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ROLE OF MACROPHAGE MIGRATION INHIBITORY FACTOR IN AGE-RELATED HEARING LOSS Q1 S. KARIYA, a* M. OKANO, a Y. MAEDA, a H. HIRAI, a
Age-related hearing loss, also known as presbycusis, is a common and serious human health concern. Approximately 10% of the population has a significant hearing loss that impairs communication, and this rate increases to 40% in those older than 65 years (Gates and Mills, 2005). Multiple factors, including noise damage, genetic susceptibility, otological disorders, systemic diseases, and exposure to ototoxic agents, affect the inner ear. The impaired central processing of acoustic information is also related to reduced hearing sensitivity, speech understanding in noisy environments, and impaired localization of sound sources in age-related hearing loss (Huang and Tang, 2010; Roth et al., 2011). Macrophage migration inhibitory factor (MIF) was originally described as a factor that inhibited the random migration of macrophages. MIF is an innate immunity molecule with ubiquitous tissue expression. However, it has now been identified as having multiple functions, such as acting as a pituitary hormone, growth factor, and pro-inflammatory cytokine (Calandra and Roger, 2003). MIF is strongly expressed in the central nervous system, but its function has not yet been defined (Fingerle-Rowson and Bucala, 2001). MIF is also observed in the peripheral nerves, and plays an important role in the acceleration of peripheral nerve regeneration and in the prevention of Schwann cell apoptosis (Nishio et al., 1999; Nihsio et al., 2002). In an experimen- Q3 tal animal study, MIF has been shown to be an essential factor in axis formation and neural development in embryos of the African clawed frog (Xenopus laevis) (Suzuki et al., 2004). A recent study showed that MIF plays a key instructional role for sensory hair cell differentiation, semicircular canal formation, and statoacoustic ganglion development in zebrafish (Danio rerio) (Shen et al., 2012). After the generation of MIF knockout mice, numerous studies reported various functions of MIF both within and outside the immune system (Calandra and Roger, 2003). MIF is considered an important factor in the pathogenesis of middle ear diseases in both humans and mice, but the role of MIF in the mammalian inner ear has not been fully defined (Kariya et al., 2003, 2008a,b). The purpose of this study was to examine the expression of MIF in the inner ear and to confirm its role in cochlear function in mice.
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Department of Otolaryngology-Head and Neck Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan
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INTRODUCTION
T. HIGAKI, a Y. NOYAMA, a T. HARUNA, a J. NISHIHIRA b AND K. NISHIZAKI a
Department of Medical Bioinformatics, Hokkaido Information University, 59-2 Nishi Nopporo, Ebetsu, Hokkaido 069-8585, Japan
Abstract—Hearing loss related to aging is the most common sensory disorder among elderly individuals. Macrophage migration inhibitory factor (MIF) is a multi-functional molecule. The aim of this study was to identify the role of macrophage MIF in the inner ear. Macrophage MIF-deficient mice (MIF/ mice) of BALB/c background and wild-type BALB/c mice were used in this study. Expression of macrophage MIF protein in the inner ear was examined by immunohistochemistry in wild-type mice (WT). The hearing function was assessed by the click-evoked auditory brainstem response in both MIF/ mice and WT at 1, 3, 6, 9, 12, and 18 months of age. Morphological examination of the cochlea was also performed using scanning electron microscopy and light microscopy. Macrophage MIF was observed in the spiral ligament, stria vascularis, Reissner’s membrane, spiral ganglion cells, saccular macula, and membranous labyrinth. The MIF/ mice had a significant hearing loss as compared with the WT at 9, 12, and 18 months of age. In the MIF/ mice, scanning electron microscopy showed that the outer cochlear hair cells were affected, but that the inner cochlear hair cells were relatively well preserved. The number of SGCs was lower in the MIF/ mice. Macrophage MIF was strongly expressed in the mouse inner ear. Older MIF/ mice showed accelerated age-related hearing loss and morphological inner ear abnormalities. These findings suggest that macrophage MIF plays an important role in the inner ear of mice. Ó 2014 Published by Elsevier Ltd. on behalf of IBRO.
Key words: macrophage migration inhibitory factor, hearing loss, cochlea, ear, hair cell, spiral ganglion cells. 14
*Corresponding author. Tel: +81-86-235-7307; fax: +81-86-2357308. E-mail address: [email protected] (S. Kariya). Q2 Abbreviations: ABR, auditory brainstem response; MIF, migration inhibitory factor; SGCs, spiral ganglion cells; SL, spiral ligament; SV, stria vascularis; WT, wild-type mice. http://dx.doi.org/10.1016/j.neuroscience.2014.08.042 0306-4522/Ó 2014 Published by Elsevier Ltd. on behalf of IBRO. 1
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EXPERIMENTAL PROCEDURES
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Animals
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BALB/c mice were used in this study. Through targeted disruption of the MIF gene, MIF-deficient mice (MIF/ mice) were generated from a BALB/c background (Honma et al., 2000). In brief, a gene-targeting vector was generated using a 6.0-kb XbaI fragment that contained all of the MIF exons subcloned. A 201-bp SacI fragment consisting of the 30 region of exon 1 and the 50 region of intron 1 was replaced with a pMC1-neo poly (A) cassette in a forward orientation relative to MIF gene transcription. A DT-A cassette was also introduced at the 30 flanking region for negative selection. R1 embryonic stem cells were transfected and subjected to positive selection. Subsequently, they generated germline chimeras with targeted disruption of the MIF gene by the aggregation method, and then generated a mouse strain deficient in the MIF gene. To achieve a pure strain of the BALB/c background on MIF/ mice, backcrossing was performed more than 10 times. Otoscopic examination was performed for all mice prior to treatment in order to ensure that the tympanic membranes were normal and that no middle ear effusion was present. Mice were deeply anesthetized using intraperitoneal injection of both ketamine (100-mg/kg body weight) and xylazine (10-mg/kg body weight). This study was performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, revised 1996. The Animal Research Control Committee of the Okayama University approved the study.
