80. [PMID:17229086] http://dx.doi.org/10.1111/j.1460-9568.2006.05225.x

[20.] Roux I, Safieddine S, Nouvian R, Grati M, Simmler MC, Bahloul A, Perfettini I, Le Gall M, Rostaing P, Hamard G, Triller A, Avan P, Moser T, Petit C. Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse. Cell. 2006;127(2):277-89. [PMID:17055430] http://dx.doi.org/10.1016/j.cell.2006.08.040

[21.] Zwaenepoel I, Mustapha M, Leibovici M, Verpy E, Goodyear R, Liu XZ, Nouaille S, Nance WE, Kanaan M, Avraham KB, Tekaia F, Loiselet J, Lathrop M, Richardson G, Petit C. Otoancorin, an inner ear protein restricted to the interface between the apical surface of sensory epithelia and their overlying acellular gels, is defective in autosomal recessive deafness DFNB22. Proc Natl Acad Sci U S A. 2002;99(9):6240-45. [PMID:11972037] http://dx.doi.org/10.1073/pnas.082515999

[22.] El Sammaa M, Linthicum FH Jr, House HP, House JW. Calcitonin as treatment for hearing loss in Paget's disease. Am J Otol. 1986;7(4):241-43. [PMID:3740234]

[23.] Bolz H, Bolz SS, Schade G, Kothe C, Mohrmann G, Hess M, Gal A. Impaired calmodulin binding of myosin-7A causes autosomal dominant hearing loss (DFNA11). Hum Mutat. 2004;24(3):274-75. [PMID:15300860] http://dx.doi.org/10.1002/humu.9272

[24.] Spencer RF, Shaia WT, Gleason AT, Sismanis A, Shapiro SM. Changes in calcium-binding protein expression in the auditory brainstem nuclei of the jaundiced Gunn rat. Hear Res. 2002;171(1-2):129-41. [PMID:12204357] http://dx.doi.org/10.1016/S0378-5955(02)00494-X

[25.] Ramakrishnan NA, Drescher MJ, Drescher DG. Direct interaction of otoferlin with syntaxin 1A, SNAP-25, and the L-type voltage-gated calcium channel Cav1.3. J Biol Chem. 2009;284(3):1364-72. [PMID:19004828] http://dx.doi.org/10.1074/jbc.M803605200

[26.] Uemaetomari I, Tabuchi K, Nakamagoe M, Tanaka S, Murashita H, Hara A. L-type voltage-gated calcium channel is involved in the pathogenesis of acoustic injury in the cochlea. Tohoku J Exp Med. 2009;218(1):41-47. [PMID:19398872] http://dx.doi.org/10.1620/tjem.218.41

[27.] Morimoto RI, Kline MP, Bimston DN, Cotto JJ. The heatshock response: regulation and function of heat-shock proteins and molecular chaperones. Essays Biochem. 1997;32: 17-29. [PMID:9493008]

[28.] Gong TW, Fairfield DA, Fullarton L, Dolan DF, Altschuler RA, Kohrman DC, Lomax MI. Induction of heat shock proteins by hyperthermia and noise overstimulation in hsf1 -/- mice. J Assoc Res Otolaryngol. 2012;13(1):29-37. [PMID:21932106] http://dx.doi.org/10.1007/s10162-011-0289-9

[29.] Ohlemiller KK, Wright JS, Dugan LL. Early elevation of cochlear reactive oxygen species following noise exposure. Audiol Neurootol. 1999;4(5):229-36. [PMID:10436315] http://dx.doi.org/10.1159/000013846

[30.] Ohlemiller KK, Dugan LL. Elevation of reactive oxygen species following ischemia-reperfusion in mouse cochlea observed in vivo. Audiol Neurootol. 1999;4(5):219-28. [PMID:10436314] http://dx.doi.org/10.1159/000013845

[31.] McFadden SL, Ohlemiller KK, Ding D, Shero M, Salvi RJ. The influence of superoxide dismutase and glutathione peroxidase deficiencies on noise-induced hearing loss in mice. Noise Health. 2001;3(11):49-64. [PMID:12689448]

[32.] Henderson D, Bielefeld EC, Harris KC, Hu BH. The role of oxidative stress in noise-induced hearing loss. Ear Hear. 2006;27(1):1-19. [PMID:16446561] http://dx.doi.org/10.1097/01.aud.0000191942.36672.f3

[33.] Ohlemiller KK, McFadden SL, Ding DL, Flood DG, Reaume AG, Hoffman EK, Scott RW, Wright JS, Putcha GV, Salvi RJ. Targeted deletion of the cytosolic Cu/Zn-superoxide dismutase gene (Sod1) increases susceptibility to noise-induced hearing loss. Audiol Neurootol. 1999;4(5):237-46. [PMID:10436316] http://dx.doi.org/10.1159/000013847

