Selective Degeneration of Synapses in the Dorsal Cochlear Nucleus of Chinchilla Following Acoustic Trauma and Effects of Antioxidant Treatment

Hearing Research 283 (2012) 1e13

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Hearing Research

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Research paper Selective degeneration of synapses in the dorsal cochlear nucleus of chinchilla following acoustic trauma and effects of antioxidant treatment

Xiaoping Du a, Kejian Chen a,1, Chul-Hee Choi a,2, Wei Li a, Weihua Cheng a, Charles Stewart a,b, Ning Hu a, Robert A. Floyd b, Richard D. Kopke a,c,d,* a Hough Ear Institute, Oklahoma, OK 73112, USA b Experimental Therapeutic Research Program, Oklahoma Medical Research Foundation, Oklahoma, OK 73104, USA c Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma, OK 73104, USA d Department of Otolaryngology, University of Oklahoma Health Sciences Center, Oklahoma, OK 73104, USA article info abstract

Article history: The purpose of this study was to reveal synaptic plasticity within the dorsal cochlear nucleus (DCN) as Received 31 May 2011 a result of noise trauma and to determine whether effective antioxidant protection to the cochlea can Received in revised form also impact plasticity changes in the DCN. Expression of synapse activity markers (synaptophysin and 18 November 2011 precerebellin) and ultrastructure of synapses were examined in the DCN of chinchilla 10 days after Accepted 30 November 2011 a 105 dB SPL octave-band noise (centered at 4 kHz, 6 h) exposure. One group of chinchilla was treated Available online 9 December 2011 with a combination of antioxidants (4-hydroxy phenyl N-tert-butylnitrone, N-acetyl-L-cysteine and acetyl-L-carnitine) beginning 4 h after noise exposure. Down-regulated synaptophysin and precerebellin expression, as well as selective degeneration of nerve terminals surrounding cartwheel cells and their primary dendrites were found in the fusiform soma layer in the middle region of the DCN of the noise exposure group. Antioxidant treatment signiﬁcantly reduced synaptic plasticity changes surrounding cartwheel cells. Results of this study provide further evidence of acoustic trauma-induced neural plasticity in the DCN and suggest that loss of input to cartwheel cells may be an important factor contributing to the emergence of hyperactivity in the DCN after noise exposure. Results further suggest that early antioxidant treatment for acoustic trauma not only rescues cochlear hair cells, but also has impact on central auditory structures. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction 2002; Heffner and Harrington, 2002; House and Brackmann, 1981; Kaltenbach, 2006; Pulec, 1995; Sziklai, 2004). Among the Acoustic trauma is the leading cause of hearing loss and tinnitus hypotheses for a central origin of tinnitus, many studies have both in the military and in the civilian population (Nelson et al., focused on the dorsal cochlear nucleus (DCN). For example, elec- 2005; Saunders and Griest, 2009). Noise-induced hearing loss is trophysiological studies have demonstrated hyperactivity in the mainly caused by hair cell damage/death through mechanical and DCN after noise exposure (Kaltenbach et al., 2005; Kaltenbach, metabolic mechanisms (Le Prell et al., 2007; Slepecky, 1986; 2006). Animals with psychophysical evidence of noise-induced Yamane et al., 1995a, b). The mechanisms of noise-induced tinnitus had elevated spontaneous activity in the DCN, suggesting tinnitus, however, are less clear. There have been many hypoth- that such hyperactivity may be related to noise-induced tinnitus eses, some focused on the auditory periphery while others (Brozoski et al., 2002; Kaltenbach et al., 2004). The same dose of emphasize the importance of changes in the CNS (Brozoski et al., noise exposure that causes hyperactivity in the DCN also causes tinnitus in animals (Heffner and Harrington, 2002). Furthermore, up-regulation of cholinergic and glutamate receptors, elevated * Corresponding author. Hough Ear Institute, P.O. Box 23206, Oklahoma, OK glutamatergic synaptic release, and depressed glutamate uptake 73123, USA. Tel.: þ1 405 639 2876; fax: þ1 405 947 6226. have been observed in both the ventral cochlear nucleus (VCN) and E-mail address: [email protected] (R.D. Kopke). DCN after noise exposure (Kaltenbach and Zhang, 2007; Muly et al., 1 Present address: Spatial Orientation Center, Naval Medical Center, San Diego, 2004). Weeks after noise exposure, expression of a glycine receptor CA, USA. a 2 Present address: Audiology & Speech-Language Pathology, College of Medical subunit (GlyR 1) decreased in the fusiform soma layer (FSL) at the Sciences, Catholic Univ. of Daegu, Kyungsansi, South Korea. middle and high frequency regions of the DCN (Wang et al., 2009).

