NeuroImage 100 (2014) 642–649
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Functional mapping of the auditory tract in rodent tinnitus model using manganese-enhanced magnetic resonance imaging Da Jung Jung a,1, Mun Han b,1, Seong-Uk Jin b, Sang Heun Lee c, Ilyong Park d, Hyun-Ju Cho e, Tae-Jun Kwon e, Hui Joong Lee f, Jin Ho Cho g, Kyu-Yup Lee a,⁎, Yongmin Chang b,f,h,⁎⁎ a
Department of Otorhinolaryngology—Head and Neck Surgery, School of Medicine, Kyungpook National University Hospital, Daegu, Republic of Korea Department of Medical and Biological Engineering, School of Medicine, Kyungpook National University Hospital, Daegu, Republic of Korea Department of Otorhinolaryngology—Head and Neck Surgery, Daegu Veterans Hospital, Daegu, Republic of Korea d Department of Biomedical Engineering, College of Medicine, Dankook University, Cheonan, Republic of Korea e Department of Biology, College of Natural Science, Kyungpook National University, Daegu, Republic of Korea f Department of Radiology, School of Medicine, Kyungpook National University Hospital, Daegu, Republic of Korea g Department of Electronic Engineering, College of IT Engineering, Kyungpook National University, Daegu, Republic of Korea h Department of Molecular Medicine, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu, Republic of Korea b c
a r t i c l e
i n f o
Article history: Accepted 23 June 2014 Available online 28 June 2014 Keywords: Salicylate Tinnitus Manganese-enhanced MRI Auditory pathway Gap prepulse inhibition of acoustic startle
a b s t r a c t Animal models of salicylate-induced tinnitus have demonstrated that salicylate modulates neuronal activity in several brain structures leading to neuronal hyperactivity in auditory and non-auditory brain areas. In addition, these animal tinnitus models indicate that tinnitus can be a perceptual consequence of altered spontaneous neural activity along the auditory pathway. Peripheral and/or central effects of salicylate can account for neuronal activity changes in salicylate-induced tinnitus. Because of this ambiguity, an in vivo imaging study would be able to address the peripheral and/or central involvement of salicylate-induced tinnitus. Therefore, in the present study, we developed a novel manganese-enhanced magnetic resonance imaging (MEMRI) method to map the in vivo functional auditory tract in a salicylate-induced tinnitus animal model by administrating manganese through the round window. We found that acute salicylate-induced tinnitus resulted in higher manganese uptake in the cochlea and in the central auditory structures. Furthermore, serial MRI scans demonstrated that the manganese signal increased in an anterograde fashion from the cochlea to the cochlear nucleus. Therefore, our in vivo MEMRI data suggest that acute salicylate-induced tinnitus is associated with higher spontaneous neural activity both in peripheral and central auditory pathways. © 2014 Elsevier Inc. All rights reserved.
Introduction Approximately 10–15% of the adult population is affected by tinnitus, the perception of a sound without an external acoustic stimulus (Henry et al., 2005; Moller, 2011). Furthermore, subjective tinnitus severely affects the quality of life of 1–3% of the population (Eggermont and Roberts, 2004). Advances in functional neuroimaging methods and development of animal models have increasingly demonstrated that an increased firing rate of neurons, enhanced neuronal synchrony, and changes in the tonotopic organization in the central auditory pathway represent the neural substrate of tinnitus (Lanting et al., 2009; Salvi ⁎ Correspondence to: K.Y. Lee, Department of Otorhinolaryngology—Head and Neck Surgery, Kyungpook National University, School of Medicine, 130 Dongdeok-ro, Jung-gu, Daegu 700-721, Republic of Korea. Fax: +82 53 423 4524. ⁎⁎ Correspondence to: Y. Chang, Department of Molecular Medicine, Kyungpook National University School of Medicine, 130 Dongdeok-ro, Jung-gu, Daegu 700-721, South Korea. Fax: +82 53 422 2677. E-mail addresses: [email protected] (K.-Y. Lee), [email protected] (Y. Chang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.neuroimage.2014.06.055 1053-8119/© 2014 Elsevier Inc. All rights reserved.
et al., 2000; Seki and Eggermont, 2003). Moreover, changes in the peripheral auditory system have also been reported (Sahley and Nodar, 2001). Therefore, a remaining challenge is to understand how peripheral neural activity communicates with the central auditory system in tinnitus. That is, while the alterations of neural activity in central or peripheral auditory system were well demonstrated, it has seldom been studied whether there is alteration in neural communication between peripheral and central auditory system in tinnitus. In vivo manganese auditory tract tracing would provide a way to map the neural communication between peripheral and central auditory system because this imaging method is capable of tracing the neural activity through bulk transport of manganese ion (Mn2+) along an axonal tract. Evidence from animal models of tinnitus suggests that tinnitus can be a perceptual consequence of altered spontaneous neural activity along the auditory pathway. Electrophysiological studies measuring the average spectrum activity from the cochlear round window in animal models of salicylate-induced tinnitus revealed increased spontaneous neural activity of single units of the cochlear nerve (Cazals et al., 1998; Evans and Borerwe, 1982; Martin et al., 1993; Stypulkowski,
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1990). These studies therefore suggest that, at least in part, salicylateinduced tinnitus is associated with dysfunction of the cochlear nerve. In addition, by blocking Ca2+ channels with nimodipine, the perception of tinnitus in an animal model was reduced, implicating Ca2+ channel activation (Liu et al., 2005, 2007). However, the functional neuroanatomy underlying these changes in cochlear function remains poorly understood. Furthermore, there have been no in vivo auditory tract tracing studies to investigate the involvement of both cochlea and central auditory system in tinnitus models. Manganese-enhanced magnetic resonance imaging (MEMRI) has been used to measure neural activity of a wide variety of neural systems in the animal brain (Koretsky and Silva, 2004; Tjälve et al., 1996). The paramagnetic manganese ions (Mn2+), used as T1-contrast agents, are well-known analogs of Ca2+, and can enter active neurons primarily via voltage-gated Ca2 + channels during neuronal depolarization (Narita et al., 1990; Watanabe et al., 2004). Several MEMRI studies have focused on the function of the auditory system (Lee et al., 2007, 2012; Watanabe et al., 2008; Yu et al., 2005, 2008); however, few studies have used MEMRI to investigate tinnitus (Brozoski et al., 2007; Holt et al., 2010). Furthermore, these few MEMRI studies on tinnitus systemically administered MnCl2 that unfortunately fails to examine the neural activity of the cochlea, a key peripheral auditory structure. Recently, Lee et al. (2012) demonstrated neural activity of the cochlea using intratympanic manganese administration. However, use of this approach, where MnCl2 is administered into the middle ear cavity through the intratympanic membrane, is limited. This is because the amount of MnCl2 reaching the cochlea is variable since it is taken up via the Eustachian tube and absorbed by the gastrointestinal tract. In the present study, we used MEMRI to investigate tinnitusassociated spontaneous neural activity, extending from peripheral (cochlea) to the central auditory system by applying Mn2+ to the round window. Specifically, we focused on tinnitus-associated alteration in bulk transport of Mn2 + between the cochlea and cochlear nucleus, which are likely the main auditory structures responsible for tinnitus in various models of tinnitus. We hypothesize that bulk transport of Mn2+ between the cochlea and cochlear nucleus increases in animals with tinnitus due to increased spontaneous neural activity. Previous MEMRI study using an animal with normal hearing demonstrated that the bulk of manganese ions in excited neurons by auditory stimulation transported through Ca2+ channels much more frequently than in neurons in control animals kept in a quiet chamber (Watanabe et al., 2008). Using a new round window Mn2+ administration, we also demonstrated that significant signal enhancement was possible without a large amount of MnCl2 that had been necessary for signal enhancement of auditory structures in previous studies. In addition, tinnitus was assessed with a gap prepulse inhibition of acoustic startle (GPIAS) method. This paradigm, which assesses the probability of whether the subject perceives a stimulus by introducing a pre-stimulus “gap” in a sound, is a recent advancement in a method for screening of tinnitus perception in animal models (Turner et al., 2006). Material and methods Subjects Ten Sprague-Dawley (SD) rats (6–7 weeks old; weighing 170–230 g) were used for this study. Before starting the study, we allowed the rats to acclimatize for 7 days. All rats were individually housed in cages maintained at 26 °C with a 12-h light/dark cycle. The rats were divided into two groups: 5 rats of group I were saline-injected and 5 rats of group II were salicylate-injected. Fig. 1 shows the paradigm of the study. On the first day, auditory brainstem response (ABR) and initial GPIAS were conducted on all rats of both groups. GPIAS test was then measured daily. Sodium salicylate or saline was injected for 4 consecutive days. On day 5, final ABR measurement was conducted and MEMRI experiments were performed. Rats with abnormal tympanic membrane or showing
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Fig. 1. Schematic diagram showing the methodology of the study. (a) Control group was treated with normal saline once a day for 4 consecutive days. The ABR test was performed on day 1 and day 5 to identify any change in hearing threshold. GPIAS was checked 3 h after saline administration. (b) 350 mg/kg/day sodium salicylate (NaSal) (i.p. dilution in normal saline, 50 mg/mL) was administered to the tinnitus group once a day for 4 consecutive days. ABR test and GPIAS were conducted at the same time schedules as the control group. On day 5, GPIAS were checked 3 h after salicylate or saline injection and ABR threshold measured 1 h after GPIAS test. Also MnCl2 were administrated through round window 2 h after ABR test and MR imaging was carried out. Therefore the entire experiments were conducted within 30 h after salicylate injection on day 5. ABR: auditory brain response, GPIAS: gap prepulse inhibition of acoustic startle.