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Immunohistochemistry
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Wild-type BALB/c mice (n = 2, 8 weeks old) were deeply anesthetized using an intraperitoneal injection of both ketamine and xylazine. The mice were decapitated, and the temporal bones were fixed in fresh 4% paraformaldehyde in phosphate-buffered saline for 24 h at 4 °C. They were then decalcified in 4% ethylenediaminetetraacetic acid in phosphate-buffered saline for 14 days at 4 °C. Immunohistochemical staining was performed on the paraffin-embedded tissues using a rabbit polyclonal MIF antibody (sc-20121; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, dilution 1:100) and 3,30 diaminobenzidine (DAB) reagent (Dako, Glostrup, Denmark) according to the manufacturer’s instructions.
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Auditory brainstem response (ABR)
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MIF/ mice (n = 5) and wild-type mice (WT) (n = 5) were used in this study. The animals were anesthetized. The stimuli used were clicks generated by a RP2.1 Enhanced Real-Time Processor (Tucker-Davis Technologies, Gainesville, FL, USA) with a plateau of 0.1 ms. A closed system with monaural stimulation was used, and clicks were delivered directly to the outer ear canal by a plastic tube. ABR was evoked with clicks, and was recorded with needle electrodes inserted through the skin (vertex to the ipsilateral retroauricle with a ground at the contralateral retroauricle). Responses were processed through a 300-Hz to 3000-
Hz bandpass filter and were averaged 1000 times using a signal processor RA16 (Tucker-Davis Technologies, Gainesville, FL, USA). Stimuli were applied in 10-dB steps with a 21-Hz stimulus repetition rate. ABR thresholds were defined as the lowest sound level at which the response peaks were clearly present in stacked waveforms. ABR thresholds were examined at 1, 3, 6, 9, 12, and 18 months of age.
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Electron microscopy
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MIF mice and WT were used in this study. The animals were anesthetized, and cochleae were collected. The tissues were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The specimens were post-fixed in 2% osmium tetroxide, dehydrated through a graded ethanol series, and then dried by critical point drying. The morphological findings of stereocilia of cochlear hair cells were investigated using scanning electron microscopy. The numbers of inner and outer hair cells were counted, and the results were expressed as the percentage of remaining hair cells, as described previously (Fetoni et al., 2009; Miller et al., 2012).
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Light microscopy
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MIF mice (n = 5) and WT (n = 5) at 9 months of age were used in this study. The mice were sacrificed and the temporal bones were collected. The tissues were fixed in fresh 4% paraformaldehyde in phosphatebuffered saline for 24 h, followed by decalcification in 4% ethylenediaminetetraacetic acid in phosphatebuffered saline for 14 days at 4 °C. Cochleae were embedded in paraffin, and were cut at a thickness of 10 lm. Hematoxylin and eosin staining was performed for histopathological study. The number of spiral ganglion cells (SGCs) in the midmodiolar section of the cochlea was counted under light microscopy according to the methods of previous studies (Willott et al., 1998; Tang et al., 2014). Briefly, the cochlea was divided into four segments (extreme base (‘hook’ region), mid-base (basal turn of mid-modiolar section), mid-apex (apical turn of mid-modiolar section, but below the apex), and apex (apex of mid-modiolar section)). The cell density of the SGCs was counted in each segment using a 100-lm 100-lm eyepiece reticle. The criterion for inclusion of SGCs was a complete nucleus.
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Statistical analysis
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Data are presented as mean ± standard deviation. For statistical analysis, the non-parametric Mann–Whitney U test was used for comparison between two groups. Significant differences were assumed at a level of P < 0.05 (IBM SPSS Statistics; IBM, New York, NY, USA).
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RESULTS
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Expression of MIF in inner ear
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We first examined the expression of MIF in the inner ear of WT. Positive immunostaining for MIF was observed in the spiral ligament, stria vascularis, spiral limbus, organ of Corti, and Reissner’s membrane. Weak staining for MIF was also seen in the SGCs in the cochlea (Fig. 1A). MIF was strongly expressed in the saccular macula, but not in the facial nerve (Fig. 1B). Immunohistological staining confirmed the presence of MIF within the crista ampullaris and cells lining the membranous labyrinth (Fig. 1C).