These results suggest an excitotoxicity mechanism in the DCN after 2. Materials and methods noise exposure. In the excitotoxicity mechanism, toxic concentra- tions of glutamate or imbalance of excitatory and inhibitory amino 2.1. Animals, noise exposure and antioxidant injection acids will lead to osmotic imbalance across the post-synaptic membrane, and result in fluid entry into cells, swelling of cells, All tissue samples used in this study were collected from our rupturing of cell membranes and degeneration (Le Prell et al., previous study except tissue samples for TEM study. Noise expo- 2007). sure, administration of antioxidants and measurement of auditory Pathological changes have been found in the central auditory brainstem responses were detailed in our previous reports (Choi system, especially in the cochlear nucleus (CN), after noise expo- et al., 2008; Du et al., 2011). In brief, the chinchilla were random- sure (Aarnisalo et al., 2000; Basta et al., 2005; Gröschel et al., 2010; ized into 3 groups (n ¼ 7 in each group, 6 for histology and 1 for Kim et al., 1997, 2004a,b,c; Morest et al., 1998; Muly et al., 2004; TEM study): Animals in the noise exposure plus carrier solution Theopold, 1975). The number or density of axons in the chinchilla (dimethyl sulfoxide, polyethylene glycol 400, and saline) and noise posterior ventral cochlear nucleus (PVCN) was decreased by about plus treatment (noise/treatment) groups were exposed to a 105 dB half (Bilak et al., 1997). However, the number of inhibitory endings sound pressure level (SPL) octave-band noise (OBN) for 6 h. only partially recovered while excitatory synapses were fully Animals in the normal control group were not exposed to noise. An restored by 24e32 weeks (Kim et al., 2004a). Excitatory terminals initial antioxidant injection was given to animals in the noise/ displayed progressive increases in neurofilaments and enlarged treatment group 4 h after noise exposure and then twice a day for synaptic vesicles in the PVCN weeks after noise exposure. Inhibi- the following 2 days. Animals in this group received 20 mg/kg of tory terminals darkened while some vesicles disintegrated and 4-HOPBN dissolved in dimethyl sulfoxide (40%), polyethylene many smaller flatter vesicles collapsed (Kim et al., 2004b, c). glycol 400 (40%), and saline (20%), 50 mg/kg of 20% NAC in water Degeneration of synapsing nerve endings of secondary neurons (containing 0.05% edetate disodium, dehydrate, PH 7.0, Hospira Inc., was found in the anterior ventral cochlear nucleus (AVCN) and lake Forest, IL), and 20 mg/kg of ALCAR (SigmaeAldrich Inc. St. octopus cell area of the PVCN (Theopold, 1975). These areas receive Louis, MO) in saline. These agents were intraperitoneally admin- afferent input from the cochlea. Reduction of neuropil volume and istered separately. Equal volumes of carrier solution were injected neuron size, and increases in neuronal packing density with into animals in the noise exposure and the normal control groups at minimal changes in neuron number were observed in the VCN and the same time points as in the noise/treatment group. ABR deep layer (DL) of the DCN, but not in layers 1 and 2 of the DCN that thresholds for both ears of each animal were measured before noise have no direct input from the cochlea (Willott et al., 1994). exposure (baseline), immediately after, and then 10 days after noise However, nerve terminals in the DCN have not been well examined exposure (Choi et al., 2008). in noise exposed animals. For noise exposure, two chinchilla at a time were placed in two In the DCN, fusiform and giant cells are the projecting cells and small wire restraint cages on a wooden plate in a sound isolation receive direct auditory nerve inputs. Cartwheel cells, major booth (Industrial Acoustics Company, New York, NY) and exposed inhibitory inter-neurons, receive synaptic inputs from the granule to 105 dB SPL OBN centered at 4 kHz for 6 h. The noise was cell-parallel fiber system and send their axons to form synapses on generated, filtered by a Tucker Davis Technologies device (TDT, fusiform cells, giant cells and also on other cartwheel cells (Davis Alachua, FL), amplified by an audio power amplifier (QSC PLX 3402, and Young, 1997; Golding and Oertel, 1996, 1997; Wouterlood Costa Mesa, CA), and transduced with an acoustic speaker and Mugnaini, 1984). Granule cells in the DCN receive (JBL 2350, Northridge, CA). The speaker was positioned directly converging inputs from both auditory and non-auditory systems above the wire cages. During noise exposure, a condenser micro- (Burian and Gestoettner, 1988; Itoh et al., 1987; Weedman et al., phone (B&K 2804, Norcross, GA) was used to continually monitor 1996). Evidence supporting the DCN’s role in tinnitus generation the noise level. has been reviewed by Kaltenbach and Godfrey (2008). Although studies suggested that the hyperactivity recorded in the DCN may 2.2. Cochlear and brain tissue processing and hair cell counting be from fusiform cells (Brozoski et al., 2002; Finlayson and Kaltenbach, 2009), some studies have suggested that cartwheel After the last ABR measurement, 18 chinchilla (6 in each group) cells may be involved (Chang et al., 2002; Kaltenbach and Zhang, were euthanized with an overdose of ketamine and xylazine, and 2007). then intracardially perfused with 0.1 M phosphate buffered saline Numerous experimental treatments have been tested in animal (PBS, pH 7.2), followed by 4% paraformaldehyde in PBS. Cochleae models of noise-induced hearing loss (Le Prell et al., 2007). The (total of 36) and brains (total of 18) were removed and post-fixed in rational for one of the most promising treatments, antioxidants, is the same fixative (overnight for the cochleae and 1 week for the based on the fact that noise exposure provokes the production of brain), and then washed in PBS and stored in the buffer at 4 C. The reactive oxygen species and reactive nitrogen species in the cochlea right cochlea from each animal was used for whole mount and (Henderson et al., 2006; Kopke et al., 2002, 2007; Ohinata et al., TRITC-phalloidin staining for hair cell counting under a fluorescent 2000; Ohlemiller et al., 1999; Yamane et al., 1995a, b). Our microscope (Olympus BX51, Melville, NY). In this study, percentage previous studies have demonstrated that an antioxidant treatment of missing OHCs and inner hair cells (IHCs) was calculated as in [4-hydroxy phenyl N-tert-butylnitrone (4-HOPBN), N-acetyl-L- previous reports (Choi et al., 2008; Eldredge et al., 1981; Harding cysteine (NAC), acetyl-L-carnitine (ALCAR)] can significantly atten- et al., 2002). The brainstem from each animal was cryoprotected uate the functional consequence of noise trauma and rescue outer in 30% sucrose in PBS at 4 C until the tissue was sitting on the hair cells (OHC) in the cochlea (Choi et al., 2008). However, effects bottom of the container, then embedded in Tissue-Tek (Sakura of the antioxidant treatment on the central auditory system have Finetek USA Inc. Torrance, CA) and serially sectioned in a coronal not yet been studied. plane with a Thermo Cryotome (Thermo Fisher Scientific, Inc. In the present study, two synapse markers: anti-synaptophysin Waltham, MA) at 18e20 mm. One section from every ten sections (Calhoun et al., 1996) and anti-precerebellin (Miura et al., 2009; from each brainstem was mounted onto a gelatin pre-coated slide Mugnaini et al., 1988) antibodies, and transmission electron (total of 10 slides). Therefore, the distance between two adjacent microscopy (TEM) were used to examine synapses in the DCN 10 sections on each slide was about 200 mm and each slide had at least days after noise exposure and antioxidant treatment. 4 sections from left and right DCNs of each animal. The sections X. Du et al. / Hearing Research 283 (2012) 1e13 3 were processed for immunohistochemical analysis (detailed below).