no proper response wave below the 20-dB threshold in ABR test were excluded from MEMRI experiments. The protocol for this experiment was carried out in compliance with the animal experiments and methods approved by the Institutional Review Board of Kyungpook National University Hospital (approval number, KNU 2012-67). Tinnitus induction using sodium salicylate Sodium salicylate (NaSal), well known as an analgesic and antiinflammatory agent, induces short-term tinnitus in humans and in animals (Cazals, 2000; Myers and Bernstein, 1965). In this study, 350 mg/kg/day of sodium salicylate was injected into rats of the tinnitus group (i.p., dilution in normal saline, 50 mg/mL) for 4 consecutive days. Rats in the control group received normal saline in a similar manner. Gap prepulse inhibition of acoustic startle reflex Tinnitus perception may be assessed using the fact that the acoustic startle reflex can be modulated by a preceding stimulus, a gap embedded in a continuous background sound (Massimo et al., 2010). A silence as a prepulse in background sound to inhibit startle response is referred to as gap prepulse inhibition of acoustic startle reflex (Massimo et al., 2010). A high GPIAS score indicates that the gap prepulse inhibits the startle response (Park et al., 2013). Otherwise, with tinnitus present throughout the silent gap, animals are unable to recognize the gap as a prepulse and thus the startle response does not decrease. Thus, a significant reduction of GPIAS value suggests tinnitus induction. In our preliminary study, GPIAS had been monitored every hour for 10 h after salicylate injection (data not shown). The salicylate-induced depression of the value of GPIAS was most constantly observed in three hours after salicylate injection. For all animals, the first GPIAS was checked on day 1 and from the second day on the GPIAS were measured 3 h after salicylate or saline administration daily until on day 5. The GPIAS was measured as previously described (Park et al., 2013). Briefly, GPIAS was checked in a startle response chamber used in a previous study (Park et al., 2013). Each rat received an acoustic stimulus made up of 30 no-gap trials and 30 gap trials at random. Conditional random inter-stimulus interval (CRISI) was utilized to separate the trials. With this method, we measured the startle response of the animals only when they were stable. By measuring the movement of the subject
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with the aid of an accelerometer, the motion artifacts could be minimized in real time. At the beginning of each session, a 2-min acclimation period was given. Each gap trial was composed of a 60-dB sound pressure level (SPL) continuous narrowband background noise that was centered at 16 kHz that rats attributed less perceptible of gap pre pulse comparable with several frequencies (Yang et al., 2007); the startle stimulus was preceded by 50-ms gap that ended 100 ms before the onset of startle stimulus. During no gap trials, the background sound was continuous without a silent period preceding the startle stimulus. Auditory brainstem response A mixture of Zoletil (Virbac Laboratories, France) and Rumpun (Bayer, Korea) were used to anesthetize the animals intramuscularly. The animals were maintained on a heating pad set at 37 °C and their body temperature was monitored using a rectal thermometer. All experiments were conducted in a soundproof room. Tucker-Davis Technologies (TDT) system was used to measure sound evoked ABRs. Briefly, needle electrodes from a head stage (RA4L1, TDT, USA) connected to a preamplifier (RA4PA, TDT) to record ABRs were inserted into the vertex (+charge), mastoid (−charge) and hind leg as ground. Acoustic stimuli were calibrated at 90-dB SPL with 4-, 8-, 16-, and 32-kHz tone bursts using calibration software (SigCalRP) in TDT system 3 and the probe microphone system. Tone burst stimuli with a 1-ms rise/fall and a 5-ms plateau or transient click stimuli at frequencies of 8, 16, and 32 kHz, generated by signal design software (SigGenRP), were applied. The signals for stimuli were generated by SigGenRP and an RP2.1 real-time processor and then transmitted in sequence through a programmable attenuator (PA5, TDT), speaker driver (ED1, TDT), and electrostatic speaker (EC1, TDT). Stimuli were generated every 36.1 ms, in 5-dB decrements at every frequency, for 500 repetitions from 90-dB SPL to the acoustic threshold. To reduce the noise caused by repetitive stimuli, the phase of the stimuli was reversed for each stimulus. Manganese administration The rats were anesthetized with an intramuscular injection of 0.03 mL xylazine (Rumpun, 20 mg/mL) and 0.15 mL ketamine (Ketalar, 50 mg/mL). A gelatin sponge slice, made of gelfoam (Cutanplast Standard®, Mascia Brunelli) was designed 0.5–1.0 mm3 sized piece which was placed under a microscope for manganese administration. After exposure of the bulla, a 2-mm diameter-hole was made on the bulla shell to approach the round window (Fig. 2). Using a 40 × surgical microscope, the round window membrane was checked and MnCl2-soaked
(0.125 mM/kg) gelfoam was placed on the round window membrane of the left ear in the animals for 20 min. Manganese-enhanced magnetic resonance imaging T1-weighted two-dimensional (2D) spin-echo images were acquired using a 3.0-T MR imager (VHi; GE Healthcare, USA). Home-built small animal radiofrequency (RF) coils were receiver-only type and the inner diameter of the coils was 75 mm. The following imaging parameters were employed for 2D spin-echo images: field of view = 50 mm × 50 mm; matrix size = 256 × 256; axial slices = 12–16; slice thickness = 1.5 mm; slice gap = 0.1 mm; repetition time (TR) = 450 ms; echo time (TE) = 13 ms; number of acquisitions (NEX) = 10. After the animals were anesthetized with an intramuscular injection of 0.03 mL xylazine (Rumpun, 20 mg/mL) and 0.15 mL ketamine (Ketalar, 50 mg/mL), the animals were placed on the magnet in a prone position with the heads firmly fixed using a RF receiver coil. During MRI scans, the animals were maintained at approximately 37 °C using a warm water blanket. After each MRI measurement, the animals were placed back in their cages with free access to food and water. The animals were kept in their cages with a quiet environment. For each scan, the animals were re-anesthetized as described above. Manganese-enhanced MR image analysis For quantitative evaluation, the signal-to-noise ratio (SNR, defined as the mean MRI signal intensity of a brain region divided by the standard deviation of the noise) was determined using software supplied by Advantage Window software (GE Healthcare, USA). To correct for possible B1 inhomogeneities between animals, the SNRs were normalized with SNR of muscle. In close accordance with resolved anatomical structures and a rat brain atlas (Paxinos and Watson, 1998), circletype regions of interest (ROIs) were manually outlined by a neuroanatomist. The selected ROIs were in the cochlea (0.15 mm2), the cochlear nerve (0.15 mm2), the ventral and dorsal portions of the cochlear nucleus (0.15 mm2), the lateral lemniscus (0.15 mm2), the inferior colliculus (0.40 mm2), and the auditory cortex (0.50 mm2). The ROI locations are shown in Supplemental Information (Fig. S1). In addition, two ROIs were located at internal capsule and hippocampus as non-auditory reference ROIs. To compare the sequential contrast enhancement patterns in the two groups, MRI studies were performed at 3, 6, 12, and 24 h after Mn2 + administration. All T1-weighted images were normalized by muscle SNR before analysis. A factorial analysis of variance (ANOVA) revealed significant group differences during the four time points in each
Fig. 2. After surgical exposure of the bulla, a 2-mm diameter-hole was made on the bulla shell to expose the round window. The gelfoam soaked with MnCl2 (0.125 mM/kg) was placed on the round window membrane for 20 min. Magnification 40× and white scale bar: 2 mm.