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Assessment of hearing threshold by ABR
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Immunohistological findings suggest the possibility of MIF functioning in the inner ear. To determine the role of MIF in mouse cochlea, click-evoked ABR was performed to evaluate the hearing threshold in MIF/ mice and WT. The typical ABR waveforms of MIF/ mice and WT are shown in Fig. 2. MIF/ mice at the age of 3 months did not show significantly impaired hearing loss in clickevoked ABR (Fig. 2A). MIF/ mice at the age of 18 months had increased hearing threshold as compared with that of WT (Fig. 2B, C). In order to investigate the effects of age, we examined ABR threshold at 1, 3, 6, 9, 12, and 18 months of age in both MIF/ mice and WT. There were no statistically significant differences in the mean hearing levels
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between MIF/ mice and WT at 1, 3, and 6 months of age by click-evoked ABR. In contrast, there was a significant increase in the levels of ABR threshold in MIF/ mice at 9, 12, and 18 months of age as compared with those of WT (Fig. 3: 9 months, P = 0.031; 12 months, P = 0.021; 18 months, P = 0.013).
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Cochlear hair cells using scanning electron microscopy
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A functional examination (ABR) revealed significant impairment of hearing in aged MIF/ mice. Next, we examined the morphological findings in the cochlea of MIF/ mice. Scanning electron microscopy showed that the outer cochlear hair cells in MIF/ mice at 9 months of age were affected. However, the inner cochlear hair cells in MIF/ mice were relatively well preserved when compared with those of WT (Fig. 4). No significant differences were observed in the loss of inner cochlear hair cells between MIF/ mice and WT. The number of outer cochlear hair cells in aged MIF/ mice was significantly less when compared with that of WT (Fig. 5: apical turn, P = 0.008; middle turn, P = 0.008; basal turn, P = 0.009). In addition, the stereocilia of the remaining outer cochlear hair cells were damaged. In MIF/ mice at 1 month of age, a slight loss of outer hair cells was observed in the middle turn and the basal turn. However, the difference in the loss of hair cells between the MIF/ mice and the WT did not reach
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Fig. 1. Immunohistological findings for macrophage migration inhibitory factor in mouse cochlea. (A–C) Positive immunostaining for macrophage migration inhibitory factor was detected in the inner ear in wild-type BALB/c mice. (D) No immunostaining for macrophage migration inhibitory factor was detected in negative controls (OC, organ of Corti; RM, Reissner’s membrane; SV, stria vascularis; SL, spiral ligament; SGC, spiral ganglion cell; S, saccule; FN, facial nerve; ML, membranous labyrinth; CA, crista ampullaris. Scale bar = 100 lm). Please cite this article in press as: Kariya S et al. Role of macrophage migration inhibitory factor in age-related hearing loss. Neuroscience (2014), http:// dx.doi.org/10.1016/j.neuroscience.2014.08.042
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Fig. 2. Typical findings for click-evoked auditory brainstem response (ABR) in (A) macrophage migration inhibitory factor-deficient mice (MIF/ mice) at 3 months of age, (B) MIF/ mice at 18 months of age, and (C) wild-type mice at 18 months of age. (Horizontal bar, 1 ms; vertical bar, 1 lv).
Fig. 3. Hearing threshold by click-evoked auditory brainstem response (ABR) in MIF/ mice (open circles, n = 5) and wild-type mice (open boxes, n = 5). MIF/ mice showed significant hearing loss at 9, 12, and 18 months of age as compared with wild-type mice (mean ± standard deviation; ⁄P < 0.05).
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statistical significance. No significant findings were observed in the inner cochlear hair cells of the MIF/ mice at 1 month of age.
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SGC counts
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Loss of SGCs is considered a major factor in hearing loss (Kariya et al., 2007; Nadol, 2010). Therefore, we evaluated the number of SGCs under light microscopy in MIF/ mice and WT. Because the apex segment of the cochlea in some mice was not suitable for counting, we counted the nuclei of SGCs in three segments (mid-apex,
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mid-base, and extreme base). The number of SGCs in the MIF/ mice was lower than that in the control WT (Fig. 6), and the cell density of SGCs was significantly lower in the MIF/ mice than in the control WT (Fig. 7: mid-apex, P = 0.009; mid-base, P = 0.004; extreme base, P = 0.009). The SV and SL in the MIF/ mice showed morphologically normal findings under light microscopy.
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DISCUSSION
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MIF is a multi-functional molecule (Calandra and Roger, 2003). The anti-MIF antibody significantly suppresses tumor growth and tumor-associated angiogenesis (Nishihira, 2000). MIF acts as a counter-regulator to down-modulate the effects of glucocorticoids on the immune and endocrine systems (Petrovsky and Bucala, 2000). Schwann cells are a potential source of MIF protein in peripheral nerves, and MIF plays a critical role in the viability of Schwann cells by suppressing apoptosisrelated proteins such as p53 (Nishio et al., 2002). The same group also reported that down-regulation of MIF prevents detrimental secondary molecular responses in the injured spinal cord (Nishio et al., 2009). MIF is required for the differentiation of neural precursor cells in embryos of X. laevis, and for inner ear development in zebrafish (Suzuki et al., 2004; Shen et al., 2012). Recombinant MIF acts as a neurotrophin in promoting neuronal survival and is expressed in both the developing and mature inner ear of chicks and mice (Bank et al., 2012). This study clearly shows that MIF is expressed in the inner ear. Our findings suggest that MIF has an important role in age-related hearing loss in mice.
