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Chlorogenic acid rescues sensorineural auditory function in a diabetic animal model
Neuroscience Letters 640 (2017) 64–69
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Chlorogenic acid rescues sensorineural auditory function in a diabetic animal model Bin Na Hong a,b , Youn Hee Nam a,c , Sang Ho Woo a,c , Tong Ho Kang a,c,∗ a b c
College of Life Sciences, Kyung Hee University, Gyeonggi 446-701, Republic of Korea Department of Audiology, Nambu University, Gwangju 506-706, Republic of Korea Graduate School of Biotechnology, Kyung Hee University, Gyeonggi 446-701, Republic of Korea
h i g h l i g h t s • CA may improve damaged peripheral and central auditory function in the DM mouse model. • CA may aid in the recovery of OHCs damage in the DM mice cochlea and otic hair cells damage in AIZL. • The efficacy of CA in diabetic sensorineural auditory dysfunctions seems to be an independent action of CA, rather than an hypoglycemiant effect.
a r t i c l e
i n f o
Article history: Received 26 August 2016 Received in revised form 24 November 2016 Accepted 12 January 2017 Available online 16 January 2017 Keywords: Diabetic mellitus Chlorogenic acid Sensorineural auditory function Mice
a b s t r a c t Recently, many studies have reported that sensorineural hearing impairment related to neurological disorders may be caused by diabetes mellitus. However, to date, only a small number of studies have investigated the treatment of sensorineural hearing impairment. In the present study, the effects of chlorogenic acid on diabetic auditory pathway impairment were evaluated by neuro-electrical physiological measurements and morphological investigations. We have shown that CA efficiently prevents the progression of auditory pathway dysfunction caused by DM using auditory brainstem responses and auditory middle latency responses in mice. Additionally, using transient-evoked otoacoustic emissions measurement and scanning electron microscope observation of hair cells in DM mice, we found that CA may aid in the recovery from outer hair cell and otic hair cell damage. In conclusion, CA has beneficial effects for the management of diabetic sensorineural auditory dysfunction. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Recently, many studies have reported that sensorineural hearing impairment is a neurological disorder possibly caused by diabetes mellitus (DM) [1]. Additionally, DM was shown to be an independent risk factor for hearing impairment in a large population-based dataset study [2]. DM causes neuropathy and microvascular damage, especially affecting the peripheral arteries and nerves, kidneys, and retinas [3]. The cochlea and auditory nerves are likewise at risk [4]. Previous studies have reported thickened vessels of the stria vascularis, atro-
Abbreviations: DM, diabetes mellitus; CA, chlorogenic acid; ABRs, auditory brainstem responses; AMLRs, auditory middle latency responses; TEOAE, transient evoked optoacoustic emission; SEM, scanning electron microscope; GLM, glimepiride; AIZL, alloxan induced zebrafish larvae. ∗ Corresponding author at: Graduate School of Biotechnology, Department of Oriental Medicine Biotechnology, Kyung Hee University, 1732, Deogyeong-daero, Giheung-gu,Yongin-si, Gyeonggi-do, 17104, Republic of Korea. E-mail address: [email protected] (T.H. Kang). http://dx.doi.org/10.1016/j.neulet.2017.01.030 0304-3940/© 2017 Elsevier B.V. All rights reserved.