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ROI (F(18,168) = 3.193, P b 0.01). The results of group differences of normalized SNR in each experiment were analyzed by t-tests. Differences in the normalized SNRs at each ROI along the auditory pathway were analyzed. P b 0.05 was accepted as the indication of statistical significance applied Bonferroni correction for multiple comparisons. Statistical analysis was performed with a commercial software package (Statistical Package for the Social Sciences 18.0 for Windows; SPSS, Chicago, IL).
Cochlear staining Inner ears were isolated from control and salicylate-treated groups at 6–7 weeks old. The ears were rapidly fixed by infusing with fixative solution (4% paraformaldehyde in phosphate-buffered saline) via the round window and immersed in the same fixative solution at 4 °C for 1 h. After fixation, the organ of Corti was dissected under a dissecting microscope (Stemi 2000-C, Carl Zeiss, Oberkochen, Germany). The dissected specimens were counter stained with Alexa Fluor 488 phalloidin to label F-actin (Molecular Probes/Invitrogen, Carlsbad, CA, USA), and were mounted with Fluoromount (Sigma-Aldrich, St. Louis, MO, USA). The specimens were visualized using a confocal laser scanning microscope (LSM700, Carl Zeiss, Oberkochen, Germany) and the ZEN 2009 program.
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Auditory brainstem response High-dose systemic salicylate can cause reversible hearing threshold shift (Crifo, 1975; Myers and Bernstein, 1965; Stebbins et al., 1973). However, some studies showed that there was no threshold shift in ABR test in salicylate treated animals (Jeremy et al., 2006; Massimo et al., 2010; Park et al., 2013). In this study, the ABR was measured in all rats prior to study on day 1 and day 5 to evaluate whether repeated salicylate injection induced temporary auditory threshold shift. On day 1, the ABR thresholds in two groups were under 25 dB SPL at all frequencies (4 kHz, 8 kHz, 16 kHz, and 32 kHz). On day 5, ABR thresholds showed slightly higher values than those on day 1 at the frequencies of 16 kHz, and 32 kHz in the salicylate-treated group. However, there were no significant statistical differences (control group: P = 0.593, 0.564, 0.655, and 0.577, the salicylate-treated group: P = 0.785, 0.063, 0.066, and 0.059) (Table 1).
Phalloidin staining of the organ of Corti Whole-mount analysis of the organ of Corti from rats in the salicylate-treated and control groups using phalloidin staining did not reveal any abnormal characteristics such as loss of hair cells, abnormal arrangement of hair cells, or degeneration of stereocilia in the former (Fig. 4).
Statistical analysis Statistical analysis was performed with a commercial software package (Statistical Package for the Social Sciences 13.0 for Windows; SPSS, Chicago, IL). The Mann–Whitney U test was used for comparison between 2 groups of GPIAS results in “Tinnitus development and GPIAS”. The Wilcoxon signed-rank test was used to compare changes for ABR thresholds between the 2 periods (day 1 and day 5) of control and salicylate-treated group.
Results Tinnitus development and GPIAS Our initial measurements of GPIAS values on the first day (baseline) were comparable between groups: 42.5 ± 4.3 in the control group and 55.3 ± 14.1 in the salicylate group (Fig. 3, Supplemental Information, Table S1; P = 0.222). As tinnitus occupied the gap space, rats treated with salicylate showed significantly lower GPIAS values than control rats from day 3 onwards (P b 0.01).
Fig. 3. Gap prepulse inhibition of acoustic startle (GPIAS). GPIAS values on the first day (baseline) are comparable between groups (n = 10): 42.5 ± 4.3 in the control group and 55.3 ± 14.1 in the salicylate group. Salicylate-exposed group shows significantly lower GPIAS values than control rats from day 3 onwards (P b 0.05). (*) significance P b 0.05. Error bars indicate one standard deviation.
Manganese-enhanced MRI via a round window approach Fig. 5a shows a clear delineation of the structures in the primary auditory pathway 3, 6, and 12 h after round window Mn2+ administration of a salicylate-treated rat. The auditory structures that were brightly labeled were the cochlea, cochlear nerve, and the ventral and dorsal cochlear nuclei. After round window administration, signal enhancements in the auditory structures were observed primarily in the ipsilateral (left) ascending auditory pathway. Fig. 5b shows signal enhancements in the auditory structures of a control rat after round window Mn2+ administration. Compared to salicylate-treated rats, signal enhancements in the cochlear nerve, ventral cochlear nucleus, and dorsal cochlear nucleus were significantly lower in the control (See also Supplemental Information, Table S2). Fig. 6 shows the pattern of sequential signal enhancement represented in normalized SNR after round window Mn2+ administration using ROIs located at the cochlea, cochlear nerve, the ventral and dorsal cochlear nucleus. Compared with the control group, the salicylateinduced tinnitus group showed significant signal enhancement after 3 h in the cochlea. Cochlear nerve, and the ventral and dorsal cochlear nucleus of the ascending auditory pathway were significant signal enhancement after 6 h. Nonetheless, the pattern of sequential signal enhancement represented in normalized SNR in the lateral lemniscus, the inferior colliculus, and the auditory cortex were not different between the groups (Supplemental Information, Fig. S2 and Table S2). As negative controls, the pattern of sequential signal enhancement represented in normalized SNR at the internal capsule and hippocampus showed no signal enhancement (Supplemental Information, Fig. S3). Furthermore, inside the cochlea of both control and salicylate-induced tinnitus groups, Mn2+ passed through the round window membrane a short time after administration and within 3 h, the Mn2+ was distributed from the perilymphatic space to the entire cochlea (Fig. 5). Only the salicylate-induced tinnitus group showed sequential signal enhancement pattern, with prolonged signal enhancement of the cochlea of up to 24 h. Although there was a lack of statistical significance, Fig. 6 showed that the peak signal enhancements proceeded sequentially from the cochlea to the cochlear nucleus, qualitatively revealing anterograde signal propagation along the ascending auditory pathway (see also Supplemental Information, Fig. S4 and Table S3).