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Fig. 4. Morphology of cochlear hair cells on scanning electron microscopy in (A) MIF/ mice and (B) wild-type mice at 9 months of age. Outer cochlear hair cells were damaged in MIF/ mice (OHC, outer cochlear hair cells; IHC, inner cochlear hair cells. Scale bar = 10 lm).
Fig. 5. Loss of outer cochlear hair cells in MIF/ mice (open bar, n = 5) and wild-type mice (black bar, n = 5) at 9 months of age. The number of remaining outer cochlear cells in MIF/ mice was significantly less as compared with that of wild-type mice (WT, wild-type mice; KO, MIF/ mice; mean ± standard deviation; ⁄⁄P < 0.01).
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The use of several animal models for the study of progressive sensorineural hearing loss has been reported (Zhou et al., 2006). The audiological examination used in this study clearly showed that the down-regulation of MIF induces accelerated age-related hearing loss in BALB/c mice. We used click-evoked ABR to determine hearing thresholds. Click-evoked ABR is widely used for estimating hearing thresholds in both humans and experimental animals but only provides data in the 1–4-kHz range (Scherf et al., 2006; Zhou et al., 2006). A limitation of this study is that the hearing assessment is not frequency specific. Although the kind of stimulus waveform used for ABR measurements was not detailed, a recent study reported that 4-week-old MIF/ mice had hearing impairments at 12, 24, and 48 kHz (Bank et al., 2012). Our data using click-evoked ABR shows that there is no significant hearing loss in MIF/ mice younger than 6 months of age. This disparity may be due to differences in the stimulus sounds of ABR, because the click-evoked ABR used in this study cannot evaluate hearing thresholds at 12, 24, and 48 kHz. We also show in this study that
Fig. 6. Histopathological findings of the cochlea in (A) MIF/ mice and (B) wild-type mice at 9 months of age. Number of spiral ganglion cells was less in MIF/ mice, as compared with that of wild-type mice (hematoxylin and eosin staining; OC, organ of Corti; RM, Reissner’s membrane; SV, stria vascularis; SL, spiral ligament; SGC, spiral ganglion cell; bar = 100 lm).
the cochleae of 4-week-old MIF/ mice show some loss of outer hair cells, although the difference does not reach statistical significance as compared with WT. Age-related
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Fig. 7. Cell density of spiral ganglion cells in MIF/ mice (open bars, n = 5) and wild-type mice (black bars, n = 5) at 9 months of age in three segments of cochlea. The number of spiral ganglion cells in MIF/ mice were significantly affected, as compared with that of wild-type mice in mid-apex, mid-base, and extreme base segments (WT, wild-type mice; KO, MIF/ mice; mean ± standard deviation; ⁄⁄ P < 0.01). 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327
hearing loss often starts in the high-frequency range and later affects lower frequencies. Our findings show that young MIF/ mice have a normal hearing function in the 1–4-kHz range. Young MIF/ mice may have a hearing loss in the high-frequency range (12, 24, and 48 kHz), as reported in the previous study (Bank et al., 2012), and that hearing loss may indicate the early onset of agerelated hearing loss in MIF/ mice. SGCs are the first neurons in the auditory pathway, and play a critical role in sensorineural hearing function (Nadol, 2010). The loss of SGCs is a significant pathological finding in age-related hearing loss (Bao and Ohlemiller, 2010). The hair bundles of the inner hair cells respond primarily to basilar membrane velocity rather than displacement. The displacement of the basilar membrane stimulates the outer hair cells by bending their stereociliary bundles against the tectorial membrane. There are more (10–20) afferent fibers from the SGCs to the inner hair cells, as compared with the outer hair cells. In contrast, the efferent fibers with cell bodies in the brainstem contact only the outer hair cells. The outer hair cells have both sensory and motor capability, and the interactive network may be part of a feedback pathway to regulate cochlear sensitivity. Acoustic information is primarily transmitted to the central nervous system through electrical signals by the inner hair cells, whereas the main task of the outer hair cells is to boost the stimulus by electromechanical feedback. These findings show that the destruction of the outer hair cells results in a greatly elevated sound threshold and a deterioration of frequency selectivity (Fettiplace and Hackney, 2006). We clearly show here that the inner cochlear hair cells in MIF/ mice are relatively well preserved, while the outer cochlear hair cells are affected. A significant decrease in the number of SGCs is also observed in MIF/ mice. Aged MIF/ mice show a significant hearing loss, and cochlear dysfunction in MIF/ mice may result, in part, from the loss of outer hair cells and SGCs.
CONCLUSION
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Aged MIF mice show an accelerated age-related hearing loss. MIF may play a significant role in the
mammalian inner ear. Our findings suggest that MIF is an essential component of normal inner ear function in mice, particularly at advanced ages.
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Acknowledgments—This work was supported by JSPS KAKENHI (Grants-in-Aid for Scientific Research from The Ministry of Education, Culture, Sports, Science and Technology of Japan; grant number, 23791900 and 25462642).