[34.] Ohlemiller KK, McFadden SL, Ding DL, Lear PM, Ho YS. Targeted mutation of the gene for cellular glutathione peroxidase (Gpx1) increases noise-induced hearing loss in mice. J Assoc Res Otolaryngol. 2000;1(3):243-54. [PMID:11545230] http://dx.doi.org/10.1007/s101620010043

[35.] Ohlemiller KK. Contributions of mouse models to understanding of age- and noise-related hearing loss. Brain Res. 2006;1091(1):89-102. [PMID:16631134] http://dx.doi.org/10.1016/j.brainres.2006.03.017

[36.] Wang Y, Wei Y, Oguntayo S, Wilkins W, Arun P, Valiyaveettil M, Song J, Long JB, Nambiar MP. Tightly coupled repetitive blast-induced traumatic brain injury: development and characterization in mice. J Neurotrauma. 2011;28(10):2171-83. [PMID:21770761] http://dx.doi.org/10.1089/neu.2011.1990

[37.] Kelley A, Athy J, Vasbinder M, Chiaramonte Rath E. The effect of blast exposure on sleep and daytime sleepiness in U.S. Marine Corps breachers. Washington (DC): U.S. Army Aeromedical Research Laboratory; 2010. Report No.: 2010-16.

[38.] Arun P, Spadaro J, John J, Gharavi RB, Bentley TB, Nambiar MP. Studies on blast traumatic brain injury using in-vitro model with shock tube. Neuroreport. 2011;22(8):379-84. [PMID:21532394] http://dx.doi.org/10.1097/WNR.0b013e328346b138

[39.] Valiyaveettil M, Alamneh Y, Oguntayo S, Wei Y, Wang Y, Arun P, Nambiar MP. Regional specific alterations in brain acetylcholinesterase activity after repeated blast exposures in mice. Neurosci Lett. 2012;506(1):141-45. [PMID:22079491] http://dx.doi.org/10.1016/j.neulet.2011.10.067

[40.] Pangrsic T, Lasarow L, Reuter K, Takago H, Schwander M, Riedel D, Frank T, Tarantino LM, Bailey JS, Strenzke N, Brose N, Muller U, Reisinger E, Moser T. Hearing requires otoferlin-dependent efficient replenishment of synaptic vesicles in hair cells. Nat Neurosci. 2010;13(7):869-76. [PMID:20562868] http://dx.doi.org/10.1038/nn.2578

[41.] Choi BY, Ahmed ZM, Riazuddin S, Bhinder MA, Shahzad M, Husnain T, Riazuddin S, Griffith AJ, Friedman TB. Identities and frequencies of mutations of the otoferlin gene (OTOF) causing DFNB9 deafness in Pakistan. Clin Genet. 2009;75(3):237-43. [PMID:19250381] http://dx.doi.org/10.1111/j.1399-0004.2008.01128.x

[42.] Reisinger E, Bresee C, Neef J, Nair R, Reuter K, Bulankina A, Nouvian R, Koch M, Buckers J, Kastrup L, Roux I, Petit C, Hell SW, Brose N, Rhee JS, Kugler S, Brigande JV, Moser T. Probing the functional equivalence of otoferlin and synaptotagmin 1 in exocytosis. J Neurosci. 2011;31(13): 4886-95. [PMID:21451027] http://dx.doi.org/10.1523/JNEUROSCI.5122-10.2011

[43.] Jovine L, Park J, Wassarman PM. Sequence similarity between stereocilin and otoancorin points to a unified mechanism for mechanotransduction in the mammalian inner ear. BMC Cell Biol. 2002;3:28. [PMID:12445334] http://dx.doi.org/10.1186/1471-2121-3-28

[44.] Ramakrishnan NA, Drescher MJ, Barretto RL, Beisel KW, Hatfield JS, Drescher DG. Calcium-dependent binding of HCN1 channel protein to hair cell stereociliary tip link protein protocadherin 15 CD3. J Biol Chem. 2009;284(5):3227-38. [PMID:19008224] http://dx.doi.org/10.1074/jbc.M806177200

[45.] Momiyama J, Hashimoto T, Matsubara A, Futai K, Namba A, Shinkawa H. Leupeptin, a calpain inhibitor, protects inner ear hair cells from aminoglycoside ototoxicity. Tohoku J Exp Med. 2006;209(2):89-97. [PMID:16707850] http://dx.doi.org/10.1620/tjem.209.89

