Chronological changes in compromised olivocochlear activity and the effect of insulin in diabetic Wistar rats

Hearing Research 270 (2010) 173e178

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Research paper

Chronological changes in compromised olivocochlear activity and the effect of insulin in diabetic Wistar rats Hung-Pin Wu a, b, c, Yueliang Leon Guo a, d, Tsun-Jen Cheng a, d, Chuan-Jen Hsu e, * a

Institute of Occupational Medicine and Industrial Hygiene, National Taiwan University, Taipei, Taiwan Department of Otolaryngology, Buddhist TzuChi General Hospital Taichung Branch, Taichung, Taiwan c School of Medicine, Tzu Chi University, Hualien, Taiwan d Department of Environmental and Occupational Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan e Department of Otolaryngology, National Taiwan University Hospital and National Taiwan University College of Medicine, No. 7, Chung-Shan S. Rd., Taipei 10002, Taiwan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 March 2010 Received in revised form 8 July 2010 Accepted 24 July 2010 Available online 1 August 2010

The aims of the present study were to investigate in diabetic rats: (1) the chronological changes of compromised medial olivocochlear bundle (MOCB) activity and auditory brainstem responses (ABR) and (2) the effect of insulin on diabetes-related hearing dysfunction. Diabetes mellitus was induced by intraperitoneal injection of streptozotocin. Thirty male Wistar rats were divided into three groups: control (C), diabetes with insulin injection (DI), and diabetes without insulin injection (DM). Click-evoked ABR, distortion product otoacoustic emission (DPOAE) and the contralateral suppression (CS) of DPOAE were measured for all animals monthly. Throughout the experiment, the thresholds of click-evoked ABR did not differ among groups. Wave III was delayed and interpeak latency IeIII was prolonged in the DM group at the age of 29 weeks (p < 0.05). The amplitudes of the CS of DPOAE were markedly decreased after the 25th week in the DM group, but not in the C and DI groups. Compared to the C group, the CS in the DI group was not attenuated at any frequency. Dysfunction of auditory efferent olivocochlear activity developed in diabetic rats presenting no evidence of hearing loss. The finding of a significant decrease of the CS of DPOAE could be used as an earlier indicator of diabetes-related hearing impairment than changes of ABRs. The time course of compromised MOCB is positively correlated with the duration of diabetes. Insulin could therefore protect against compromised MOCB. Ó 2010 Elsevier B.V. All rights reserved.

1. Introduction Diabetes mellitus is a heterogeneous group of metabolic disorders characterized by chronic hyperglycemia. There have been reports that patients with diabetes have greater hearing loss than those without this disease (Austin et al., 2009; Cullen and Cinnamond, 1993; Frisina et al., 2006). Auditory impairment may compromise the cochlear, retrocochlear and/or central auditory pathways in diabetic patients (Di Leo et al., 1997; Elamin et al., 2005; Huang et al., 1992; Pessin et al., 2008; Sasaki et al., 1997; Stolk and Boomsma, 1999). Several studies have attempted to

Abbreviations: ABR, Auditory brainstem response; ANOVA, Analysis of variance; CS, Contralateral suppression; DPOAE, Distortion product otoacoustic emission; IDDM, Insulin-dependent diabetes mellitus; FSR, Full suppression ratio; MEM, Middle ear muscle; MOCB, Medial olivocochlear bundle; OHC, Outer hair cell; peSPL, Peak sound pressure level; SE, Standard error; SPL, Sound pressure level; STZ, Streptozotocin; TEOAE, Transient evoked otoacoustic emission. * Corresponding author. Tel.: þ886 2 23123456x65220; fax: þ886 2 23410905. E-mail address: [email protected] (C.-J. Hsu). 0378-5955/$ e see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2010.07.008

identify the source of hearing loss in diabetic patients, but currently the location of the lesions, and the mechanism underlying the deficit, remain controversial. The sensorineural hearing loss is reported to be mild, symmetric, and predominantly high-frequency in type 1 and type 2 diabetes (Austin et al., 2009; Elamin et al., 2005; Hirose, 2008; Pessin et al., 2008; Ren et al., 2009). Speech discrimination abilities in diabetic patients have also been measured, and the results are controversial (Cullen and Cinnamond, 1993; Frisina et al., 2006; Huang et al., 1992; Pessin et al., 2008). One comparison study that recruited 44 insulindependent diabetes mellitus (IDDM) patients and 38 controls revealed no difference in speech discrimination between the groups(Cullen and Cinnamond, 1993). Another comparison study with 86 subjects found poor speech discrimination ability in diabetics (Huang et al., 1992). These studies were limited due to the small numbers of participants. Aside from hearing loss and decreased speech discrimination, disorders involving central auditory cognitive processing could also occur in diabetic patients. To investigate the association of diabetes with functionality measured along the central auditory pathway, numerous authors