2.3. Biomarker expression analysis in brainstem using immunohistochemical staining

The sections were washed with PBS, blocked in 1% bovine serum albumin (fraction V) and 1% normal goat serum in PBS and per- meabilized in 0.2% triton X-100 in PBS (PBS/T). The tissue was incubated with mouse anti-synaptophysin IgG (a synapse marker, 1:1000, Chemicon International, Inc. Temecula, CA), or rabbit anti- precerebellin antibody (a marker for cartwheel cell synapses, 1:15, AnaSpec, Inc. San Jose, CA), or rabbit anti-PEP-19 IgG (a marker for cartwheel cells, 1:1000, kindly provided by Dr. James Morgan, St. Jude Children’s Research Hospital), or rabbit anti-NeuN IgG (a neuron marker, 1:500, Chemicon International, Inc. Temecula, CA) overnight. After PBS/T washing, biotinylated anti-mouse IgG or anti-rabbit IgG (1:200, Vector Laboratories, Inc. Burlingame, CA) was applied to the slides for 1 h, and Vectastain ABC and DAB kits Fig. 1. An example of low magnification of the DCN (synaptophysin immunostaining (Vector Laboratories, Inc. Burlingame, CA) were then used for the and methyl green nuclear counter-staining). The DCN was divided into three parts immunolabeling visualization. Immunopositive cells had a brown (dashed lines): the medial third (medial), the middle third (middle) and the lateral reaction product. Methyl green was used for nuclear counter- third (lateral). The rectangles indicate the locations that images were taken for quantification of immunostaining. Scale bar ¼ 500 mm. staining. Negative control was conducted by omitting the primary antibodies. immunostaining density. Bivariate correlation (SPSS 14.0 for fi 2.4. Quanti cation of biomarker immunostaining and statistical windows) was used to determine the correlation between hair cell analysis loss in the cochlea and immunostaining density in the DCN. A p-value of less than 0.05 was considered to be significant in the A tonotopic organization has been delineated along the statistical analyses. mediolateral axis of the DCN. Low frequencies are found laterally with high frequencies medially (Friauf, 1992; Rouiller et al., 1992; Ryan et al., 1988; Saint Marie et al., 1999; Yajima and Hayashi, 2.5. Transmission electron microscopic study of DCN 1989). Based on these data, the DCN of the chinchilla was divided into three parts: the medial third (medial), the middle third Changed expression of biomarkers in the DCN may be caused (middle) and the lateral third (lateral). Images taken from these by degeneration of neurons, nerve terminals, and/or synapses in regions were used for quantification of immunostaining. A modi- the DCN. To test this hypothesis, 3 chinchilla (one from each fied two-dimensional quantification method (Idrizbegovic et al., group) were used for the TEM study. ABR measurement, noise 1998) was employed to count positive immuno-stained cells in exposure and antioxidant treatment were performed as described the DCN. A color camera (DP70) attached to an Olympus micro- above. Ten days after noise exposure, the animals were intracar- scope (BX51) and DPController and DPManager programs dially perfused with PBS, followed by 4% freshly depolymerized (Olympus, Melville, NY) were used to obtain images. The three paraformaldehyde and 0.125% glutaradehyde in PBS, and then 4% regions of the DCN were first identified at a low power magnifi- freshly depolymerized paraformaldehyde. The brainstems were cation (4), an image was then captured using a higher power dissected out and post-fixed in the same fixative overnight at magnification (20) from each region. The superficial edge of DCN 4 C. Sections were cut on a vibratome at 40e50 mm and the was included in the image to ensure that a similar depth of DCN middle region of DCN was dissected out under a dissecting was enclosed in the quantification, which contained the molecular microscope and used for TEM. The tissues were post-fixed in 1% layer (ML) and FSL and superficial part of the DL (Fig. 1). All DCN Osmium for 90 min and dehydrated in a dilution series of ethanol sections (4e6 sections from 2 DCN) on each slide from each chin- and propylene oxide and then slowly infused with resin. The chilla were used in quantification. The distance between two tissues were embedded in an epon/araldite mixture and cured for sections was about 200 mm to ensure non-duplicate counting. The 48 h in an oven to harden. Blocks were then cut perpendicularly total number of positive cells within each image was counted by to the tissue plane into 100 nm thick sections on a Leica UC6 using ImageJ software (National Institutes of Health). Only dark Ultramicrotome (Leica Microsystems Inc.). The sections were laid brown stained cells were counted. down on glow discharged formvar coated 75 Cu mesh grids and The cell counting was conducted by a technician who was lead/uranyl acetate stained for contrast then imaged in a Hitatchi unaware of the identity of each slide. The ABR threshold shifts and H-7600 TEM (Hitachi High Technologies America, Inc). We the cell counting data were tested for normal distribution by focused on cartwheel cells in the TEM study since the expression Kurtosis and Skewness (SPSS 14.0 for windows). The value region of the synaptic markers was down-regulated only in the vicinity between þ2and2 was considered to be a normal distribution. of cartwheel cells (detailed in Results). Cartwheel cells were The distribution of ABR threshold shifts and all cell counts were identified using the established criteria reported previously normal. One way ANOVA (SPSS 14.0 for windows) and a post hoc (Wouterlood and Mugnaini, 1984). The diameter of cartwheel test (Tukey HSD) were used to determine if there were statistically cells is about 9e10 mm. The cartwheel cell body has a smooth significant differences among three groups, between different contour. The nucleus contains evenly dispersed chromatin and DCN regions in the same group and between each of the two has at least one indentation (Fig. 2). Five to seven cartwheel cells groups (normal control vs. noise exposure, normal control vs. were identified in each group. Once a cartwheel cell was identi- noise/treatment, noise exposure vs. noise/treatment) in fied with low magnification (i.e. 3500), higher magnification 4 X. Du et al. / Hearing Research 283 (2012) 1e13

Fig. 2. An example of a cartwheel cell in the DCN imaged by TEM. The diameter of cartwheel cells is about 9e10 mm. The cartwheel cell body has a smooth contour. The nucleus contains evenly dispersed chromatin and an indentation (arrow). The scale bar ¼ 2 mm.

(i.e. 8000) was used to take images of synapses surrounding the cell body and the primary dendrites that arise directly from the cell body.