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(Accepted 26 August 2014) (Available online xxxx)
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ROLE OF MACROPHAGE MIGRATION INHIBITORY FACTOR IN AGE-RELATED HEARING LOSS Q1 S. KARIYA, a* M. OKANO, a Y. MAEDA, a H. HIRAI, a
Age-related hearing loss, also known as presbycusis, is a common and serious human health concern. Approximately 10% of the population has a significant hearing loss that impairs communication, and this rate increases to 40% in those older than 65 years (Gates and Mills, 2005). Multiple factors, including noise damage, genetic susceptibility, otological disorders, systemic diseases, and exposure to ototoxic agents, affect the inner ear. The impaired central processing of acoustic information is also related to reduced hearing sensitivity, speech understanding in noisy environments, and impaired localization of sound sources in age-related hearing loss (Huang and Tang, 2010; Roth et al., 2011). Macrophage migration inhibitory factor (MIF) was originally described as a factor that inhibited the random migration of macrophages. MIF is an innate immunity molecule with ubiquitous tissue expression. However, it has now been identified as having multiple functions, such as acting as a pituitary hormone, growth factor, and pro-inflammatory cytokine (Calandra and Roger, 2003). MIF is strongly expressed in the central nervous system, but its function has not yet been defined (Fingerle-Rowson and Bucala, 2001). MIF is also observed in the peripheral nerves, and plays an important role in the acceleration of peripheral nerve regeneration and in the prevention of Schwann cell apoptosis (Nishio et al., 1999; Nihsio et al., 2002). In an experimen- Q3 tal animal study, MIF has been shown to be an essential factor in axis formation and neural development in embryos of the African clawed frog (Xenopus laevis) (Suzuki et al., 2004). A recent study showed that MIF plays a key instructional role for sensory hair cell differentiation, semicircular canal formation, and statoacoustic ganglion development in zebrafish (Danio rerio) (Shen et al., 2012). After the generation of MIF knockout mice, numerous studies reported various functions of MIF both within and outside the immune system (Calandra and Roger, 2003). MIF is considered an important factor in the pathogenesis of middle ear diseases in both humans and mice, but the role of MIF in the mammalian inner ear has not been fully defined (Kariya et al., 2003, 2008a,b). The purpose of this study was to examine the expression of MIF in the inner ear and to confirm its role in cochlear function in mice.
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Department of Otolaryngology-Head and Neck Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan
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INTRODUCTION
T. HIGAKI, a Y. NOYAMA, a T. HARUNA, a J. NISHIHIRA b AND K. NISHIZAKI a
Department of Medical Bioinformatics, Hokkaido Information University, 59-2 Nishi Nopporo, Ebetsu, Hokkaido 069-8585, Japan
Abstract—Hearing loss related to aging is the most common sensory disorder among elderly individuals. Macrophage migration inhibitory factor (MIF) is a multi-functional molecule. The aim of this study was to identify the role of macrophage MIF in the inner ear. Macrophage MIF-deficient mice (MIF/ mice) of BALB/c background and wild-type BALB/c mice were used in this study. Expression of macrophage MIF protein in the inner ear was examined by immunohistochemistry in wild-type mice (WT). The hearing function was assessed by the click-evoked auditory brainstem response in both MIF/ mice and WT at 1, 3, 6, 9, 12, and 18 months of age. Morphological examination of the cochlea was also performed using scanning electron microscopy and light microscopy. Macrophage MIF was observed in the spiral ligament, stria vascularis, Reissner’s membrane, spiral ganglion cells, saccular macula, and membranous labyrinth. The MIF/ mice had a significant hearing loss as compared with the WT at 9, 12, and 18 months of age. In the MIF/ mice, scanning electron microscopy showed that the outer cochlear hair cells were affected, but that the inner cochlear hair cells were relatively well preserved. The number of SGCs was lower in the MIF/ mice. Macrophage MIF was strongly expressed in the mouse inner ear. Older MIF/ mice showed accelerated age-related hearing loss and morphological inner ear abnormalities. These findings suggest that macrophage MIF plays an important role in the inner ear of mice. Ó 2014 Published by Elsevier Ltd. on behalf of IBRO.
Key words: macrophage migration inhibitory factor, hearing loss, cochlea, ear, hair cell, spiral ganglion cells. 14
*Corresponding author. Tel: +81-86-235-7307; fax: +81-86-2357308. E-mail address: [email protected] (S. Kariya). Q2 Abbreviations: ABR, auditory brainstem response; MIF, migration inhibitory factor; SGCs, spiral ganglion cells; SL, spiral ligament; SV, stria vascularis; WT, wild-type mice. http://dx.doi.org/10.1016/j.neuroscience.2014.08.042 0306-4522/Ó 2014 Published by Elsevier Ltd. on behalf of IBRO. 1
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EXPERIMENTAL PROCEDURES
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Animals
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BALB/c mice were used in this study. Through targeted disruption of the MIF gene, MIF-deficient mice (MIF/ mice) were generated from a BALB/c background (Honma et al., 2000). In brief, a gene-targeting vector was generated using a 6.0-kb XbaI fragment that contained all of the MIF exons subcloned. A 201-bp SacI fragment consisting of the 30 region of exon 1 and the 50 region of intron 1 was replaced with a pMC1-neo poly (A) cassette in a forward orientation relative to MIF gene transcription. A DT-A cassette was also introduced at the 30 flanking region for negative selection. R1 embryonic stem cells were transfected and subjected to positive selection. Subsequently, they generated germline chimeras with targeted disruption of the MIF gene by the aggregation method, and then generated a mouse strain deficient in the MIF gene. To achieve a pure strain of the BALB/c background on MIF/ mice, backcrossing was performed more than 10 times. Otoscopic examination was performed for all mice prior to treatment in order to ensure that the tympanic membranes were normal and that no middle ear effusion was present. Mice were deeply anesthetized using intraperitoneal injection of both ketamine (100-mg/kg body weight) and xylazine (10-mg/kg body weight). This study was performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, revised 1996. The Animal Research Control Committee of the Okayama University approved the study.