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Table 1 Hearing thresholds measured by auditory brainstem response (ABR). ABR was recorded 1 day prior to the study and on day 5, before Mn2+ injections, for both groups. ABRs at all 4 frequencies, measured on all days, were not statistically different. Control
Click sound Tone burst 4 K Tone burst 8 K Tone burst 16 K Tone burst 32 K
Salicylate
P-value
Day 1 (A)
Day 5 (B)
Day 1 (C)
Day 5 (D)
A and B
C and D
12.0 14.0 16.0 18.0 21.0
14.0 18.0 17.0 19.0 23.0
11.0 15.0 14.0 18.0 21.0
17.0 15.0 24.0 30.0 31.0
0.480 0.593 0.564 0.655 0.577
0.131 0.785 0.063 0.066 0.059
± ± ± ± ±
2.7 4.2 4.2 4.5 4.2
± ± ± ± ±
4.2 9.7 4.5 2.2 5.7
Discussion In the current study, we developed an in vivo auditory tract tracing method to investigate a salicylate-induced tinnitus model by administering Mn2+ through the round window. To our knowledge, this study is the first to demonstrate functional auditory tract tracing from periphery (cochlea) to central auditory structures in a rat tinnitus model. The main technical advance achieved in the current study is the anterograde auditory tract tracing made possible by using a new round window focal administration method. The time-normalized SNR curve of Mn2 + enhancement in auditory structures clearly demonstrated that the neural signal measured with Mn2+ activity propagated from the cochlea to the cochlear nucleus through the cochlear nerve. Previous MEMRI studies using systemic injection showed tinnitus-related Mn2+ enhancement in auditory (dorsal cochlear nucleus) and non-auditory brain structures (Brozoski et al., 2007; Holt et al., 2010). However, previous MEMRI studies using systemic injection failed to show Mn2+ enhancement in the cochlea. Limited blood flow to the cochlea (Angelborg et al., 1988), together with the blood–labyrinth barrier, hampers the delivery of Mn2+ from blood to cochlear tissues (Juhn et al., 2001). Recently, Lee et al (2012) demonstrated in vivo auditory tract tracing in healthy rats using an intratympanic approach for Mn2+ administration. However, intratympanic administration, may lead to MnCl2 absorption by the gastrointestinal tract possibly via the Eustachian tube. To overcome these obstacles, we applied Mn2+ through the round window and discovered that there was significant signal enhancement without a large amount of MnCl2, which has previously been necessary for signal enhancement of auditory structures. In accordance with previous studies (Holt et al., 2010; Turner et al., 2006), the current study also confirmed the tinnitus generation in the salicylate-treated rats using the GPIAS measurement. Mn2 +
± ± ± ± ±
5.5 7.1 5.5 4.5 6.5
± ± ± ± ±
2.7 8.7 6.5 9.4 7.4
enhancement of cochlear labeling in the salicylate group lasted longer than in the control group. This prolonged Mn2+ enhancement in the cochlea of salicylate-treated rats may reflect the increased excitatory effects of glutamate in the cochlea. Sodium salicylate is known to develop the excitatory effects of glutamate at cochlear N-methyl-Daspartate (NMDA) receptors and activation of cochlear NMDA receptors has been proposed to play a key role in salicylate-induced tinnitus (Guitton et al., 2003). More specifically, salicylate may act on cochlear fast synaptic transmission via the activation of NMDA receptors, accounting for the occurrence of tinnitus (Guitton et al., 2003). That is, fast synaptic transmission via the activation of NMDA receptors increases voltage-gated Ca2+ channel activity and thus increases Mn2+ accumulation in neurons through overstimulated Ca2+ channels due to salicylate-induced tinnitus. Mn2+ ion is known to be accumulated primarily in the endoplasmic reticulum (ER) (Narita et al., 1990; Pautler et al., 1998). In the ER, Mn2+ ion is packaged for bulk transport and transported along microtubules to the synaptic cleft where it is released and taken up by the next neuron in the neural pathway (Pautler and Koretsky, 2001). Therefore, it might be possible that the increase of Mn2 + accumulation in neurons in animal with salicylate-induced tinnitus makes more frequent bulk transport than in neurons in control animal. This interpretation is in line with previous evidence that in excited neurons by auditory stimulation, the bulk of manganese ions transported through Ca2+ channels much more frequently than in neurons in control mice kept in a quiet chamber (Watanabe et al., 2008). However, further study is warranted to investigate in detail the mechanism for alteration in bulk transport of manganese ions in tinnitus. In addition, our results demonstrated significant increases in Mn2+ enhancement of the cochlear nerve, and the ventral and dorsal portions of the cochlear nucleus in the salicylate group over 12 h. These enhancement patterns over time without external sound stimulation clearly
Fig. 4. Photomicrograph showing phalloidin staining of the organ of Corti whole-mount from a control and a salicylate-treated rat. White scale bars: 20 μm.
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Fig. 5. MEMRI shows signal enhancement represented in normalized SNR in auditory structures 3, 6, and 12 h after round window Mn2+ administration in a salicylate-treated rat and in a control rat. The enhanced auditory structures were the cochlea, cochlear nerve, ventral and dorsal cochlear nucleus. Signal enhancements in the auditory structures were observed primarily in the ipsilateral (left) ascending auditory pathway after round window administration.
showed that increased spontaneous neural signal propagation along the auditory tract occurs in the salicylate-induced tinnitus group. One possible mechanism for this increase in Mn2+ activity is that salicylate influences the pre- and postsynaptic conductance of several transmembrane ion channels by modulating neurotransmitter activity (Lu et al., 2011). Because this mechanism depends on voltage-gated Ca2+ channel activity, increased excitatory synaptic transmission may contribute to
an increased Mn2+ accumulation in auditory structures. Furthermore, while a previous salicylate-induced tinnitus MEMRI study (Holt et al., 2010) using systemic Mn2 + injection showed signal enhancement only at the dorsal portion of the cochlear nucleus, our results using focal round window administration demonstrated the qualitative signal enhancement from the cochlear nerve to the ventral and to the dorsal portions of the cochlear nucleus. Therefore, our in vivo MEMRI data
Fig. 6. The pattern of sequential signal enhancement represented in normalized SNR after round window Mn2+ administration using ROIs located at the cochlea, cochlear nerve, ventral and dorsal cochlear nucleus of the left (ipsilateral) auditory pathway in both salicylate-treated group and in a control group. (*) significance P b 0.05, (**) significance P b 0.01.
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seem to support the hypothesis that increased spontaneous activity of neurons in the central auditory system underlies tinnitus. Salicylate has been reported to increase the spontaneous activity of the auditory nerve, the inferior colliculus, and the auditory cortex (Evans and Borerwe, 1982; Manabe et al., 1997; Ochi and Eggermont, 1996; Stypulkowski, 1990). However, cell recordings of non-auditory structures showed no change in spontaneous activity (Jastreboff and Sasaki, 1986) suggesting the selectivity of salicylate on auditory pathways. In the present study, we demonstrated significant Mn2+ enhancement over the cochlear nucleus along the central auditory tract with a Mn2 + dose of only 0.125 mM/kg. However, Mn2 + enhancements in the lateral lemniscus, the inferior colliculus, and the auditory cortex were not statistically different between the salicylate group and the control group, likely because of the low dose we employed. There were two reasons for choosing a small amount of Mn2+ in the present study. First, high doses of Mn2+ are associated with possible Mn2+ neurotoxicity (Silva et al., 2004). Because the current study is the first in vivo imaging attempt to investigate cochlear activity in a salicylate-induced tinnitus model, it was necessary to use as little Mn2+ as possible to minimize any possible neurotoxic effect of Mn2+ on the cochlea. Second, due to its paramagnetic properties, a high dose of Mn2+ at the round window can possibly produce an image artifact in the cochlea and the cochlear nucleus. However, to evaluate neural activity in higher central auditory structures and possibly in non-auditory brain structures, further investigations are warranted to determine the optimal Mn2 + dose to avoid toxicity and image artifacts. Finally, there are some limitations in the present study. First, the MnCl2 uptake into the cochlea was slightly different across animals after round window administration due to the individual size difference of round windows. Second, although an expert determined the ROIs based on resolved anatomical structures and a rat brain atlas, it remained subject to operator bias across animals. In summary, we developed a novel MEMRI method with Mn2+ administration through the round window to map the in vivo functional auditory tract in a salicylate-induced tinnitus animal model. The present study demonstrated that salicylate-induced tinnitus showed higher Mn2+ uptake in the cochlea and in the central auditory pathway. The serial MRI scans also demonstrated that the Mn2+ signal traveled from the cochlea to the cochlear nucleus. Therefore, our MEMRI data suggest that salicylate-induced tinnitus is associated with greater spontaneous neural activity in both peripheral and central auditory pathways. Acknowledgment This work was supported by the Ministry of Health & Welfare, Republic of Korea (A092106). This work was also supported by Basic Science Research Program through the National Research Foundation (NRF) of Korea (2012-005117 to H.J. Lee) and the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A111774 to K.Y. Lee). Conflict of interest statement The authors declare that there are no conflicts of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.neuroimage.2014.06.055. References Angelborg, C., Hillerdal, M., Hultcrantz, E., Larsen, H.C., 1988. The microsphere method for studies of inner ear blood blow. ORL J. Otorhinolaryngol Relat. Spec. 50, 355–362. Brozoski, T.J., Ciobanu, L., Bauer, C.A., 2007. Central neural activity in rats with tinnitus evaluated with manganese-enhanced magnetic resonance imaging (MEMRI). Hear. Res. 228, 168–179.