phy of the stria vascularis, and loss of outer hair cells (OHCs) in the cochlea [5]. Auditory neural changes have also been demonstrated by histologic studies revealing auditory nerve demyelination and spiral ganglion loss [6]. But few studies on preventing or treating diabetic sensorineural hearing impairments have been reported. Accordingly, this study investigated the anti-diabetic sensorineural auditory dysfunction efficacy of chlorogenic acid (CA) administration in a DM mouse model and a zebrafish model of alloxan-induced neuromast damage. To demonstrate the effect of CA in diabetic sensorineural auditory dysfunction, we performed electrophysiological auditory functional studies in a DM mouse model. Neurological evaluation of the auditory brainstem responses (ABRs) served to assess the integrity of the peripheral auditory nerve and the lower part of the brain. Furthermore, assessment of the auditory middle latency responses (AMLRs) provided useful insight into the neurological function of the higher central auditory nervous system. We also performed transient evoked otoacoustic emissions (TEOAEs) testing to directly measure cochlear function and used scanning
B.N. Hong et al. / Neuroscience Letters 640 (2017) 64–69
electron microscope (SEM) observation to directly measure the outer hair cells in DM mice. We confirmed the ameliorative action of CA using live image observation of neuromasts and otic hair cells in a zebrafish larvae model of alloxan-induced neuromast damage. Finally, in a comparative experiment with hypoglycemic agents, we verified whether the efficacy of CA was related to its glucoselowering effects.
2. Materials and methods All of the experimental procedures described herein were performed in accordance with the Principles of Laboratory Animal Care (NIH publication, #80-23, revised 1996) and the Animal Care and Use Guidelines of Nambu University, Republic of Korea. Seven-week-old male Lepr (+/+)C57BL/KsJ (dbdb) mice and Lepr(+/−)C57BL/KsJ mice (dbh) as the appropriate control were purchased from Jung-Ang Lab Animal (Seoul, Republic of Korea). The mice were housed individually at a 12-h light/dark cycle with food and water ad libitum. Normal hearing mice were divided into four groups (n = 10/group) as follows: eight-week-old adult male Lepr(+/−)C57BL/KsJ (dbh) littermates (Nor), Lepr (+/+)C57BL/KsJ (dbdb) mice (DM), and Lepr (+/+)C57BL/KsJ (dbdb) mice treated with 10 mg/kg (C10) and 20 mg/kg (C20) CA. CA treatments were performed once daily for 8 weeks beginning in mice that were 8 weeks old. Body weights and blood glucose levels were evaluated in 16week-old mice of four groups (n = 10/group) using a method described by Hong and Kang [7]. At 16 weeks, the ABR, AMLR, and TEOAE measurements of four groups (n = 10/group) were measured on anesthetized mice after i.m. administration of xylazine 0.43 mg/kg (Bayer Korea) and ketamine 4.57 mg/kg (Yuhan Co., Korea) using a method described by Hong and Kang [7]. Peripheral auditory functions were assessed through measurements of ABRs and AMLRs using GSI Audera (Viasys Healthcare Inc., USA) and cochlear function was assessed with TEOAEs using ILO analyzer (Otodynamics Co. Ltd., UK). After the auditory tests, all mice of four groups (n = 10/group) were sacrificed and the cochleae were prepared for scanning electron microscopy (SEM) measurements, as described previously [8]. The hair cells of the organ of Corti were observed using a Hitachi S-500 SEM (Tokyo, Japan). Adult zebrafish or larvae maintenance and embryo production were performed as described previously [9]. The zebrafish larvae were divided into four groups (n = 10/group) as follows: no treated zebrafish larvae (Nor), alloxan-induced zebrafish larvae (AIZL) (DM), AIZL treated with 10 M glimepiride (GLM), and AIZL treated with 10 M CA (CA). Five days post-fertilization (dpf), the zebrafish larvae of DM, GLM, and CA groups were treated with 100 M alloxan for 15 min, and then placed in 0.03% sea salt solution. After 6 h, the zebrafish larvae of GLM or CA groups, respectively, were treated with 10 M glimepiride (GLM) or 10 M CA for 12 h. Following treatment, the zebrafish larvae of four groups (n = 10/group) were stained with 0.1% YO-PRO for 30 min to identify apoptotic cells and the morphologies of the lateral line neuromasts and otic hair cells were analyzed under a fluorescence microscope using a method described [9]. Data were analyzed using the Prism 5 Statistical Software package (GraphPad, San Diego, CA, USA). All data are expressed as mean ± standard error of the mean. Statistical comparisons between all groups were performed using a one-way ANOVA test with a Dunnett post hoc test. P values of <0.05, 0.01, and 0.001 were considered significant.