3. Results

3.1. Antioxidant treatment reduced hearing loss and hair cell loss Fig. 3. Average ABR threshold shifts at each frequency (A), low frequencies (0.5e1 kHz) and high frequencies (2e8 kHz, B) 10 days after noise exposure. Hearing threshold shifts were found in both the noise exposure and the noise/treatment groups at all The average ABR threshold shifts were 42.6 dB in the noise frequencies with greater shifts in the high frequencies (2e8 kHz). Significant reduc- exposure group and 19.79 dB in the noise/treatment group tions in threshold shifts were found in the noise/treatment group at all frequencies, (ANOVA, number of ears ¼ 12 in each group, df ¼ 1 and 142 especially at high frequencies compared to the noise exposure group (***, ** and * (between and within groups), F value ¼ 70.33, p < 0.001). The indicate p < 0.001, <0.01 and <0.05, respectively. Error bars represent standard error of the means. Number of ears ¼ 12 in each group). antioxidant treatment reduced hearing loss by 14.48 dB at low frequencies (0.5e1 kHz, 25.21 vs. 10.73 dB, ANOVA, number of ¼ ears ¼ 12 in each group, df ¼ 1 and 46, F value ¼ 17.26, p < 0.001) loss among the groups (ANOVA, number of cochleae 6ineach ¼ ¼ < and 26.98 dB at high frequencies (2e8 kHz, 51.30 vs. 24.32 dB, group, df 2and102,F value 28.19, p 0.001). There was fi ANOVA, number of ears ¼ 12 in each group, df ¼ 1 and 94, F a signi cant difference between the noise exposure and noise/ value ¼ 95.07, p < 0.001, Fig. 3). treatment groups in the mean percentages of IHC loss in the < As shown in the cytocochleogram in Fig. 4, reduced OHC loss cochleae (Tukey HSD, 1.06% vs. 1.90%, p 0.001). The majority of was found in the noise/treatment group compared to the noise missing IHCs in the noise exposure group were located at the exposure group. Average percentages of missing OHCs were 42.3% region ranging from 3.33 to 14.53 kHz (Fig. 4B). Average in the noise exposure group, 22.17% in the noise/treatment group percentages of missing IHCs in regions corresponding to cochlear and 4.04% in the normal control group (number of cochleae ¼ 6in frequencies ranging from 2 to 8 kHz were 2.50% in the noise each group, df ¼ 2and102,F value ¼ 63.48, p < 0.001). The exposure group, 1.13% in the noise/treatment group and 0.39% in ¼ majority of missing OHCs in the noise exposure group were the normal control group (ANOVA, number of cochleae 6ineach ¼ ¼ < located at region ranging from 1.59 to 14.53 kHz (Fig. 4A). Average group, df 2and69,F value 5.49, p 0.01). There was fi percentages of missing OHCs in the regions corresponding to a signi cant difference between the noise exposure and noise/ cochlear frequencies ranging from 2 to 8 kHz were 68% in the treatment groups in the average IHC loss at the high frequency e < fi noise exposure group, 27.67% in the noise/treatment group and region (2 8 kHz, Tukey HSD, p 0.05). However, signi cant IHC e 2.56% in the normal control group (ANOVA, number of loss was also found in the apical turn (0.13 0.49 kHz) in the noise cochleae ¼ 6 in each group, df ¼ 2and69,F value ¼ 27.30, exposure group. Reduced IHC loss was also found in this region in p < 0.001). There was a significant difference between the noise the noise/treatment group compared to the noise exposure group < exposure and noise/treatment groups in the average OHC loss at (1.28% vs. 2.35%, Tukey HSD, p 0.01). the high frequency region (2e 8 kHz, Tukey HSD, p < 0.001). Reduced IHC loss was also found in the noise/treatment group 3.2. Antioxidant treatment restored expression of synaptophysin in compared to the noise exposure group. Average percentages of the DCN missing IHCs were 1.90% in the noise exposure group, 1.06% in the noise/treatment group and 0.45% in the normal control group. At least two types of positive synaptophysin staining were Significant differences were found in the mean percentages of IHC identified in the DCN: small dots in the ML, FSL and DL (arrowheads X. Du et al. / Hearing Research 283 (2012) 1e13 5

3.3. Antioxidant treatment restored expression of precerebellin in the DCN

To reveal the neuron types whose synaptic endings may have degenerated following AAT, anti-precerebellin antibody was used to label cartwheel cell synapses because precerebellin is highly abundant in parallel fiberePurkinje cell synaptic clefts in cerebellum (Miura et al., 2009; Matsuda et al., 2010), and its active form, cerebellin, is highly enriched in the post-synaptic spine of cerebellar Purkinje cells in cerebellum and cartwheel cells in the DCN (Mugnaini et al., 1988). Examples of precerebellin immunostaining images from the middle region of the DCN of the normal control, the noise exposure and the noise/treatment groups by light microscopy are shown in Fig. 6AeC. Positive precerebellin staining is found in the FSL and DL of the DCN. Medium size, strong circle-like staining is found in the FSL (arrows in Fig. 6), representing synapses of cartwheel cells (Mugnaini et al., 1988). A few dense, solid smaller size positive dots are found in the DL (arrowheads in Fig. 6), although the population of inputs it represents is not clear. Only medium size, circle-like positive stained cells were counted in the present study. A decreased number of positive precerebellin stained cells was found in the middle region of the DCN of the noise exposure group. More positive precerebellin stained cells are found in this region of the Fig. 4. Cytocochleograms representing mean percentage of missing OHCs (A) and IHCs DCN of the noise/treatment group. Significant differences are (B) at the corresponding frequency on the basilar membrane in the control, the noise exposure and the noise/treatment groups. Reduced OHC and IHC loss was found in the found only in the middle region of the DCN among the groups noise/treatment group compared to the noise exposure group. There was a significant (ANOVA, number of DCN samples ¼ 12 in each group, df ¼ 2and difference between these two groups in average OHC loss (p < 0.001) and IHC loss 63, F value ¼ 5.97, p < 0.01), and between the normal control and < (p 0.001) in the cochleae. Error bars represent standard error of the means. Number noise exposure groups (Tukey, HSD, p < 0.01); as well as between of cochleae ¼ 6 in each group. the noise exposure and noise/treatment groups (Tukey, HSD, p < 0.05), but not between the normal control and noise/treat- in Fig. 5), and larger strongly staining dots in the FSL (arrows in ment groups (Tukey, HSD, p > 0.05). There was no significant Fig. 5), representing the small and the single larger terminal bou- difference among the three groups in the lateral (ANOVA, number tons, respectively (Gil-Loyzaga et al., 1998). Much weaker syn- of DCN samples ¼ 12 in each group, df ¼ 2and61,F value ¼ 1.15, aptophysin immunostaining was found in the middle region of the p > 0.05) and medial (ANOVA, number of DCN samples ¼ 12 in DCN of the noise exposure group, and both types of immuno- each group, df ¼ 2and61,F value ¼ 0.79, p > 0.05) regions staining were decreased in all three layers (Fig. 5B). Compared to (Fig. 6D). These results indicate that noise exposure used in the the noise exposure group, more positive synaptophysin stained present study inhibited expression of precerebellin in the DCN granules and dots were found in the DCN of the noise/treatment with a regional specific pattern, probably reflecting degeneration group (Fig. 5C). The number of small and single larger terminal of synaptic endings surrounding cartwheel cells following AAT. boutons were counted in the DCN and statistically analyzed. The antioxidant treatment significantly restored its expression in Significant differences were found in the middle region of the DCN the DCN. among the groups (ANOVA, number of DCN samples ¼ 12 in each Higher synaptophysin and precerebellin immunodensities in group, df ¼ 2 and 53, F value ¼ 13.56, p < 0.01), and between the the middle region of both normal controls and noise exposed normal control and the noise exposure groups (Tukey HSD, animals (Figs. 5D and 6D) may mirror the highest neuron density in p < 0.01), between the noise exposure and the noise/treatment this region (Figs. 7D and 8, Spirou et al., 1993). groups (Tukey HSD, p < 0.01), but not between the normal control and noise/treatment groups (Tukey HSD, p > 0.05). There was no 3.4. Noise exposure did not change the number of cartwheel cells in significant difference among three groups in the lateral (ANOVA, the DCN number of DCN samples ¼ 12 in each group, df ¼ 2 and 55, F value ¼ 0.13, p > 0.05) and medial (ANOVA, number of DCN Lower expression of synaptophysin and precerebellin in the samples ¼ 12 in each group, df ¼ 2 and 51, F value ¼ 0.46, p > 0.05) DCN may be caused by a decreased number of cartwheel cells. To regions (Fig. 5D). These results indicate that noise exposure used in exclude this possibility, anti-PEP-19 antibody, a marker for cart- the present study inhibited expression of synaptophysin in the DCN wheel cells (Berrebi and Spirou, 1998), was used to quantify the with a regional specific pattern, probably reflecting degeneration of number of cartwheel cells in the DCN. Positive PEP-19 stained cells synaptic endings following acute acoustic trauma (AAT, Muly et al., were mainly found in the FSL (arrows in Fig. 7AeC) with a few in 2002), and that antioxidant treatment significantly restores its the DL (arrowheads in Fig. 7AeC) of the DCN of the normal control, expression in the DCN. Positive synaptophysin staining was also the noise exposure, and the noise/treatment groups. PEP-19 posi- found in the VCN as reported (Gil-Loyzaga et al., 1998). However, tive cells in the DL may represent the misplaced cartwheel cells or the noise exposure did not change its expression in the VCN (data giant cells (Berrebi and Mugnaini, 1991; Berrebi and Spirou, 1998). not shown). Only tiny granular like positive staining was found in The results of PEP-19 immunostaining density measurement are inferior colliculus (IC) without obvious differences between groups shown in Fig. 7D. No significant difference is found among the (data not shown). Very weak or no signifi cant positive staining was groups in each region of the DCN (ANOVA, number of DCN detected in the lateral superior olive (LSO) or medial superior olive samples ¼ 12 in each group, df ¼ 2 and 39, F value ¼ 0.52e1.07, all (MSO, data not shown). p > 0.05). 6 X. Du et al. / Hearing Research 283 (2012) 1e13