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Immunohistochemistry
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Wild-type BALB/c mice (n = 2, 8 weeks old) were deeply anesthetized using an intraperitoneal injection of both ketamine and xylazine. The mice were decapitated, and the temporal bones were fixed in fresh 4% paraformaldehyde in phosphate-buffered saline for 24 h at 4 °C. They were then decalcified in 4% ethylenediaminetetraacetic acid in phosphate-buffered saline for 14 days at 4 °C. Immunohistochemical staining was performed on the paraffin-embedded tissues using a rabbit polyclonal MIF antibody (sc-20121; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, dilution 1:100) and 3,30 diaminobenzidine (DAB) reagent (Dako, Glostrup, Denmark) according to the manufacturer’s instructions.
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Auditory brainstem response (ABR)
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MIF/ mice (n = 5) and wild-type mice (WT) (n = 5) were used in this study. The animals were anesthetized. The stimuli used were clicks generated by a RP2.1 Enhanced Real-Time Processor (Tucker-Davis Technologies, Gainesville, FL, USA) with a plateau of 0.1 ms. A closed system with monaural stimulation was used, and clicks were delivered directly to the outer ear canal by a plastic tube. ABR was evoked with clicks, and was recorded with needle electrodes inserted through the skin (vertex to the ipsilateral retroauricle with a ground at the contralateral retroauricle). Responses were processed through a 300-Hz to 3000-
Hz bandpass filter and were averaged 1000 times using a signal processor RA16 (Tucker-Davis Technologies, Gainesville, FL, USA). Stimuli were applied in 10-dB steps with a 21-Hz stimulus repetition rate. ABR thresholds were defined as the lowest sound level at which the response peaks were clearly present in stacked waveforms. ABR thresholds were examined at 1, 3, 6, 9, 12, and 18 months of age.
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Electron microscopy
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MIF mice and WT were used in this study. The animals were anesthetized, and cochleae were collected. The tissues were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The specimens were post-fixed in 2% osmium tetroxide, dehydrated through a graded ethanol series, and then dried by critical point drying. The morphological findings of stereocilia of cochlear hair cells were investigated using scanning electron microscopy. The numbers of inner and outer hair cells were counted, and the results were expressed as the percentage of remaining hair cells, as described previously (Fetoni et al., 2009; Miller et al., 2012).
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Light microscopy
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MIF mice (n = 5) and WT (n = 5) at 9 months of age were used in this study. The mice were sacrificed and the temporal bones were collected. The tissues were fixed in fresh 4% paraformaldehyde in phosphatebuffered saline for 24 h, followed by decalcification in 4% ethylenediaminetetraacetic acid in phosphatebuffered saline for 14 days at 4 °C. Cochleae were embedded in paraffin, and were cut at a thickness of 10 lm. Hematoxylin and eosin staining was performed for histopathological study. The number of spiral ganglion cells (SGCs) in the midmodiolar section of the cochlea was counted under light microscopy according to the methods of previous studies (Willott et al., 1998; Tang et al., 2014). Briefly, the cochlea was divided into four segments (extreme base (‘hook’ region), mid-base (basal turn of mid-modiolar section), mid-apex (apical turn of mid-modiolar section, but below the apex), and apex (apex of mid-modiolar section)). The cell density of the SGCs was counted in each segment using a 100-lm 100-lm eyepiece reticle. The criterion for inclusion of SGCs was a complete nucleus.
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Statistical analysis
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Data are presented as mean ± standard deviation. For statistical analysis, the non-parametric Mann–Whitney U test was used for comparison between two groups. Significant differences were assumed at a level of P < 0.05 (IBM SPSS Statistics; IBM, New York, NY, USA).
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RESULTS
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Expression of MIF in inner ear
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We first examined the expression of MIF in the inner ear of WT. Positive immunostaining for MIF was observed in the spiral ligament, stria vascularis, spiral limbus, organ of Corti, and Reissner’s membrane. Weak staining for MIF was also seen in the SGCs in the cochlea (Fig. 1A). MIF was strongly expressed in the saccular macula, but not in the facial nerve (Fig. 1B). Immunohistological staining confirmed the presence of MIF within the crista ampullaris and cells lining the membranous labyrinth (Fig. 1C).