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3. Results To investigate the anti-diabetes efficacy of CA, we evaluated body weights and blood glucose levels in 16-week-old mice (Fig. 1). The DM group exhibited significantly increased body weights and blood glucose levels compared with the Normal group. However, the body weights in the C20 group showed significantly decreased compared with DM group. Mice in the CA treatment groups exhibited decreased blood glucose levels compared with DM group. These results demonstrate that CA improved the hyperglycemia observed in the diabetic mouse model. To detect any improvement mediated by CA in peripheral auditory function damaged by DM, hearing threshold and latency tests were performed using ABRs. Hearing thresholds or latencies in response to clicks, 4-kHz TBs and 8-kHz TBs in the DM group were significantly higher or delayed compared with the Normal group, respectively. Hearing thresholds or latencies of the CA treatment groups decreased significantly compared to the DM group (Fig. 2AF). These data indicate that CA may improve damaged peripheral auditory function in the DM mouse model. In order to investigate any protective effects of CA against abnormalities in central auditory functions, AMLR amplitudes and latencies were measured. Pa latencies and the Na-Pa amplitudes of the AMLRs in the DM group were significantly delayed and lower compared with the Normal groups. However, in the CA treatment groups, Pa latencies decreased and Na-Pa amplitudes increased compared with DM group to an extent similar to that observed in the Normal group. These data indicate that the central auditory pathway may become affected in diabetic mice over time, whereas CA may suppress central auditory pathway dysfunction in DM mice models (Fig. 3A, B). To assess the efficacy of CA against the normality of cochlear function and morphological differences in the DM mouse model, TEOAE tests and SEM measurements were performed. TEOAE SNRs of the DM group were significantly lower compared with the Normal group. The results of the morphological analysis of hair cells in the organ of Corti performed using SEM were similar to the physiological findings. Damage to the stereocilia of OHCs occurred in the DM mice. In the CA treatment groups, TEOAE SNRs were increased significantly in a dose-dependent manner excluding 4 kHz TB stimulus (Fig. 4A, B, C). Also, the morphologic results of OHCs in mice treated with CA were similar to those of the Normal mice (Fig. 4D). The concordance between these morphological and physiological data suggests that CA may aid in the recovery of OHC damage in the cochlea. In order to morphologically confirm the efficacy of CA, lateral line neuromasts and otic hair cells were observed in AIZL using live images obtained with a fluorescence microscope. In AIZL, damage to lateral line neuromasts and otic hair cells was observed. However, in the CA-treated zebrafish, number of trunk neuromasts increased significantly compared with the number in DM zebrafish (Fig. 5A, B). The number of otic hair cells also increased and the increased distance between each hair cell decreased significantly compared with DM zebrafish (Fig. 5C–E). We confirmed the ameliorative action of CA on alloxan induced damage to neuromast and otic hair cells. In order to determine whether the effect of the CA was mediated through a glucose-lowering effect, a comparative experiment using zebrafish larvae treated with GLM, a hypoglycemic agent, was performed. GLM-treated zebrafish larvae showed no significant difference compare with animals in the alloxan-treated group with respect to the number of trunk neuromasts and the number of otic hair cells or the increased distance between hair cells.
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B.N. Hong et al. / Neuroscience Letters 640 (2017) 64–69
Body weight
weight (g)
60 40
*** ***
20 0 Normal
DM
C10
C20
blood glucose level (mg/dl)
Blood glucose level
80
400 300 200
* **
100 0 Normal
DM
C10
C20
Fig. 1. Body weights and glucose levels in diabetic mice. Body weights and blood glucose levels were measured in the following groups: dbh mice (Normal), db/db mice (DM), db/db mice treated with chlorogenic acid 10 mg/kg (C10), and db/db mice treated with chlorogenic acid 20 mg/kg (C20). Data are shown as means ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate significant differences from the DM group.