3.5. Noise exposure did not change the number of NeuN positive F value ¼ 0.112e3.313, all p > 0.05). Relatively large variability was cells in the DCN found between animals in the NeuN immunostaining density measurement, which may be responsible for high standard devia- To determine whether any changes occurred in the number of tions and for not detected significant differences between groups. other types of neurons in the DCN, anti-NeuN antibody was used to More animals in each group may be needed to decrease the label the majority of neurons in the DCN (Sekiya et al., 2009). variability. Positive NeuN stained cells were found in all three layers of the DCN of the normal control, the noise exposure, and the noise/treatment 3.6. Relationship between hair cell loss in the cochlea and groups. The results of the NeuN immunostaining density expression of synaptophysin or precerebellin in the DCN measurement are shown in Fig. 8. There was a trend of an increase in the mean neuron density with noise exposure and treatment in Altered expression of synaptophysin and precerebellin in the the middle region of the DCN with a slight decreased neuron DCN might be associated with hair cell loss in the cochlea. To test density in the lateral and medial regions of the DCN after noise this hypothesis, correlation between the mean percentage of OHC exposure and treatment. However, no significant differences were or IHC loss in the frequency region of 1e5 kHz in the cytoco- found among the groups in any of the three regions of the DCN chleogram and the mean density of synaptophysin or precer- (ANOVA, number of DCN samples ¼ 12 in each group, df ¼ 2 and ebellin immunostaining in the middle region of the DCN for 53e58, F value ¼ 0.13e0.81, all p > 0.05). Compared to the normal individual animals in the noise exposure and noise/treatment control group, the neuron density was increased by 9.35% in the groups was examined. The middle region of the DCN of chinchilla noise exposure group and 13.76% in the noise/treatment group in represents approximately the 1e5 kHz region in the tonotopic the middle region, were slightly decreased by 7.82% and 6.72% in map of chinchilla (Kaltenbach and Saunders, 1987; Kaltenbach, the noise exposure group, and 1.27% and 2.39% in the noise/treat- personal communication). There was no significant correlation ment group in the lateral and medial regions, respectively. No between the hair cell loss in the frequency region of 1e5 kHz and significant differences were found between the groups in the the immunostaining density (OHC loss and synaptophysin percentages in any of the three regions of the DCN (ANOVA, density: r ¼ 0.293, p ¼ 0.262; OHC loss and precerebellin density: number of DCN samples ¼ 12 in each group, df ¼ 1 and 33e37, r ¼ 0.346, p ¼ 0.224; IHC loss and synaptophysin density:

Fig. 5. Examples of synaptophysin immunostaining images obtained from the middle region of the DCN of the normal control (A), the noise exposure (B) and the noise/treatment (C) groups by light microscopy. Positive synaptophysin staining was found in the FSL and DL of the DCN (arrows and arrowheads in AeC). Positive stained dots were counted and statistically analyzed (D). Signiﬁcant differences were found only among the groups in the middle region of the DCN, but not in the lateral or medial regions (D). In the middle region, signiﬁcant differences were found between the normal control and the noise exposure groups (p < 0.01), as well as between the noise exposure and the noise/treatment groups (p < 0.01), but not between the normal control and noise/treatment groups (p > 0.05). ** in D indicates p < 0.01. Error bars represent standard error of the means. Scale bar ¼ 200 mm in C for AeC. X. Du et al. / Hearing Research 283 (2012) 1e13 7

Fig. 6. Examples of precerebellin immunostaining images obtained from the middle region of the DCN of the normal control (A), the noise exposure (B) and the noise/treatment (C) groups by light microscopy. Positive precerebellin stained cells were found in the FSL (arrows in AeC) and the DL (arrowheads in AeC) of the DCN. Positive stained cells in the FSL were counted and statistically analyzed (D). Significant differences were found among the groups in the middle region of the DCN only. In the middle region, significant differences were found between the normal control and the noise exposure groups (p < 0.01); as well as between the noise exposure and the noise/treatment groups (p < 0.05). ** and * in D indicate p < 0.01, <0.05, respectively. Error bars represent standard error of the means. Scale bar ¼ 200 mm in C for AeC. r ¼ 0.264, p ¼ 0.284; IHC loss and precerebellin density: r ¼ 0.573, vesicles were found in the nerve terminals on the cartwheel cell p ¼ 0.089). There was no significant correlation between the mean primary dendrites of the noise exposure group (arrowheads in percentages of OHC or IHC loss in the all frequency regions in Fig. 10E), but not in the normal control and the noise/treatment the cytocochleogram and the immunostaining density either groups (Fig. 10D and F). These pathologic changes in the nerve (data not shown). terminals may represent the intermediate stages of degeneration observed in the PVCN after a similar dose of noise exposure 3.7. Results of TEM study (Kim et al., 2004b).