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evaluated auditory brainstem response (ABR) latencies within the lower auditory pathways in subjects with type 1 and type 2 diabetes mellitus (Di Leo et al., 1997; Durmus et al., 2004; KonradMartin et al., 2010; Lisowska et al., 2001; Pessin et al., 2008; Ren et al., 2009; Vaughan et al., 2007). Increased latencies for waves III and V and wave intervals IeIII and IeV, as well as reduced amplitudes of wave V, were associated with diabetes. Central auditory dysfunction was indicated when abnormal ABR measures were adjusted for hearing in diabetic patients. Therefore, it is inferred that diabetes could impair auditory function through at least two mechanisms: one that is probably cochlear, and another that occurs within the central auditory system above the auditory nerve (Di Leo et al., 1997; Konrad-Martin et al., 2010; Lisowska et al., 2001; Pessin et al., 2008). Medial auditory efferent fibers (medial olivocochlear bundle, MOCB) originate from neurons adjacent to the medial and ventral regions of the superior olivary complex and preferentially innervate outer hair cells (OHCs) contralaterally via myelinated fibers (Guinan et al., 1983; Mountain, 1980). The activation of the MOCB has been shown to enhance the discrimination of signals and the perception of speech sounds amidst noise (Hienz et al., 1998; Kawase and Liberman, 1993; Kumar and Vanaja, 2004; Micheyl and Collet, 1996). The compromised activity of the MOCB is considered to result in poor speech perception in noisy environments. Recently, focus has shifted to determining whether there is a dysfunction of the efferent auditory system in diabetic subjects, especially those without hearing loss (Namyslowski et al., 2001; Ugur et al., 2009). These two recent cross-sectional reports offer a positive correlation between diabetes and compromised MOCB activity but lack any evidence of evolutionary adaptations in the MOCB. These human studies also lack certain medical information, especially the hyperglycemic status and the use of anti-hyperglycemic therapy among diabetic subjects. To further elucidate the casual association between diabetes and MOCB activity, we used a chemically induced diabetic animal model to imitate the status of insulin-dependent diabetes mellitus (IDDM) in humans (Jacobs et al., 1998). The aims of the present study were to investigate, in diabetic rats, (1) the evolution of compromised MOCB activity and auditory brainstem responses and (2) the effect of insulin on diabetes-related hearing dysfunction.

2. Materials and methods 2.1. Animals and induction of diabetes Male Wistar rats at the age of four weeks, weighing 90e110 g, were obtained from the National Taiwan University animal center for use in this experiment. The animals were housed in independent ventilation cages and were allowed free access to water and food. The lights were turned on from 6:30 am to 6:30 pm, and the temperature was maintained at 21  1  C. The weights and blood glucose levels of all the animals were measured weekly. All exposures and tests were performed during the daytime. The rats were fasted overnight, and diabetes was induced using 65 mg/kg of streptozotocin (STZ) in 0.1 mol citrate-buffered solution (pH 4.5, Sigma Chemicals), injected intraperitoneally when the animals were six weeks old. For all animals, blood glucose concentrations were measured in the morning via tail puncture (Glucocard Memory 2; Menarini Diagnostics, Florence, Italy) to confirm diabetic status. Rats with blood glucose levels under 200 mg/dl at the 48th hour were excluded from the study (Serdaroglu et al., 2005; Wu et al., 2009).

2.2. Auditory brainstem response (ABR) The measurements were carried out in a soundproof booth. Animals were anesthetized through an intraperitoneal injection with a mixture of ketamine hydrochloride (30 mg/kg) and xylazine hydrochloride (5 mg/kg). The auditory status of all of the rats was evaluated using click-evoked auditory brainstem responses (AEP, Navigator Pro; Bio-logic System Corp, Mundelein, IL). Elevations of the click-evoked ABR threshold have been shown to provide a reliable indicator of the degree of cochlear hearing loss for experimental animals (Hsu et al., 1998; Wassick and Yonovitz, 1985). The stimulation signals were channeled through plastic tubes into the animals’ ear canals. Click stimuli at a rate of 57.7/s were used to evoke the ABR. The ABR measurements were performed as previously described (Chen et al., 2005). The threshold was defined as the lowest intensity level of sound at which a response was still visible. We also measured the latencies of ABR waveforms in response to an 85 dB peak sound pressure level (dB peSPL) stimulus at a rate of 11.1/s. The prolongation of interpeak wave latencies and the delay of the absolute latencies of waveforms could potentially reveal a lesion site in the auditory pathway.