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NeuroImage journal homepage: www.elsevier.com/locate/ynimg
Functional mapping of the auditory tract in rodent tinnitus model using manganese-enhanced magnetic resonance imaging Da Jung Jung a,1, Mun Han b,1, Seong-Uk Jin b, Sang Heun Lee c, Ilyong Park d, Hyun-Ju Cho e, Tae-Jun Kwon e, Hui Joong Lee f, Jin Ho Cho g, Kyu-Yup Lee a,⁎, Yongmin Chang b,f,h,⁎⁎ a
Department of Otorhinolaryngology—Head and Neck Surgery, School of Medicine, Kyungpook National University Hospital, Daegu, Republic of Korea Department of Medical and Biological Engineering, School of Medicine, Kyungpook National University Hospital, Daegu, Republic of Korea Department of Otorhinolaryngology—Head and Neck Surgery, Daegu Veterans Hospital, Daegu, Republic of Korea d Department of Biomedical Engineering, College of Medicine, Dankook University, Cheonan, Republic of Korea e Department of Biology, College of Natural Science, Kyungpook National University, Daegu, Republic of Korea f Department of Radiology, School of Medicine, Kyungpook National University Hospital, Daegu, Republic of Korea g Department of Electronic Engineering, College of IT Engineering, Kyungpook National University, Daegu, Republic of Korea h Department of Molecular Medicine, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu, Republic of Korea b c
a r t i c l e
i n f o
Article history: Accepted 23 June 2014 Available online 28 June 2014 Keywords: Salicylate Tinnitus Manganese-enhanced MRI Auditory pathway Gap prepulse inhibition of acoustic startle
a b s t r a c t Animal models of salicylate-induced tinnitus have demonstrated that salicylate modulates neuronal activity in several brain structures leading to neuronal hyperactivity in auditory and non-auditory brain areas. In addition, these animal tinnitus models indicate that tinnitus can be a perceptual consequence of altered spontaneous neural activity along the auditory pathway. Peripheral and/or central effects of salicylate can account for neuronal activity changes in salicylate-induced tinnitus. Because of this ambiguity, an in vivo imaging study would be able to address the peripheral and/or central involvement of salicylate-induced tinnitus. Therefore, in the present study, we developed a novel manganese-enhanced magnetic resonance imaging (MEMRI) method to map the in vivo functional auditory tract in a salicylate-induced tinnitus animal model by administrating manganese through the round window. We found that acute salicylate-induced tinnitus resulted in higher manganese uptake in the cochlea and in the central auditory structures. Furthermore, serial MRI scans demonstrated that the manganese signal increased in an anterograde fashion from the cochlea to the cochlear nucleus. Therefore, our in vivo MEMRI data suggest that acute salicylate-induced tinnitus is associated with higher spontaneous neural activity both in peripheral and central auditory pathways. © 2014 Elsevier Inc. All rights reserved.
Introduction Approximately 10–15% of the adult population is affected by tinnitus, the perception of a sound without an external acoustic stimulus (Henry et al., 2005; Moller, 2011). Furthermore, subjective tinnitus severely affects the quality of life of 1–3% of the population (Eggermont and Roberts, 2004). Advances in functional neuroimaging methods and development of animal models have increasingly demonstrated that an increased firing rate of neurons, enhanced neuronal synchrony, and changes in the tonotopic organization in the central auditory pathway represent the neural substrate of tinnitus (Lanting et al., 2009; Salvi ⁎ Correspondence to: K.Y. Lee, Department of Otorhinolaryngology—Head and Neck Surgery, Kyungpook National University, School of Medicine, 130 Dongdeok-ro, Jung-gu, Daegu 700-721, Republic of Korea. Fax: +82 53 423 4524. ⁎⁎ Correspondence to: Y. Chang, Department of Molecular Medicine, Kyungpook National University School of Medicine, 130 Dongdeok-ro, Jung-gu, Daegu 700-721, South Korea. Fax: +82 53 422 2677. E-mail addresses: [email protected] (K.-Y. Lee), [email protected] (Y. Chang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.neuroimage.2014.06.055 1053-8119/© 2014 Elsevier Inc. All rights reserved.
et al., 2000; Seki and Eggermont, 2003). Moreover, changes in the peripheral auditory system have also been reported (Sahley and Nodar, 2001). Therefore, a remaining challenge is to understand how peripheral neural activity communicates with the central auditory system in tinnitus. That is, while the alterations of neural activity in central or peripheral auditory system were well demonstrated, it has seldom been studied whether there is alteration in neural communication between peripheral and central auditory system in tinnitus. In vivo manganese auditory tract tracing would provide a way to map the neural communication between peripheral and central auditory system because this imaging method is capable of tracing the neural activity through bulk transport of manganese ion (Mn2+) along an axonal tract. Evidence from animal models of tinnitus suggests that tinnitus can be a perceptual consequence of altered spontaneous neural activity along the auditory pathway. Electrophysiological studies measuring the average spectrum activity from the cochlear round window in animal models of salicylate-induced tinnitus revealed increased spontaneous neural activity of single units of the cochlear nerve (Cazals et al., 1998; Evans and Borerwe, 1982; Martin et al., 1993; Stypulkowski,
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1990). These studies therefore suggest that, at least in part, salicylateinduced tinnitus is associated with dysfunction of the cochlear nerve. In addition, by blocking Ca2+ channels with nimodipine, the perception of tinnitus in an animal model was reduced, implicating Ca2+ channel activation (Liu et al., 2005, 2007). However, the functional neuroanatomy underlying these changes in cochlear function remains poorly understood. Furthermore, there have been no in vivo auditory tract tracing studies to investigate the involvement of both cochlea and central auditory system in tinnitus models. Manganese-enhanced magnetic resonance imaging (MEMRI) has been used to measure neural activity of a wide variety of neural systems in the animal brain (Koretsky and Silva, 2004; Tjälve et al., 1996). The paramagnetic manganese ions (Mn2+), used as T1-contrast agents, are well-known analogs of Ca2+, and can enter active neurons primarily via voltage-gated Ca2 + channels during neuronal depolarization (Narita et al., 1990; Watanabe et al., 2004). Several MEMRI studies have focused on the function of the auditory system (Lee et al., 2007, 2012; Watanabe et al., 2008; Yu et al., 2005, 2008); however, few studies have used MEMRI to investigate tinnitus (Brozoski et al., 2007; Holt et al., 2010). Furthermore, these few MEMRI studies on tinnitus systemically administered MnCl2 that unfortunately fails to examine the neural activity of the cochlea, a key peripheral auditory structure. Recently, Lee et al. (2012) demonstrated neural activity of the cochlea using intratympanic manganese administration. However, use of this approach, where MnCl2 is administered into the middle ear cavity through the intratympanic membrane, is limited. This is because the amount of MnCl2 reaching the cochlea is variable since it is taken up via the Eustachian tube and absorbed by the gastrointestinal tract. In the present study, we used MEMRI to investigate tinnitusassociated spontaneous neural activity, extending from peripheral (cochlea) to the central auditory system by applying Mn2+ to the round window. Specifically, we focused on tinnitus-associated alteration in bulk transport of Mn2 + between the cochlea and cochlear nucleus, which are likely the main auditory structures responsible for tinnitus in various models of tinnitus. We hypothesize that bulk transport of Mn2+ between the cochlea and cochlear nucleus increases in animals with tinnitus due to increased spontaneous neural activity. Previous MEMRI study using an animal with normal hearing demonstrated that the bulk of manganese ions in excited neurons by auditory stimulation transported through Ca2+ channels much more frequently than in neurons in control animals kept in a quiet chamber (Watanabe et al., 2008). Using a new round window Mn2+ administration, we also demonstrated that significant signal enhancement was possible without a large amount of MnCl2 that had been necessary for signal enhancement of auditory structures in previous studies. In addition, tinnitus was assessed with a gap prepulse inhibition of acoustic startle (GPIAS) method. This paradigm, which assesses the probability of whether the subject perceives a stimulus by introducing a pre-stimulus “gap” in a sound, is a recent advancement in a method for screening of tinnitus perception in animal models (Turner et al., 2006). Material and methods Subjects Ten Sprague-Dawley (SD) rats (6–7 weeks old; weighing 170–230 g) were used for this study. Before starting the study, we allowed the rats to acclimatize for 7 days. All rats were individually housed in cages maintained at 26 °C with a 12-h light/dark cycle. The rats were divided into two groups: 5 rats of group I were saline-injected and 5 rats of group II were salicylate-injected. Fig. 1 shows the paradigm of the study. On the first day, auditory brainstem response (ABR) and initial GPIAS were conducted on all rats of both groups. GPIAS test was then measured daily. Sodium salicylate or saline was injected for 4 consecutive days. On day 5, final ABR measurement was conducted and MEMRI experiments were performed. Rats with abnormal tympanic membrane or showing
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Fig. 1. Schematic diagram showing the methodology of the study. (a) Control group was treated with normal saline once a day for 4 consecutive days. The ABR test was performed on day 1 and day 5 to identify any change in hearing threshold. GPIAS was checked 3 h after saline administration. (b) 350 mg/kg/day sodium salicylate (NaSal) (i.p. dilution in normal saline, 50 mg/mL) was administered to the tinnitus group once a day for 4 consecutive days. ABR test and GPIAS were conducted at the same time schedules as the control group. On day 5, GPIAS were checked 3 h after salicylate or saline injection and ABR threshold measured 1 h after GPIAS test. Also MnCl2 were administrated through round window 2 h after ABR test and MR imaging was carried out. Therefore the entire experiments were conducted within 30 h after salicylate injection on day 5. ABR: auditory brain response, GPIAS: gap prepulse inhibition of acoustic startle.