[46.] Abaamrane L, Raffin F, Schmerber S, Sendowski I. Intracochlear perfusion of leupeptin and z-VAD-FMK: influence of antiapoptotic agents on gunshot-induced hearing loss. Eur Arch Otorhinolaryngol. 2011;268(7):987-93. [PMID:21246210] http://dx.doi.org/10.1007/s00405-011-1487-0

[47.] Kathiresan T, Harvey M, Orchard S, Sakai Y, Sokolowski B. A protein interaction network for the large conductance Ca(2+)-activated K(+) channel in the mouse cochlea. Mol Cell Proteomics. 2009;8(8):1972-87. [PMID:19423573] http://dx.doi.org/10.1074/mcp.M800495-MCP200

[48.] Scherer EQ, Herzog M, Wangemann P. Endothelin-1induced vasospasms of spiral modiolar artery are mediated by rho-kinase-induced Ca(2+) sensitization of contractile apparatus and reversed by calcitonin gene-related peptide. Stroke. 2002;33(12):2965-71. [PMID:12468798] http://dx.doi.org/10.1161/01.STR.0000043673.22993.FD

[49.] Elgoyhen AB, Katz E, Fuchs PA. The nicotinic receptor of cochlear hair cells: a possible pharmacotherapeutic target? Biochem Pharmacol. 2009;78(7):712-19. [PMID:19481062] http://dx.doi.org/10.1016/j.bcp.2009.05.023

[50.] Fetoni AR, Ralli M, Sergi B, Parrilla C, Troiani D, Paludetti G. Protective effects of N-acetylcysteine on noiseinduced hearing loss in guinea pigs. Acta Otorhinolaryngol Ital. 2009;29(2):70-75. [PMID:20111615]

[51.] Le T, Keithley EM. Effects of antioxidants on the aging inner ear. Hear Res. 2007;226(1-2):194-202. [PMID:16843623] http://dx.doi.org/10.1016/j.heares.2006.04.003

[52.] Koliatsos VE, Cernak I, Xu L, Song Y, Savonenko A, Crain BJ, Eberhart CG, Frangakis CE, Melnikova T, Kim H, Lee D. A mouse model of blast injury to brain: initial pathological, neuropathological, and behavioral characterization. J Neuropathol Exp Neurol. 2011;70(5):399-416. [PMID:21487304] http://dx.doi.org/10.1097/NEN.0b013e3182189f06

[53.] Cernak I, Merkle AC, Koliatsos VE, Bilik JM, Luong QT, Mahota TM, Xu L, Slack N, Windle D, Ahmed FA. The pathobiology of blast injuries and blast-induced neurotrauma as identified using a new experimental model of injury in mice. Neurobiol Dis. 2011;41(2):538-51. [PMID:21074615] http://dx.doi.org/10.1016/j.nbd.2010.10.025

[54.] Pun PB, Kan EM, Salim A, Li Z, Ng KC, Moochhala SM, Ling EA, Tan MH, Lu J. Low level primary blast injury in rodent brain. Front Neurol. 2011;2:19. [PMID:21541261] http://dx.doi.org/10.3389/fneur.2011.00019 Submitted for publication September 30, 2011. Accepted in revised form February 7, 2012.

Manojkumar Valiyaveettil, PhD; (1) * Yonas Alamneh, MS; (1) Stacy-Ann Miller, BS; (2) Rasha Hammamieh, PhD; (2) Ying Wang, MD; (1) Peethambaran Arun, PhD; (1) Yanling Wei, MD; (1) Samuel Oguntayo; (1) Madhusoodana P. Nambiar, PhD (1) *

(1) Blast-Induced Neurotrauma Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, Silver Spring, MD; (2) United States Army Center for Environmental Health Research, United States Army Medical Research and Materiel Command, Fort Detrick, MD

* Address all correspondence to Manojkumar Valiyaveettil, PhD, or Madhusoodana P. Nambiar, PhD; 503 Robert Grant Ave, Walter Reed Army Institute of Research, Silver Spring, MD 20910; 301-319-9679, ext 7307; fax: 301-319-9404. Emails: madhusoodana.nambiar@amedd.army.mil, m.valiyaveettil@amedd.army.mil

http://dx.doi.org/10.1682/JRRD.2011.09.0182

** Arun P, Valiyaveettil M, Biggemann L, Alamneh Y, Wei Y, Oguntayo S, Wang Y, Long JB, Nambiar MP. Modulation of hearing related proteins in the brain and inner ear following repeated blast exposures. Intervent Med Appl Sci. Forthcoming.
Table 1.