2.3. Distortion product otoacoustic emission (DPOAE) The auditory status of each rat was also evaluated using distortion product otoacoustic emissions (DPOAEs, Smart Tag; Intelligent Hearing Systems, Miami, FL, USA). The stimulus parameters were chosen according to published data on DPOAEs (Probst et al., 1991; Tan et al., 2001; Wu et al., 2010, 2009). The spectral resolution of each frequency data point was 4.88 Hz. The tested frequency range of f 2 was 1.1e17.7 kHz with four frequencies per octave. The two primaries, L1 and L2, were set at 65 and 55 dB SPL, respectively. The amplitudes of DPOAEs were measured at selected frequencies. Data are described with respect to f 2 frequency, as the generator site of the 2f 1-f 2 distortion product was most closely correlated with the f 2 frequency position in the cochlea (Brown and Kemp, 1984).

2.4. Contralateral suppression of DPOAEs Amplitudes of DPOAEs were measured with and without contralateral noise stimuli. Contralateral suppression (CS) was defined as the difference in dB between two DPOAE recordings, calculated by subtracting DPOAE amplitudes, with contralateral noise stimulation from DPOAE amplitude in the absence of sound. Therefore, suppression is indicated by positive values. Broadband white noise at the 60 dB level was tested in each ear of the animal before the procedure. This stimulation was insufficient to generate middle ear muscle (MEM) reflexes. To determine whether the CS of DPOAE originated as MOCB activity rather than MEM reflexes, we conducted a pilot study. Five male Wistar rats (6 weeks old) were ventilated via a tracheostomy tube; tubocurarine, a muscle relaxant, was injected intramuscularly (1 mg/kg). The CS assay was used to compare values before and after paralysis with those obtained using tubocurarine. The paralyzed MEM did not significantly affect the CS amplitude at any frequency (Fig. 1). The results suggest that under 60 dB broadband white noise stimulation, the MEM plays a limited role in the CS of DPOAE. When CS was present with a value of 3 dB at six or more frequencies between 5247 Hz and 17,672 Hz, we accepted that DPOAE was fully suppressed in that particular ear by the given noise. The full suppression ratio (FSR) is the ratio of fully suppressed ears in each group.

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2.5. Experimental procedures Thirty healthy, four-week-old, male Wistar rats with clickevoked ABR thresholds of less than 15 dB peSPL were employed in this experiment. They were divided into three groups: 10 control rats (C), 10 diabetic rats treated with insulin injection (DI) and 10 diabetic rats without insulin injection (DM). Insulin (2 IU, Insulatard HM, Novo Nordisk A/S, Denmark) was injected daily and subcutaneously in the DI group from the age of ten weeks. The ABRs, DPOAEs and CS of DPOAEs were measured every month as described above. The Tzu Chi General Hospital Animal Care and Use Committee approved all of the experimental protocols.

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3.2. Chronological ABR changes 2.6. Statistical analysis Statistical analysis was performed using JMP 5.0 and SPSS 13.0 software (Chicago, Illinois). The values of the three experimental groups, including the time course of each group, were analyzed using analyses of variance (ANOVA) assuming equal variance, followed by post-hoc analysis using Tukey’s HSD test. The results were expressed as means and standard errors (SE). Statistical significance was determined at p < 0.05.

Through the experiment, the thresholds of click-evoked ABR in each group did not differ among groups (Fig. 3). The ABR threshold was not elevated in the DM group at the end of the experiment. The chronological changes in absolute wave latencies and interpeak wave latencies of ABR are shown in Fig. 4. Compared with the control group, the DM group exhibited delayed wave III latency and prolonged interpeak IeIII latency at the 29th week (p < 0.05). 3.3. Chronological DPOAE changes

3.1. Body weight and blood glucose changes The blood glucose level of the non-diabetic control group (C) was 103.8  6.3 (SE) mg/dl. After the induction of diabetes at the age of six weeks, the blood glucose level increased significantly in the diabetic groups, i.e., DI and DM (Fig. 2a). The blood glucose level of the DM group gradually increased from 105.9  5.7 to 350.4  28.9 (SE) mg/dL when the rats were 29 weeks old. The blood glucose level of the DI group was reduced to 163.9  20.2 (SE) mg/dl at the age of 29 weeks. In general, the blood glucose level of the DM group was higher than that of the DI group, and the level in the DI group was higher than that in the C group after the age of 10 weeks (p < 0.05). Additionally, at the age of 29 weeks, the average body weight of the C group was 531.9  2.9 (SE) g, while average body weights in the DI and DM groups were 498.4  4.5 (SE) g and 429.9  15.8 (SE) g, respectively. Throughout the experimental period, the body weight growth rates of the DM and DI groups were much lower than those of the C group after the induction of diabetes (Fig. 2b).