no proper response wave below the 20-dB threshold in ABR test were excluded from MEMRI experiments. The protocol for this experiment was carried out in compliance with the animal experiments and methods approved by the Institutional Review Board of Kyungpook National University Hospital (approval number, KNU 2012-67). Tinnitus induction using sodium salicylate Sodium salicylate (NaSal), well known as an analgesic and antiinflammatory agent, induces short-term tinnitus in humans and in animals (Cazals, 2000; Myers and Bernstein, 1965). In this study, 350 mg/kg/day of sodium salicylate was injected into rats of the tinnitus group (i.p., dilution in normal saline, 50 mg/mL) for 4 consecutive days. Rats in the control group received normal saline in a similar manner. Gap prepulse inhibition of acoustic startle reflex Tinnitus perception may be assessed using the fact that the acoustic startle reflex can be modulated by a preceding stimulus, a gap embedded in a continuous background sound (Massimo et al., 2010). A silence as a prepulse in background sound to inhibit startle response is referred to as gap prepulse inhibition of acoustic startle reflex (Massimo et al., 2010). A high GPIAS score indicates that the gap prepulse inhibits the startle response (Park et al., 2013). Otherwise, with tinnitus present throughout the silent gap, animals are unable to recognize the gap as a prepulse and thus the startle response does not decrease. Thus, a significant reduction of GPIAS value suggests tinnitus induction. In our preliminary study, GPIAS had been monitored every hour for 10 h after salicylate injection (data not shown). The salicylate-induced depression of the value of GPIAS was most constantly observed in three hours after salicylate injection. For all animals, the first GPIAS was checked on day 1 and from the second day on the GPIAS were measured 3 h after salicylate or saline administration daily until on day 5. The GPIAS was measured as previously described (Park et al., 2013). Briefly, GPIAS was checked in a startle response chamber used in a previous study (Park et al., 2013). Each rat received an acoustic stimulus made up of 30 no-gap trials and 30 gap trials at random. Conditional random inter-stimulus interval (CRISI) was utilized to separate the trials. With this method, we measured the startle response of the animals only when they were stable. By measuring the movement of the subject
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with the aid of an accelerometer, the motion artifacts could be minimized in real time. At the beginning of each session, a 2-min acclimation period was given. Each gap trial was composed of a 60-dB sound pressure level (SPL) continuous narrowband background noise that was centered at 16 kHz that rats attributed less perceptible of gap pre pulse comparable with several frequencies (Yang et al., 2007); the startle stimulus was preceded by 50-ms gap that ended 100 ms before the onset of startle stimulus. During no gap trials, the background sound was continuous without a silent period preceding the startle stimulus. Auditory brainstem response A mixture of Zoletil (Virbac Laboratories, France) and Rumpun (Bayer, Korea) were used to anesthetize the animals intramuscularly. The animals were maintained on a heating pad set at 37 °C and their body temperature was monitored using a rectal thermometer. All experiments were conducted in a soundproof room. Tucker-Davis Technologies (TDT) system was used to measure sound evoked ABRs. Briefly, needle electrodes from a head stage (RA4L1, TDT, USA) connected to a preamplifier (RA4PA, TDT) to record ABRs were inserted into the vertex (+charge), mastoid (−charge) and hind leg as ground. Acoustic stimuli were calibrated at 90-dB SPL with 4-, 8-, 16-, and 32-kHz tone bursts using calibration software (SigCalRP) in TDT system 3 and the probe microphone system. Tone burst stimuli with a 1-ms rise/fall and a 5-ms plateau or transient click stimuli at frequencies of 8, 16, and 32 kHz, generated by signal design software (SigGenRP), were applied. The signals for stimuli were generated by SigGenRP and an RP2.1 real-time processor and then transmitted in sequence through a programmable attenuator (PA5, TDT), speaker driver (ED1, TDT), and electrostatic speaker (EC1, TDT). Stimuli were generated every 36.1 ms, in 5-dB decrements at every frequency, for 500 repetitions from 90-dB SPL to the acoustic threshold. To reduce the noise caused by repetitive stimuli, the phase of the stimuli was reversed for each stimulus. Manganese administration The rats were anesthetized with an intramuscular injection of 0.03 mL xylazine (Rumpun, 20 mg/mL) and 0.15 mL ketamine (Ketalar, 50 mg/mL). A gelatin sponge slice, made of gelfoam (Cutanplast Standard®, Mascia Brunelli) was designed 0.5–1.0 mm3 sized piece which was placed under a microscope for manganese administration. After exposure of the bulla, a 2-mm diameter-hole was made on the bulla shell to approach the round window (Fig. 2). Using a 40 × surgical microscope, the round window membrane was checked and MnCl2-soaked
(0.125 mM/kg) gelfoam was placed on the round window membrane of the left ear in the animals for 20 min. Manganese-enhanced magnetic resonance imaging T1-weighted two-dimensional (2D) spin-echo images were acquired using a 3.0-T MR imager (VHi; GE Healthcare, USA). Home-built small animal radiofrequency (RF) coils were receiver-only type and the inner diameter of the coils was 75 mm. The following imaging parameters were employed for 2D spin-echo images: field of view = 50 mm × 50 mm; matrix size = 256 × 256; axial slices = 12–16; slice thickness = 1.5 mm; slice gap = 0.1 mm; repetition time (TR) = 450 ms; echo time (TE) = 13 ms; number of acquisitions (NEX) = 10. After the animals were anesthetized with an intramuscular injection of 0.03 mL xylazine (Rumpun, 20 mg/mL) and 0.15 mL ketamine (Ketalar, 50 mg/mL), the animals were placed on the magnet in a prone position with the heads firmly fixed using a RF receiver coil. During MRI scans, the animals were maintained at approximately 37 °C using a warm water blanket. After each MRI measurement, the animals were placed back in their cages with free access to food and water. The animals were kept in their cages with a quiet environment. For each scan, the animals were re-anesthetized as described above. Manganese-enhanced MR image analysis For quantitative evaluation, the signal-to-noise ratio (SNR, defined as the mean MRI signal intensity of a brain region divided by the standard deviation of the noise) was determined using software supplied by Advantage Window software (GE Healthcare, USA). To correct for possible B1 inhomogeneities between animals, the SNRs were normalized with SNR of muscle. In close accordance with resolved anatomical structures and a rat brain atlas (Paxinos and Watson, 1998), circletype regions of interest (ROIs) were manually outlined by a neuroanatomist. The selected ROIs were in the cochlea (0.15 mm2), the cochlear nerve (0.15 mm2), the ventral and dorsal portions of the cochlear nucleus (0.15 mm2), the lateral lemniscus (0.15 mm2), the inferior colliculus (0.40 mm2), and the auditory cortex (0.50 mm2). The ROI locations are shown in Supplemental Information (Fig. S1). In addition, two ROIs were located at internal capsule and hippocampus as non-auditory reference ROIs. To compare the sequential contrast enhancement patterns in the two groups, MRI studies were performed at 3, 6, 12, and 24 h after Mn2 + administration. All T1-weighted images were normalized by muscle SNR before analysis. A factorial analysis of variance (ANOVA) revealed significant group differences during the four time points in each
Fig. 2. After surgical exposure of the bulla, a 2-mm diameter-hole was made on the bulla shell to expose the round window. The gelfoam soaked with MnCl2 (0.125 mM/kg) was placed on the round window membrane for 20 min. Magnification 40× and white scale bar: 2 mm.