At least two types of axo-somatic synaptic terminals were 4. Discussion found surrounding cartwheel cells in the DCN of all 3 groups of chinchilla: PVD (pleomorphic vesicles, dense) and PVL (pleomor- The results of this study suggest that antioxidant treatment phic vesicles, lucent, Fig. 9) as previously reported in the DCN of rat can provide protection not only to the cochlea, but also to the (Wouterlood and Mugnaini, 1984). No obvious pathologic changes central auditory system. Significant hearing protection with were seen in the PVD synaptic terminals of all 3 groups of chin- antioxidants was found in all frequencies, especially at high chilla. However, enlarged vesicles were seen in the PVL of noise frequencies (2e8 kHz). Consistent with ABR results, significant exposure and noise/treatment groups (arrow heads in Fig. 9Eand OHC and IHC protection with antioxidant treatment was found in F), but not in the PVL of the normal control group (Fig. 9D). The size all frequency regions, especially at high frequency regions (1.59 or of the enlarged vesicles was approximately 130 500 nm. Nor- 3.33e14.53 kHz, Fig. 4). These results indicate that antioxidant mally, the size of the vesicles is around 25e45 nm in diameter (Kim treatment significantly reduced both OHC and IHC loss from AAT. et al., 2004b). Furthermore, the normal convexity of synaptic Hair cell protection from the antioxidants in the cochlea is prob- membranes, as shown in the PVL terminals of the normal control ably associated with inhibition of free radical formation and and the noise/treatment groups (arrows in Fig. 9D and F), was migration of mononuclear phagocytes (Du et al., 2011). The replaced by a flattened and lower density contour in the noise mechanisms involved in central protection, however, are not clear. exposed chinchilla (empty arrow in Fig. 9E). Similar pathologic They could be direct protection to the CNS neurons by antioxi- changes were also found in the nerve terminals on cartwheel cell dants or an indirect effect by promoting hair cell survival in the primary dendrites of the noise exposure group (Fig. 10). Enlarged cochlea. 8 X. Du et al. / Hearing Research 283 (2012) 1e13

Fig. 7. Examples of PEP-19 immunostaining images obtained from the middle region of the DCN of the normal control (A), the noise exposure (B) and the noise/treatment (C) groups by light microscopy. Positive PEP-19 stained cells were found in the FSL (arrows in AeC) and the DL (arrowheads in AeC) of the DCN. Positive stained cells in the FSL were counted and statistically analyzed (D). No signiﬁcant differences were found among the groups in all regions of the DCN (p > 0.05). Error bars represent standard error of the means. Scale bar ¼ 200 mm in C for AeC.

4.1. Effects of noise exposure and antioxidant treatment on synapse secretion, which may cause synaptic integrity damage in the DCN. biomarker expression in the DCN Taken together these results indicate synapse degeneration in the DCN after noise exposure. Antioxidant treatment significantly Synaptophysin is a 38 kDa calcium-binding glycoprotein widely restored precerebellin and synaptophysin expression in the DCN, found in presynaptic membranes and vesicles (Jahn et al., 1985; suggesting antioxidants reduced synapse degeneration in the DCN. Wiedenmann and Franke, 1985) and is used as a marker for It is unclear whether antioxidants provided a direct protection on synaptic reorganization after denervation (Benson et al., 1997; DCN neurons or an indirect protection by promoting hair cell Brock and O’Callaghan, 1987; Masliah et al., 1991; Walaas et al., survival in the cochlea. However, hyperactivity in the DCN after 1988) and for quantification of synapses (Calhoun et al., 1996). noise exposure was not dependent on cochlear activity (Zacharek Decreased expression of synaptophysin was found in the VCN following AAT or cochlear ablation (Benson et al., 1997; Fuentes- Santamaría et al., 2007; Muly et al., 2002). Precerebellin (Cbln1), the precursor of the brain-specific neuropeptide cerebellin, is a synapse organizer for both pre- and post-synaptic components and is involved in synaptic integrity, plasticity, as well as synapse maintenance in the cerebellum (Hirai et al., 2005; Ito-Ishida et al., 2008; Matsuda et al., 2010; Miura et al., 2009). The levels of precerebellin and its active form, cerebellin, closely parallel synapse formation between granule cells and Purkinje cells (Slemmon et al., 1985; Urade et al., 1991). It is unclear whether precerebellin plays similar roles in the DCN. However, Purkinje cells in the cerebellum and cartwheel cells in the DCN share many features including histology, immunocytochemistry and electrophysiology (Berrebi et al., 1990). The characteristics of their synapses may also be analogous (Wouterlood and Mugnaini, 1984; Portfors and Roberts, Fig. 8. The results of the NeuN immunostaining density measurement. No significant 2007). The decreased precerebellin expression could be the result differences were found among the groups in all regions of the DCN (p > 0.05). Error of inhibited precerebellin synthesis and/or increased precerebellin bars represent standard error of the means. X. Du et al. / Hearing Research 283 (2012) 1e13 9

Fig. 9. Example of nerve terminals surrounding cartwheel cells in the DCN of a normal control (A, D), noise exposed (B, E) and noise/treatment (C F) chinchilla. The images in panels DeF are the higher magniﬁcation of the images in panels AeC, respectively. Two types of terminals, PVL and PVD, were found around cartwheels in the DCN of the normal control (A), noise exposure (B) noise/treatment (C, PVD is not shown in C) groups. No obvious changes were seen in the PVD synaptic terminals of all 3 groups of chinchilla. However, huge vesicles were seen in the PVL of the noise exposure and the noise/treatment groups (arrowheads in E, F), but not seen in the PVL of the normal control group (D). The size of the huge vesicles was around 130 500 nm in both the noise exposure and the noise/treatment groups. Furthermore, the normal convexity of synaptic membranes as shown in the normal control and noise/treatment groups (arrows in D and F) was replaced by a ﬂattened and lower density contour in the noise exposure chinchilla (empty arrow in E). The scale bars ¼ 500 nm (the bar in C for AeC; and in F for DeF, N stands for nucleus and M for mitochondrion).