Chronological measurements of the DPOAEs showed that throughout the experiment, the amplitudes of DPOAEs in each group were similar to their original values (Fig. 5). No decrease of

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DPOAE amplitude with time was observed for any frequency among all three groups.

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3.4. Chronological CS of DPOAE changes The chronological values of contralateral suppression between 5247 and 17,672 Hz were markedly decreased after the 25th week in the DM group, but not in the C and DI groups (Fig. 6). In addition, the CS of DPOAEs with time was demonstrated for all groups (Fig. 7). The CS was significantly attenuated between 7247 and 10,510 Hz at the 25th week in the DM group (p < 0.05). CS also declined significantly between 5247 and 12,503 Hz at the 29th week in the DM group as compared to the C group (p < 0.05). In contrast, the CS in the DI group was not attenuated at any frequency by the end of the experiment when compared to the C group. The FSR was significantly lower in the DM group (20%, 4 of 20 ears) than in the C (70%, 14 of 20 ears) or DI groups (55%, 11 of 20 ears) after the 25th week (p < 0.05). The FSR did not differ significantly between the C and DI groups during the experimental period. These data suggest that diabetes may compromise the efferent activities of the MOCB in diabetic subjects presenting no evidence of hearing loss. A compromised MOCB is associated with the long-term evolution of diabetes, and insulin could have a protective effect on diabetesrelated dysfunction of the efferent auditory system.

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Fig. 4. Chronological changes in the absolute latencies of waves I (a), II (b), III (c), and IV (d), as well as in the interpeak latency IeIII (e) of ABR waveforms, in each group of Wistar rats. C: C group; -: DI group; ,: DM group. Error bars indicate standard error. *DM > DI; #DM > C (by ANOVA and Tukey’s HSD test).

The results of this study demonstrate that the changes in the contralateral suppression of DPOAEs precede the changes in ABR latencies and thresholds in the DM group with normal hearing thresholds, but not in the DI group. This suggests that dysfunction of the auditory efferent MOCB might develop in diabetic subjects presenting no evidence of hearing loss. The contralateral

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Fig. 7. Comparison of chronological changes in the contralateral suppression of DPOAE for different frequencies among groups of Wistar rats. C: C group; -: DI group; ,: DM group. Error bars indicate standard error. *DI > DM; #C > DM (by ANOVA and Tukey’s HSD test).

suppression of DPOAE could be used instead of changes in ABRs as an earlier indicator of the central manifestation of diabetes-related hearing impairment. In addition, insulin could effectively preserve the activity of the MOCB in diabetic subjects. ABR latencies and interpeak latencies vary among diabetic subjects in different studies (Di Leo et al., 1997; Konrad-Martin et al., 2010; Lisowska et al., 2001; Pessin et al., 2008). The present study also demonstrated a delayed wave III and prolonged interpeak latency IeIII at the age of 29 weeks in the DM group with normal hearing thresholds but not in the DI group (Fig. 4). Changes in ABR wave latencies are consistent with the hypothesis that there is a primary diabetic effect on the auditory pathways above the cochlea in the absence of pure tone hearing loss (Konrad-Martin et al., 2010; Lisowska et al., 2001; Pessin et al., 2008). Interpeak latency IeIII is often taken as a measure of peripheral conduction time from the auditory nerve to the vicinity of the cochlear nucleus in humans (Konrad-Martin et al., 2010). Because wave III is affected by multiple generators in the brainstem, it is reasonable to infer that the MOCB could be compromised in diabetic hyperglycemic rats with normal hearing thresholds. The present study demonstrated a failure of the contralateral suppression of DPOAE in the DM group, which supports the hypothesis that diabetes might compromise the MOCB. Hearing impairment was considered to be related with the duration of diabetes and/or the severity of diabetes in some reports (Cullen and Cinnamond, 1993; Elamin et al., 2005; Hirose, 2008;