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ROI (F(18,168) = 3.193, P b 0.01). The results of group differences of normalized SNR in each experiment were analyzed by t-tests. Differences in the normalized SNRs at each ROI along the auditory pathway were analyzed. P b 0.05 was accepted as the indication of statistical significance applied Bonferroni correction for multiple comparisons. Statistical analysis was performed with a commercial software package (Statistical Package for the Social Sciences 18.0 for Windows; SPSS, Chicago, IL).
Cochlear staining Inner ears were isolated from control and salicylate-treated groups at 6–7 weeks old. The ears were rapidly fixed by infusing with fixative solution (4% paraformaldehyde in phosphate-buffered saline) via the round window and immersed in the same fixative solution at 4 °C for 1 h. After fixation, the organ of Corti was dissected under a dissecting microscope (Stemi 2000-C, Carl Zeiss, Oberkochen, Germany). The dissected specimens were counter stained with Alexa Fluor 488 phalloidin to label F-actin (Molecular Probes/Invitrogen, Carlsbad, CA, USA), and were mounted with Fluoromount (Sigma-Aldrich, St. Louis, MO, USA). The specimens were visualized using a confocal laser scanning microscope (LSM700, Carl Zeiss, Oberkochen, Germany) and the ZEN 2009 program.
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Auditory brainstem response High-dose systemic salicylate can cause reversible hearing threshold shift (Crifo, 1975; Myers and Bernstein, 1965; Stebbins et al., 1973). However, some studies showed that there was no threshold shift in ABR test in salicylate treated animals (Jeremy et al., 2006; Massimo et al., 2010; Park et al., 2013). In this study, the ABR was measured in all rats prior to study on day 1 and day 5 to evaluate whether repeated salicylate injection induced temporary auditory threshold shift. On day 1, the ABR thresholds in two groups were under 25 dB SPL at all frequencies (4 kHz, 8 kHz, 16 kHz, and 32 kHz). On day 5, ABR thresholds showed slightly higher values than those on day 1 at the frequencies of 16 kHz, and 32 kHz in the salicylate-treated group. However, there were no significant statistical differences (control group: P = 0.593, 0.564, 0.655, and 0.577, the salicylate-treated group: P = 0.785, 0.063, 0.066, and 0.059) (Table 1).
Phalloidin staining of the organ of Corti Whole-mount analysis of the organ of Corti from rats in the salicylate-treated and control groups using phalloidin staining did not reveal any abnormal characteristics such as loss of hair cells, abnormal arrangement of hair cells, or degeneration of stereocilia in the former (Fig. 4).
Statistical analysis Statistical analysis was performed with a commercial software package (Statistical Package for the Social Sciences 13.0 for Windows; SPSS, Chicago, IL). The Mann–Whitney U test was used for comparison between 2 groups of GPIAS results in “Tinnitus development and GPIAS”. The Wilcoxon signed-rank test was used to compare changes for ABR thresholds between the 2 periods (day 1 and day 5) of control and salicylate-treated group.
Results Tinnitus development and GPIAS Our initial measurements of GPIAS values on the first day (baseline) were comparable between groups: 42.5 ± 4.3 in the control group and 55.3 ± 14.1 in the salicylate group (Fig. 3, Supplemental Information, Table S1; P = 0.222). As tinnitus occupied the gap space, rats treated with salicylate showed significantly lower GPIAS values than control rats from day 3 onwards (P b 0.01).
Fig. 3. Gap prepulse inhibition of acoustic startle (GPIAS). GPIAS values on the first day (baseline) are comparable between groups (n = 10): 42.5 ± 4.3 in the control group and 55.3 ± 14.1 in the salicylate group. Salicylate-exposed group shows significantly lower GPIAS values than control rats from day 3 onwards (P b 0.05). (*) significance P b 0.05. Error bars indicate one standard deviation.
Manganese-enhanced MRI via a round window approach Fig. 5a shows a clear delineation of the structures in the primary auditory pathway 3, 6, and 12 h after round window Mn2+ administration of a salicylate-treated rat. The auditory structures that were brightly labeled were the cochlea, cochlear nerve, and the ventral and dorsal cochlear nuclei. After round window administration, signal enhancements in the auditory structures were observed primarily in the ipsilateral (left) ascending auditory pathway. Fig. 5b shows signal enhancements in the auditory structures of a control rat after round window Mn2+ administration. Compared to salicylate-treated rats, signal enhancements in the cochlear nerve, ventral cochlear nucleus, and dorsal cochlear nucleus were significantly lower in the control (See also Supplemental Information, Table S2). Fig. 6 shows the pattern of sequential signal enhancement represented in normalized SNR after round window Mn2+ administration using ROIs located at the cochlea, cochlear nerve, the ventral and dorsal cochlear nucleus. Compared with the control group, the salicylateinduced tinnitus group showed significant signal enhancement after 3 h in the cochlea. Cochlear nerve, and the ventral and dorsal cochlear nucleus of the ascending auditory pathway were significant signal enhancement after 6 h. Nonetheless, the pattern of sequential signal enhancement represented in normalized SNR in the lateral lemniscus, the inferior colliculus, and the auditory cortex were not different between the groups (Supplemental Information, Fig. S2 and Table S2). As negative controls, the pattern of sequential signal enhancement represented in normalized SNR at the internal capsule and hippocampus showed no signal enhancement (Supplemental Information, Fig. S3). Furthermore, inside the cochlea of both control and salicylate-induced tinnitus groups, Mn2+ passed through the round window membrane a short time after administration and within 3 h, the Mn2+ was distributed from the perilymphatic space to the entire cochlea (Fig. 5). Only the salicylate-induced tinnitus group showed sequential signal enhancement pattern, with prolonged signal enhancement of the cochlea of up to 24 h. Although there was a lack of statistical significance, Fig. 6 showed that the peak signal enhancements proceeded sequentially from the cochlea to the cochlear nucleus, qualitatively revealing anterograde signal propagation along the ascending auditory pathway (see also Supplemental Information, Fig. S4 and Table S3).