et al., 2002). Indeed, antioxidants (L-carnitine, vitamin E) targeting (Kim et al., 2004b). It is more likely that the PVL surrounding the ongoing oxidative damage can provide direct protection to cartwheel cells are inhibitory, which may originate from other synapses in the CNS (Elinos-Calderón et al., 2009; Urano et al., cartwheel cells and other inhibitory inter-neurons, such as stellate 1997). Therefore, protection of DCN synapses may come directly cells in the VCN, vertical cells in the DCN, or neurons in the per- from antioxidants. iolivary nucleus, lateral lemniscus and IC (Rubio and Juiz, 2004). Thus, cartwheel cells in the DCN may lose their inhibitory inputs 4.2. Signiﬁcance of synapse degeneration in the DCN and effects of from other cartwheel cells and/or other inhibitory inter-neurons antioxidant treatment after noise exposure. Consistent with this explanation, no hyperactivity was recorded from fusiform cells in the DCN after noise The results of precerebellin immunostaining and TEM imaging exposure (Chang et al., 2002). Therefore, cartwheel cells may be in the present study indicate that synapse degeneration occurred one major source of the hyperactivity recorded in the DCN after selectively in the vicinity of cartwheel cells in the middle region of a 127 dB SPL 10 kHz tone exposure for 4 h (Chang et al., 2002; the DCN after noise exposure. Most synaptic endings (4 out of 5) Kaltenbach and Zhang, 2007) although these data may be in contacting cartwheel cells are glycinergic or GABA-ergic and contrast to Brozoski et al. (2002), who did show elevated activity presumably inhibitory (Rubio and Juiz, 2004). Although both PVL in fusiform cells in the DCN after an 80 dB SPL 4 kHz tone expo- and PVD could be either glycinergic/GABA-ergic or glutamatergic sure for 30e60 min. in the DCN, most of the PVL and PVD are GABA-ergic in the PVCN Cartwheel cells receive excitatory inputs from granule cells and (Rubio and Juiz, 2004; Wouterlood and Mugnaini, 1984). We send inhibitory outputs to fusiform cells and other cartwheel cells, found signs of synapse degeneration only in the PVL that contact modulating their activity (Berrebi and Mugnaini, 1991; Davis and the cartwheel cell bodies and its primary dendrites in the DCN Young, 1997; Rubio and Juiz, 2004; Wouterlood and Mugnaini, and reduced synapse degeneration in the antioxidant treated 1984). An alternative explanation of the current results is that DCN. These PVL have symmetrical synapses (arrows in Fig. 9D, F, degenerated synapses surrounding cartwheel cells may originate 10D and F) which are thought to be inhibitory (De Felipe et al., from granule cells. If this is the case, fusiform cells may lose the 1997). Further, the PVL terminals contain only pleomorphic and inhibitory inputs from cartwheel cells and become a source of ﬂattened vesicles (Fig. 9DeF), presumed to be inhibitory endings hyperactivity that was recorded in the DCN after noise exposure 10 X. Du et al. / Hearing Research 283 (2012) 1e13

Fig. 10. Example of nerve terminals on cartwheel cell primary dendrites in the DCN of the normal control (A, D), noise exposed (B, E) and noise/treatment (C, F) chinchilla. The images in panels DeF are the higher magniﬁcation of the images in panels AeC, respectively. Two types of terminals, PVL and PVD, were also found around the cartwheel primary dendrites in the DCN of the normal control (A), the noise exposure (B) and the noise/treatment (C) groups (the PVD is not shown in C and D). No obvious changes were seen in the PVD synaptic terminals of all 3 groups of chinchilla (data not shown). However, huge vesicles were seen in the primary dendrite and the PVL of the noise exposure group (arrowheads in E), but not in the normal control (A) or in the noise/treatment groups (F). The size of the huge vesicles was around 100e300 nm in the primary dendrite and 75 120 nm in the PVL. Furthermore, the normal convexity of the synaptic membranes as shown in the normal control and the noise/treatment groups (arrows in D and F) was replaced by a ﬂattened and lower density contour in the noise exposure chinchilla (empty arrow in E). The scale bars ¼ 500 nm (the bar in C for AeC; and in F for DeF, D1 stands for primary dendrite and M for mitochondrion).