Pessin et al., 2008). In contrast, hearing loss was not related to the duration of diabetes and/or the severity of diabetes in other reports (Austin et al., 2009; Cullen and Cinnamond, 1993; KonradMartin et al., 2010). Significant changes in ABR interpeak latencies were often detected in the diabetic subjects who had normal hearing thresholds (Di Leo et al., 1997; Konrad-Martin et al., 2010; Lisowska et al., 2001; Pessin et al., 2008). The present study confirms this finding. In this study, no significant difference in ABR thresholds or DPOAE amplitudes was found at any frequency among any of the groups (Figs. 3 and 5). This result suggests a lack of hearing loss during the experimental period in the DM group or the DI group. However, wave III latencies and interpeak latencies IeIII were significantly prolonged from the age of 29 weeks in the DM group. The amplitudes of the contralateral suppression of DPOAE were significantly decreased from the age of 25 weeks in the DM group. In addition, no significant changes in ABR latencies or the contralateral suppression of DPOAE were demonstrated in the DI group when hyperglycemia was noted between the ages of 6 weeks and 10 weeks. These features indicate that the chronological change in diabetes-associated hearing impairment is positively correlated with the duration of exposure to hyperglycemia rather than with the blood glucose level. Previous studies on the contralateral suppression of TEOAEs demonstrated similar decreased amplitudes in diabetic subjects (Namyslowski et al., 2001; Ugur et al., 2009). However, Ugur et al. (Ugur et al., 2009) found no correlation between the mean duration of disease and the OAE

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parameters or between HbA1c blood levels and the degree of TEOAE suppression in the diabetic group. Prolonged wave latencies of auditory brainstem responses and decreased MOCB activity may indicate altered central neural transmission, which has been attributed to decreased electroconductive properties of the myelin sheath. This impairment results from metabolic changes caused by diabetes (Namyslowski et al., 2001). In addition, mitochondrial dysfunction has been proposed as a central mediator in diabetes complications, especially in neurons and endothelial cells (Nishikawa et al., 2000; Sasaki et al., 1997). Notably, insulin can prevent the depolarization of the mitochondrial inner membrane in sensory neurons of IDDM rats in the presence of sustained hyperglycemia (Huang et al., 2003). In the present study, the average blood glucose level was significantly higher in the DI group than in the C group. Hyperglycemia was addressed by a daily bolus injection with insulin. However, the contralateral suppression of DPOAE was not attenuated in the DI group. The insulin-mediated preservation of MOCB activity involves the control of glucose levels as well as the prevention of inner mitochondrial membrane depolarization. The compromised health of the rats due to the substantial decrease in body weight may have been responsible for the observed auditory changes. However, this chemically induced diabetes animal model that imitates insulin-dependent diabetes mellitus can also result in delayed growth. Therefore, the observed auditory changes may have resulted from chemically induced diabetes alone or from diabetes in conjunction with poor health. This study revealed that diabetes compromised MOCB activity and implies that the ability to discriminate speech amidst noise might be impaired in the context of diabetes (Frisina et al., 2006; Kumar and Vanaja, 2004; Micheyl and Collet, 1996). However, a large-scale investigation of speech perception amidst noise and related effects on MOCB activity in diabetic humans will be necessary to clarify this issue. 5. Conclusions The present study demonstrated that olivocochlear efferent auditory activity was compromised in diabetic subjects presenting no evidence of hearing loss. A significant decrease in the CS of DPOAE could be used instead of changes in ABRs as an earlier indicator of diabetes-related hearing impairment. The time course of MOCB compromise is positively correlated with the duration of diabetes. Insulin may exert a protective effect in such a context. Acknowledgements The authors thank the Core Lab of Taipei Tzu Chi General Hospital for animal care and Ms. Bin-Yu Chen for statistical assistance. The authors declare that they have no competing financial interests. References Austin, D.F., Konrad-Martin, D., Griest, S., McMillan, G.P., McDermott, D., Fausti, S., 2009. Diabetes-related changes in hearing. Laryngoscope 119, 1788e1796. Brown, A.M., Kemp, D.T., 1984. Suppressibility of the 2f1ef2 stimulated acoustic emissions in gerbil and man. Hear. Res. 13, 29e37. Chen, Y.S., Tseng, F.Y., Liu, T.C., Lin-Shiau, S.Y., Hsu, C.J., 2005. Involvement of nitric oxide generation in noise-induced temporary threshold shift in guinea pigs. Hear. Res. 203, 94e100. Cullen, J.R., Cinnamond, M.J., 1993. Hearing loss in diabetics. J. Laryngol. Otol. 107, 179e182.

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