List of auditory-related genes significantly altered in hippocampus
after repeated blast exposures in mice.

Gene             GenBank
Symbol        Accession No.                 Gene Product

Otoa            NM_139310     Otoancorin
Cabp2           NM_013878     Calcium binding protein 2
Sod3            NM_011435     Superoxide dismutase 3
Hspb8           NM_030704     Heat shock protein 8
Hsf5          NM_001045527    Heat shock transcription factor member 5
Otof            NM_031875     Otoferlin
Calca           NM_007587     Calcitonin/calcitonin-related
                                polypeptide alpha
Pcdha4          NM_007766     Protocadherin alpha 4

Gene
Symbol        Fold Change   p-Value

Otoa             +3.4        0.04
Cabp2            +1.6        0.003
Sod3             +2.0        0.03
Hspb8            +1.3        0.02
Hsf5             +1.8        0.02
Otof             -1.8        0.04
Calca            -1.9        0.04
Pcdha4           -1.3        0.001

Table 2.

List of auditory-related genes significantly altered in cerebellum
after repeated blast exposures in mice.

                 GenBank
Gene Symbol   Accession No.             Gene Product

Pcdhb20         NM_053145     Protocadherin beta 20
Pcdha4          NM_007766     Protocadherin alpha 4
S100a7a         NM_199422     S100 calcium binding protein A7A
Cacng1          NM_007582     Calcium channel, voltage-dependent,
                                gamma subunit 1
Cacna2d1        NM_009784     Calcium channel, alpha 2, delta
                                subunit 1
Efcab2          NM_026626     EF-hand calcium binding domain 2
LOC641192       XM 918536     Similar to heat shock protein 8

Gene Symbol   Fold Change   p-Value

Pcdhb20          +1.2        0.03
Pcdha4           +1.2        0.03
S100a7a          +1.4        0.03
Cacng1           -1.2        0.003
Cacna2d1         -1.1        0.049
Efcab2           -1.1        0.01
LOC641192        -1.1        0.046

Table 3.

List of auditory-related genes significantly altered in frontal
cortex after repeated blast exposures in mice.

                 GenBank
Gene Symbol   Accession No.                 Gene Product

Capn3         NM_007601       Calpain 3
S100a3        NM_011310       S100 calcium binding protein A3
Camkk1        NM_018883       Calcium/calmodulin-dependent protein
                                kinase kinase 1
Efcab4a       NM_001025103    EF-hand calcium binding domain 4A alpha
                                polypeptide 7
Pcdhb11       NM_053136       Protocadherin beta 11
Calr          NM_00759        Calreticulin

Gene Symbol   Fold Change   p-Value

Capn3            +1.5        0.03
S100a3           +1.4        0.03
Camkk1           +1.2        0.01
Efcab4a          +1.4        0.04
Pcdhb11          -2.2        0.004
Calr             -1.2        0.04

Table 4.

List of auditory-related genes significantly altered in midbrain
after repeated blast exposures in mice.

                 GenBank
Gene Symbol   Accession No.                Gene Product

Cdh24         NM_199470       Cadherin-like 24
Capn9         NM_023709       Calpain 9
S100a3        NM_011310       S100 calcium binding protein A3
Gpx4          NM_008162       Glutathione peroxidase 4
Ccs           NM_016892       Copper chaperone for superoxide
                                dismutase
Kcnmb3        NM_171828       Calcium activated potassium channel
                                beta 3
Pcdh8         NM_021543       Protocadherin 8
Cadh12        NM_001008420    Cadherin 12
Cabp7         NM_138948       Calcium binding protein 7
Cacna1d       NM_028981       Calcium channel, voltage-dependent,
                                L type, alpha 1D subunit
Camk2g        NM_178597       Calcium/calmodulin-dependent protein
                                kinase 2 gamma
Hspb2         NM_178597       Heat shock protein 2
Chrna7        NM_007390       Cholinergic receptor, nicotinic alpha
                                polypeptide 7
Stc2          NM_011491       Stanniocalcin 2

Gene Symbol   Fold Change   p-Value

Cdh24            +1.8        0.02
Capn9            +2.1        0.03
S100a3           +1.2        0.048
Gpx4             +1.1        0.02
Ccs              +1.1        0.03
Kcnmb3           +2.1        0.03
Pcdh8            -1.4        0.02
Cadh12           -1.7        0.01
Cabp7            -2.2        0.04
Cacna1d          -1.6        0.03
Camk2g           -1.1        0.03
Hspb2            -1.3        0.04
Chrna7           -1.5        0.02
Stc2             -1.3        0.04
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