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Assessment of hearing threshold by ABR
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Immunohistological findings suggest the possibility of MIF functioning in the inner ear. To determine the role of MIF in mouse cochlea, click-evoked ABR was performed to evaluate the hearing threshold in MIF/ mice and WT. The typical ABR waveforms of MIF/ mice and WT are shown in Fig. 2. MIF/ mice at the age of 3 months did not show significantly impaired hearing loss in clickevoked ABR (Fig. 2A). MIF/ mice at the age of 18 months had increased hearing threshold as compared with that of WT (Fig. 2B, C). In order to investigate the effects of age, we examined ABR threshold at 1, 3, 6, 9, 12, and 18 months of age in both MIF/ mice and WT. There were no statistically significant differences in the mean hearing levels
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between MIF/ mice and WT at 1, 3, and 6 months of age by click-evoked ABR. In contrast, there was a significant increase in the levels of ABR threshold in MIF/ mice at 9, 12, and 18 months of age as compared with those of WT (Fig. 3: 9 months, P = 0.031; 12 months, P = 0.021; 18 months, P = 0.013).
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Cochlear hair cells using scanning electron microscopy
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A functional examination (ABR) revealed significant impairment of hearing in aged MIF/ mice. Next, we examined the morphological findings in the cochlea of MIF/ mice. Scanning electron microscopy showed that the outer cochlear hair cells in MIF/ mice at 9 months of age were affected. However, the inner cochlear hair cells in MIF/ mice were relatively well preserved when compared with those of WT (Fig. 4). No significant differences were observed in the loss of inner cochlear hair cells between MIF/ mice and WT. The number of outer cochlear hair cells in aged MIF/ mice was significantly less when compared with that of WT (Fig. 5: apical turn, P = 0.008; middle turn, P = 0.008; basal turn, P = 0.009). In addition, the stereocilia of the remaining outer cochlear hair cells were damaged. In MIF/ mice at 1 month of age, a slight loss of outer hair cells was observed in the middle turn and the basal turn. However, the difference in the loss of hair cells between the MIF/ mice and the WT did not reach
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Fig. 1. Immunohistological findings for macrophage migration inhibitory factor in mouse cochlea. (A–C) Positive immunostaining for macrophage migration inhibitory factor was detected in the inner ear in wild-type BALB/c mice. (D) No immunostaining for macrophage migration inhibitory factor was detected in negative controls (OC, organ of Corti; RM, Reissner’s membrane; SV, stria vascularis; SL, spiral ligament; SGC, spiral ganglion cell; S, saccule; FN, facial nerve; ML, membranous labyrinth; CA, crista ampullaris. Scale bar = 100 lm). Please cite this article in press as: Kariya S et al. Role of macrophage migration inhibitory factor in age-related hearing loss. Neuroscience (2014), http:// dx.doi.org/10.1016/j.neuroscience.2014.08.042
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Fig. 2. Typical findings for click-evoked auditory brainstem response (ABR) in (A) macrophage migration inhibitory factor-deficient mice (MIF/ mice) at 3 months of age, (B) MIF/ mice at 18 months of age, and (C) wild-type mice at 18 months of age. (Horizontal bar, 1 ms; vertical bar, 1 lv).
Fig. 3. Hearing threshold by click-evoked auditory brainstem response (ABR) in MIF/ mice (open circles, n = 5) and wild-type mice (open boxes, n = 5). MIF/ mice showed significant hearing loss at 9, 12, and 18 months of age as compared with wild-type mice (mean ± standard deviation; ⁄P < 0.05).
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statistical significance. No significant findings were observed in the inner cochlear hair cells of the MIF/ mice at 1 month of age.
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SGC counts
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Loss of SGCs is considered a major factor in hearing loss (Kariya et al., 2007; Nadol, 2010). Therefore, we evaluated the number of SGCs under light microscopy in MIF/ mice and WT. Because the apex segment of the cochlea in some mice was not suitable for counting, we counted the nuclei of SGCs in three segments (mid-apex,
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mid-base, and extreme base). The number of SGCs in the MIF/ mice was lower than that in the control WT (Fig. 6), and the cell density of SGCs was significantly lower in the MIF/ mice than in the control WT (Fig. 7: mid-apex, P = 0.009; mid-base, P = 0.004; extreme base, P = 0.009). The SV and SL in the MIF/ mice showed morphologically normal findings under light microscopy.
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DISCUSSION
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MIF is a multi-functional molecule (Calandra and Roger, 2003). The anti-MIF antibody significantly suppresses tumor growth and tumor-associated angiogenesis (Nishihira, 2000). MIF acts as a counter-regulator to down-modulate the effects of glucocorticoids on the immune and endocrine systems (Petrovsky and Bucala, 2000). Schwann cells are a potential source of MIF protein in peripheral nerves, and MIF plays a critical role in the viability of Schwann cells by suppressing apoptosisrelated proteins such as p53 (Nishio et al., 2002). The same group also reported that down-regulation of MIF prevents detrimental secondary molecular responses in the injured spinal cord (Nishio et al., 2009). MIF is required for the differentiation of neural precursor cells in embryos of X. laevis, and for inner ear development in zebrafish (Suzuki et al., 2004; Shen et al., 2012). Recombinant MIF acts as a neurotrophin in promoting neuronal survival and is expressed in both the developing and mature inner ear of chicks and mice (Bank et al., 2012). This study clearly shows that MIF is expressed in the inner ear. Our findings suggest that MIF has an important role in age-related hearing loss in mice.