(Brozoski et al., 2002; Finlayson and Kaltenbach, 2009; Kaltenbach (Caspary et al., 2005). Therefore, hyperactivity recorded in the DCN and Zhang, 2007). Consistent with this explanation, significant might originate from both cartwheel cells and fusiform cells. In decreases of glycine receptor subunits and an anchoring protein addition, whether the hyperactivity recorded in the DCN plays any were found in the DCN of rats with behavioral evidence of tinnitus important role in noise induced-tinnitus remains unclear. For after noise exposure (Wang et al., 2009). However, granule cell- example, bilateral ablation of DCN did not affect the psychophys- parallel fiber synapses on cartwheel cells are mainly on their ical evidence of noise induced-tinnitus (Brozoski and Bauer, 2005). dendrites, especially on dendrites located in the ML. Another The DCN inhibitory circuits formed by vertical, cartwheel and possibility is that the same process that causes loss of inhibition of D-multipolar cells are believed to preserve signal in background cartwheel cells may also cause loss of inhibition of fusiform cells. noise (Caspary et al., 2005). Our results may explain one of the The current study could not rule out this possibility due to lack of symptoms of noise induced-hearing loss, difficulty hearing in specific markers for fusiform cells. noisy environments. Antioxidants may help this symptom if this is Hyperactivity can be recorded in several parts of the auditory the case. system, such as the cochlear nucleus, IC and auditory cortex after noise exposure (Kaltenbach and Saunders, 1987; Kaltenbach et al., 4.3. Tonotopic synapse degeneration in the DCN and effects of 2004; Salvi et al., 2000; Seki and Eggermont, 2003). If the cart- antioxidant treatment wheel cells indeed receive less inhibition and increase their activity, how would this lead to hyperactivity elsewhere in the Previous studies delineated a tonotopic organization along the auditory system? Cartwheel cells axons synapse with other cart- mediolateral axis of the DCN. Low frequencies were found later- wheel cells and fusiform cells within the DCN (Berrebi and ally with high frequencies medially (Friauf, 1992; Rouiller et al., Mugnaini, 1991; Manis et al., 1994). However, fusiform cells 1992; Saint Marie et al., 1999; Yajima and Hayashi, 1989). receive their major inhibitory signals from vertical cells in the DL. Degeneration of synapses was found only in the middle region of Altered inhibitory signals from cartwheel cells would not likely the DCN in the present study. This specific regional pattern was alter fusiform cell spontaneous activity and its output to the IC also found in the DCN when rats were exposed to 116 dB SPL OBN X. Du et al. / Hearing Research 283 (2012) 1e13 11 centered at 17 kHz for 1 h. Changed expression of GlyR a1 protein since NeuN immunostaining may not stain all types of neurons in and mRNA was found only in the FSL in the middle and high the DCN. frequency regions of the DCN (Wang et al., 2009). This phenomenon may be caused by the specific frequencies of OBN. We used 4.4. Correlation between hair cell loss in the cochlea and synapse OBN centered at 4 kHz, which is lower than that in the report by biomarker expression in the DCN Wang et al. (2009). Consistent with our observation, axonal degeneration in the DL of the DCN was largely located in the It has been hypothesized that the pathological and/or elec- middle and lower frequency regions after exposing chinchilla to trophysiological changes in the central auditory system may be 108 dB SPL OBN centered at 4 kHz (Kim et al., 1997). A recent a consequence of hair cell injury in the cochlea after noise study has suggested that cartwheel cells respond well to tonal exposure or ototoxic drug administration (Kaltenbach et al., 2002; stimuli although they do not receive direct auditory nerve input Kim et al., 1997; Melamed et al., 2000; Muly et al., 2004; Salvi (Portfors and Roberts, 2007). This observation may explain the et al., 2000). Cochlear nuclei primarily receive two types of tonotopic degeneration of synapses surrounding cartwheel cells afferents from the cochlea: type I from IHCs and type II from in the DCN. OHCs. One mechanism that may trigger the pathological and/or Axon and synapse degeneration were found in the central electrophysiological changes in the central auditory system is auditory system after relatively low level octave-band noise expo- changes in the balance of inputs from these two types of afferents sure (108 dB SPL for 3 h, Muly et al., 2002; Kim et al., 1997, (Tonndorf, 1987). Strong correlation was found between loss of 2004a,b,c). Degeneration of axonal and synaptic endings in the primary afferent dendrites in the cochleae and noise induced- CN could result from a dystrophic mechanism or an excitotoxic tinnitus (Bauer et al., 2007). In the CNS, correlation was also mechanism or a trans-synaptic process (Kim et al., 1997, 2004b). found between axon degeneration in the VCN and IHC loss, but Degeneration of synaptic terminals surrounding cartwheel cells not between OHC loss and axon degeneration in the DCN and their primary dendrites may be caused by trans-synaptic (Kim et al., 1997). Furthermore, OHC loss is associated with degeneration because cartwheel cells do not have direct auditory increased activity in the DCN (Kaltenbach et al., 2002; Melamed input. However, cartwheel cells receive excitatory terminals from et al., 2000) while IHC loss is associated with cochlear nerve granular cells that have both auditory and somatosensory projec- loss and normal activity in the inferior colliculus (Kiang et al., tions (Burian and Gestoettner, 1988; Davis and Young, 1997; Itoh 1976; Salvi et al., 2000). In the present study, no significant et al., 1987; Weedman et al., 1996) and excitotoxicity could be correlation was found between the OHC or IHC loss in the cochlea one mechanism. Some antioxidants (i.e. ALCAR, PBN) are capable of and the synaptophysin or precerebellin immunostaining density reducing excitotoxicity-induced neuronal injury and death in the in the DCN. Consistent with our results, no correlation was found CNS (Elinos-Calderón et al., 2009; Schulz et al., 1995; Tastekin et al., between IHC or OHC loss and noise induced-tinnitus in rats 2005; Zanelli et al., 2005). Therefore, these antioxidants may (Bauer et al., 2007). Noise exposure used in the present study reduce synapse degeneration in the DCN by inhibiting caused widespread OHC loss, as well as significant IHC loss in the excitotoxicity. cochlea. The correlation between OHC loss accompanied by In contrast, neuron degeneration or death seems to be seen only damage to IHCs and hyperactivity in the DCN was weak as in the central auditory system of animals exposed to intense previously reported (Kaltenbach et al., 2002). Therefore, more octave-band noise (130 dB SPL for 5 h, Aarnisalo et al., 2000)or studies are needed to address this issue in the future. For impulse noise (198e202 dB or 40 shots of an alarm pistol, Säljö example, one could study pathological and electrophysiological et al., 2002; Theopold, 1975). The results of NeuN immunostain- changes in the DCN using noise exposure or ototoxic drugs that ing indicate that noise exposure used in the present study did not cause only OHC loss or IHC loss in the cochlea. cause neuron loss in the DCN at this time point of observation (10 days after noise exposure), although a previous study has 4.5. Conclusion suggested that cell density was decreased by 30e39% in the DCN 1 week after 3 h of 115 dB SPL broad band noise exposure (4e20 kHz, Results of this study provided further evidence that acoustic Gröschel et al., 2010). Expression of the cartwheel cell specific trauma can cause neural plasticity in the central auditory struc- marker (PEP-19) or the cellular morphology of cartwheel cells (TEM tures. Cartwheel cells as a major type of inhibitory inter-neuron study) were not changed in the present study, suggesting no cart- have been suggested to be a source of DCN hyperactivity recorded wheel cell loss or cartwheel cell body degeneration occurred in the from animals following acoustic trauma. This may be related to DCN 10 days after noise exposure. Unexpectedly, there was a trend reduced inhibitory synapses on their somata. The reduced inhibi- of the mean increase of neuron density with noise exposure and tion may be underlying hyperactivity after acoustic trauma. treatment in the middle region of the DCN (Fig. 8), suggesting Whether this neural plastic change following noise exposure is cochlear injury induced by noise exposure and/or antioxidants related to the mechanisms of some symptoms of noise induced- used in the present study may alter neurogenesis in the DCN hearing loss, i.e. difficulty hearing in noisy environments and (Zheng et al., 2011). Impaired neurogenesis was found in the tinnitus, will require further studies. Antioxidant treatment soon hippocampus 10 weeks after noise exposure (Kraus et al., 2010). In after noise not only rescued cochlear hair cells, but also reduced the the present study, there was a trend of increased neuron density in brainstem plasticity changes in the DCN. the middle region but slight decreased neuron density in the lateral and medial regions of the DCN after noise exposure, suggesting Acknowledgments a possible noise-induced neurogenesis with regional effects in the DCN. There was a trend of less neural loss in the lateral and medial This study was supported by grants (N00014-08-1-0484) from regions and a trend of increased neural density in the middle region the Office of Naval Research (ONR) and Integris Health, Oklahoma of the DCN in the treated subjects, implying the treatment may City, Oklahoma (RDK). The authors would like to thank Joe Wil- have impact on neurogenesis in the DCN. A previous study has kerson in Core Facility for Imaging at the Oklahoma Medical suggested a decreased cell density in the DCN after noise exposure Research Foundation for assistance in TEM. The authors wish like to although specific cell type(s) involved were not identified (Gröschel thank Dr. James Kaltenbach for his thoughtful review of and et al., 2010). Other neuron markers should be used in future studies suggestions for this manuscript. 12 X. Du et al. / Hearing Research 283 (2012) 1e13

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