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Table 1 Hearing thresholds measured by auditory brainstem response (ABR). ABR was recorded 1 day prior to the study and on day 5, before Mn2+ injections, for both groups. ABRs at all 4 frequencies, measured on all days, were not statistically different. Control
Click sound Tone burst 4 K Tone burst 8 K Tone burst 16 K Tone burst 32 K
Salicylate
P-value
Day 1 (A)
Day 5 (B)
Day 1 (C)
Day 5 (D)
A and B
C and D
12.0 14.0 16.0 18.0 21.0
14.0 18.0 17.0 19.0 23.0
11.0 15.0 14.0 18.0 21.0
17.0 15.0 24.0 30.0 31.0
0.480 0.593 0.564 0.655 0.577
0.131 0.785 0.063 0.066 0.059
± ± ± ± ±
2.7 4.2 4.2 4.5 4.2
± ± ± ± ±
4.2 9.7 4.5 2.2 5.7
Discussion In the current study, we developed an in vivo auditory tract tracing method to investigate a salicylate-induced tinnitus model by administering Mn2+ through the round window. To our knowledge, this study is the first to demonstrate functional auditory tract tracing from periphery (cochlea) to central auditory structures in a rat tinnitus model. The main technical advance achieved in the current study is the anterograde auditory tract tracing made possible by using a new round window focal administration method. The time-normalized SNR curve of Mn2 + enhancement in auditory structures clearly demonstrated that the neural signal measured with Mn2+ activity propagated from the cochlea to the cochlear nucleus through the cochlear nerve. Previous MEMRI studies using systemic injection showed tinnitus-related Mn2+ enhancement in auditory (dorsal cochlear nucleus) and non-auditory brain structures (Brozoski et al., 2007; Holt et al., 2010). However, previous MEMRI studies using systemic injection failed to show Mn2+ enhancement in the cochlea. Limited blood flow to the cochlea (Angelborg et al., 1988), together with the blood–labyrinth barrier, hampers the delivery of Mn2+ from blood to cochlear tissues (Juhn et al., 2001). Recently, Lee et al (2012) demonstrated in vivo auditory tract tracing in healthy rats using an intratympanic approach for Mn2+ administration. However, intratympanic administration, may lead to MnCl2 absorption by the gastrointestinal tract possibly via the Eustachian tube. To overcome these obstacles, we applied Mn2+ through the round window and discovered that there was significant signal enhancement without a large amount of MnCl2, which has previously been necessary for signal enhancement of auditory structures. In accordance with previous studies (Holt et al., 2010; Turner et al., 2006), the current study also confirmed the tinnitus generation in the salicylate-treated rats using the GPIAS measurement. Mn2 +
± ± ± ± ±
5.5 7.1 5.5 4.5 6.5
± ± ± ± ±
2.7 8.7 6.5 9.4 7.4
enhancement of cochlear labeling in the salicylate group lasted longer than in the control group. This prolonged Mn2+ enhancement in the cochlea of salicylate-treated rats may reflect the increased excitatory effects of glutamate in the cochlea. Sodium salicylate is known to develop the excitatory effects of glutamate at cochlear N-methyl-Daspartate (NMDA) receptors and activation of cochlear NMDA receptors has been proposed to play a key role in salicylate-induced tinnitus (Guitton et al., 2003). More specifically, salicylate may act on cochlear fast synaptic transmission via the activation of NMDA receptors, accounting for the occurrence of tinnitus (Guitton et al., 2003). That is, fast synaptic transmission via the activation of NMDA receptors increases voltage-gated Ca2+ channel activity and thus increases Mn2+ accumulation in neurons through overstimulated Ca2+ channels due to salicylate-induced tinnitus. Mn2+ ion is known to be accumulated primarily in the endoplasmic reticulum (ER) (Narita et al., 1990; Pautler et al., 1998). In the ER, Mn2+ ion is packaged for bulk transport and transported along microtubules to the synaptic cleft where it is released and taken up by the next neuron in the neural pathway (Pautler and Koretsky, 2001). Therefore, it might be possible that the increase of Mn2 + accumulation in neurons in animal with salicylate-induced tinnitus makes more frequent bulk transport than in neurons in control animal. This interpretation is in line with previous evidence that in excited neurons by auditory stimulation, the bulk of manganese ions transported through Ca2+ channels much more frequently than in neurons in control mice kept in a quiet chamber (Watanabe et al., 2008). However, further study is warranted to investigate in detail the mechanism for alteration in bulk transport of manganese ions in tinnitus. In addition, our results demonstrated significant increases in Mn2+ enhancement of the cochlear nerve, and the ventral and dorsal portions of the cochlear nucleus in the salicylate group over 12 h. These enhancement patterns over time without external sound stimulation clearly
Fig. 4. Photomicrograph showing phalloidin staining of the organ of Corti whole-mount from a control and a salicylate-treated rat. White scale bars: 20 μm.
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Fig. 5. MEMRI shows signal enhancement represented in normalized SNR in auditory structures 3, 6, and 12 h after round window Mn2+ administration in a salicylate-treated rat and in a control rat. The enhanced auditory structures were the cochlea, cochlear nerve, ventral and dorsal cochlear nucleus. Signal enhancements in the auditory structures were observed primarily in the ipsilateral (left) ascending auditory pathway after round window administration.
showed that increased spontaneous neural signal propagation along the auditory tract occurs in the salicylate-induced tinnitus group. One possible mechanism for this increase in Mn2+ activity is that salicylate influences the pre- and postsynaptic conductance of several transmembrane ion channels by modulating neurotransmitter activity (Lu et al., 2011). Because this mechanism depends on voltage-gated Ca2+ channel activity, increased excitatory synaptic transmission may contribute to
an increased Mn2+ accumulation in auditory structures. Furthermore, while a previous salicylate-induced tinnitus MEMRI study (Holt et al., 2010) using systemic Mn2 + injection showed signal enhancement only at the dorsal portion of the cochlear nucleus, our results using focal round window administration demonstrated the qualitative signal enhancement from the cochlear nerve to the ventral and to the dorsal portions of the cochlear nucleus. Therefore, our in vivo MEMRI data
Fig. 6. The pattern of sequential signal enhancement represented in normalized SNR after round window Mn2+ administration using ROIs located at the cochlea, cochlear nerve, ventral and dorsal cochlear nucleus of the left (ipsilateral) auditory pathway in both salicylate-treated group and in a control group. (*) significance P b 0.05, (**) significance P b 0.01.
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seem to support the hypothesis that increased spontaneous activity of neurons in the central auditory system underlies tinnitus. Salicylate has been reported to increase the spontaneous activity of the auditory nerve, the inferior colliculus, and the auditory cortex (Evans and Borerwe, 1982; Manabe et al., 1997; Ochi and Eggermont, 1996; Stypulkowski, 1990). However, cell recordings of non-auditory structures showed no change in spontaneous activity (Jastreboff and Sasaki, 1986) suggesting the selectivity of salicylate on auditory pathways. In the present study, we demonstrated significant Mn2+ enhancement over the cochlear nucleus along the central auditory tract with a Mn2 + dose of only 0.125 mM/kg. However, Mn2 + enhancements in the lateral lemniscus, the inferior colliculus, and the auditory cortex were not statistically different between the salicylate group and the control group, likely because of the low dose we employed. There were two reasons for choosing a small amount of Mn2+ in the present study. First, high doses of Mn2+ are associated with possible Mn2+ neurotoxicity (Silva et al., 2004). Because the current study is the first in vivo imaging attempt to investigate cochlear activity in a salicylate-induced tinnitus model, it was necessary to use as little Mn2+ as possible to minimize any possible neurotoxic effect of Mn2+ on the cochlea. Second, due to its paramagnetic properties, a high dose of Mn2+ at the round window can possibly produce an image artifact in the cochlea and the cochlear nucleus. However, to evaluate neural activity in higher central auditory structures and possibly in non-auditory brain structures, further investigations are warranted to determine the optimal Mn2 + dose to avoid toxicity and image artifacts. Finally, there are some limitations in the present study. First, the MnCl2 uptake into the cochlea was slightly different across animals after round window administration due to the individual size difference of round windows. Second, although an expert determined the ROIs based on resolved anatomical structures and a rat brain atlas, it remained subject to operator bias across animals. In summary, we developed a novel MEMRI method with Mn2+ administration through the round window to map the in vivo functional auditory tract in a salicylate-induced tinnitus animal model. The present study demonstrated that salicylate-induced tinnitus showed higher Mn2+ uptake in the cochlea and in the central auditory pathway. The serial MRI scans also demonstrated that the Mn2+ signal traveled from the cochlea to the cochlear nucleus. Therefore, our MEMRI data suggest that salicylate-induced tinnitus is associated with greater spontaneous neural activity in both peripheral and central auditory pathways. Acknowledgment This work was supported by the Ministry of Health & Welfare, Republic of Korea (A092106). This work was also supported by Basic Science Research Program through the National Research Foundation (NRF) of Korea (2012-005117 to H.J. Lee) and the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A111774 to K.Y. Lee). Conflict of interest statement The authors declare that there are no conflicts of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.neuroimage.2014.06.055. References Angelborg, C., Hillerdal, M., Hultcrantz, E., Larsen, H.C., 1988. The microsphere method for studies of inner ear blood blow. ORL J. Otorhinolaryngol Relat. Spec. 50, 355–362. Brozoski, T.J., Ciobanu, L., Bauer, C.A., 2007. Central neural activity in rats with tinnitus evaluated with manganese-enhanced magnetic resonance imaging (MEMRI). Hear. Res. 228, 168–179.
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