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Fig. 4. Morphology of cochlear hair cells on scanning electron microscopy in (A) MIF/ mice and (B) wild-type mice at 9 months of age. Outer cochlear hair cells were damaged in MIF/ mice (OHC, outer cochlear hair cells; IHC, inner cochlear hair cells. Scale bar = 10 lm).
Fig. 5. Loss of outer cochlear hair cells in MIF/ mice (open bar, n = 5) and wild-type mice (black bar, n = 5) at 9 months of age. The number of remaining outer cochlear cells in MIF/ mice was significantly less as compared with that of wild-type mice (WT, wild-type mice; KO, MIF/ mice; mean ± standard deviation; ⁄⁄P < 0.01).
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The use of several animal models for the study of progressive sensorineural hearing loss has been reported (Zhou et al., 2006). The audiological examination used in this study clearly showed that the down-regulation of MIF induces accelerated age-related hearing loss in BALB/c mice. We used click-evoked ABR to determine hearing thresholds. Click-evoked ABR is widely used for estimating hearing thresholds in both humans and experimental animals but only provides data in the 1–4-kHz range (Scherf et al., 2006; Zhou et al., 2006). A limitation of this study is that the hearing assessment is not frequency specific. Although the kind of stimulus waveform used for ABR measurements was not detailed, a recent study reported that 4-week-old MIF/ mice had hearing impairments at 12, 24, and 48 kHz (Bank et al., 2012). Our data using click-evoked ABR shows that there is no significant hearing loss in MIF/ mice younger than 6 months of age. This disparity may be due to differences in the stimulus sounds of ABR, because the click-evoked ABR used in this study cannot evaluate hearing thresholds at 12, 24, and 48 kHz. We also show in this study that
Fig. 6. Histopathological findings of the cochlea in (A) MIF/ mice and (B) wild-type mice at 9 months of age. Number of spiral ganglion cells was less in MIF/ mice, as compared with that of wild-type mice (hematoxylin and eosin staining; OC, organ of Corti; RM, Reissner’s membrane; SV, stria vascularis; SL, spiral ligament; SGC, spiral ganglion cell; bar = 100 lm).
the cochleae of 4-week-old MIF/ mice show some loss of outer hair cells, although the difference does not reach statistical significance as compared with WT. Age-related
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Fig. 7. Cell density of spiral ganglion cells in MIF/ mice (open bars, n = 5) and wild-type mice (black bars, n = 5) at 9 months of age in three segments of cochlea. The number of spiral ganglion cells in MIF/ mice were significantly affected, as compared with that of wild-type mice in mid-apex, mid-base, and extreme base segments (WT, wild-type mice; KO, MIF/ mice; mean ± standard deviation; ⁄⁄ P < 0.01). 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327
hearing loss often starts in the high-frequency range and later affects lower frequencies. Our findings show that young MIF/ mice have a normal hearing function in the 1–4-kHz range. Young MIF/ mice may have a hearing loss in the high-frequency range (12, 24, and 48 kHz), as reported in the previous study (Bank et al., 2012), and that hearing loss may indicate the early onset of agerelated hearing loss in MIF/ mice. SGCs are the first neurons in the auditory pathway, and play a critical role in sensorineural hearing function (Nadol, 2010). The loss of SGCs is a significant pathological finding in age-related hearing loss (Bao and Ohlemiller, 2010). The hair bundles of the inner hair cells respond primarily to basilar membrane velocity rather than displacement. The displacement of the basilar membrane stimulates the outer hair cells by bending their stereociliary bundles against the tectorial membrane. There are more (10–20) afferent fibers from the SGCs to the inner hair cells, as compared with the outer hair cells. In contrast, the efferent fibers with cell bodies in the brainstem contact only the outer hair cells. The outer hair cells have both sensory and motor capability, and the interactive network may be part of a feedback pathway to regulate cochlear sensitivity. Acoustic information is primarily transmitted to the central nervous system through electrical signals by the inner hair cells, whereas the main task of the outer hair cells is to boost the stimulus by electromechanical feedback. These findings show that the destruction of the outer hair cells results in a greatly elevated sound threshold and a deterioration of frequency selectivity (Fettiplace and Hackney, 2006). We clearly show here that the inner cochlear hair cells in MIF/ mice are relatively well preserved, while the outer cochlear hair cells are affected. A significant decrease in the number of SGCs is also observed in MIF/ mice. Aged MIF/ mice show a significant hearing loss, and cochlear dysfunction in MIF/ mice may result, in part, from the loss of outer hair cells and SGCs.
CONCLUSION
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Aged MIF mice show an accelerated age-related hearing loss. MIF may play a significant role in the
mammalian inner ear. Our findings suggest that MIF is an essential component of normal inner ear function in mice, particularly at advanced ages.
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Acknowledgments—This work was supported by JSPS KAKENHI (Grants-in-Aid for Scientific Research from The Ministry of Education, Culture, Sports, Science and Technology of Japan; grant number, 23791900 and 25462642).
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(Accepted 26 August 2014) (Available online xxxx)
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