Fig. 2. ABR hearing thresholds in response to stimulation with clicks, 4-kHz TBs, or 8-kHz TBs were measured (A,C,E). Wave I and IV absolute latencies of ABRs after stimulation with either clicks, 4-kHz TBs, or 8-kHz TBs were measured (B,D,F). Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate significant differences from the DM group.
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Fig. 3. Pa latencies (A) and Na-Pa amplitudes (B) of the AMLRs were measured. Data are expressed as mean ± SEM. * p < 0.05 and ** p < 0.01 indicate significant differences from the DM group.
Fig. 4. Ratios of the signal intensity to noise for TEOAEs in the analyzed response to 2-kHz TBs, 3-kHz TBs and 4-kHz TBs were measured (A,B,C). Results of SEM in Normal, DM, and C20 animals at 16 weeks of age (D). Data are expressed as mean ± SEM. *p < 0.05, ** p < 0.01 and *** p < 0.001 indicate a significant difference from the DM group.
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Fig. 5. Number of trunk neuromasts (A), live images of lateral line neuromasts (B), number of otic hair cells (C), the increased distance between hair cells (D), and live images of otic hair cells (E) in zebrafish larvae. Data are expressed as mean ± SEM. * p < 0.05 and *** p < 0.001 indicate a significant difference from the DM group. ## p < 0.01 and. ### p < 0.001 indicate a significant difference from the NOR group.
4. Discussion This comprehensive neuro-electrophysiological and morphological study demonstrates for the first time that CA ameliorates the sensorineural auditory dysfunction caused by DM in the db/db mouse model. The db/db mouse was used as spontaneous type 2 diabetic animal model. It becomes hyperphagic and hyperinsulinemic, insulin resistant early in life (within 2 weeks of age), then develops obesity at the age of 3–4 weeks [10]. To investigate the therapeutic actions of CA in diabetic sensorineural auditory dysfunction, auditory functions have been assessed by ABRs, AMLRs, and TEOAEs. Increased ABR or AMLR latencies in diabetic mice have been interpreted as evidence of neuropathy, central and/or peripheral, as the basis of auditory dysfunction [11]. The availability of TEOAEs to directly investigate
cochlear function, particularly outer hair cell function, has also suggested possible impairment in this organ [12]. CA resulted in auditory function improvement in hair cells of the cochlear and peripheral and central auditory pathways damaged by DM. The reduced hearing thresholds in ABRs of the CA treated group are an exemplary result showing an improvement in peripheral hearing function. Additionally, the decreased latencies of the CA treated group in ABRs demonstrated that the auditory nervous system damage caused by DM was suppressed. Also, the decreased latencies and increased amplitudes in AMLRs of the CA treated group indicated an improvement in the auditory central nervous system. In order to morphologically confirm the efficacy of CA, lateral line neuromasts and otic hair cells were observed in AIZL using live images. Alloxan is a cytotoxic agent that causes pancreatic -cell
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necrosis, which in turn decreases -cell mass and blocks insulin secretion to induce diabetes [10]. Physiologically, the lateral line neuromasts of zebrafish are a system of sense organs similar to human peripheral nerves. Also, otic hair cells of zebrafish are very similar to inner ear hair cells [13]. In this study, the observation of lateral line neuromasts in zebrafish might be support data of peripheral auditory nerves in mice. Unlike the results of TEOAE on CA efficacy in mice, there was no significant CA improvement effect on otic hair cell numbers in zebrafish. Morphological results of SEM showed that OHC damaged by DM improved by CA in mice. Likewise, otic hair cells damaged by DM seems to have improved morphologically by CA in zebrafish. Future studies will require morphological observation of otic hair cells rather than otic hair cell numbers in zebrafish. Evidence for the pathophysiological mechanisms by which diabetes may influence sensorineural hearing impairment continues to evolve. Numerous studies have demonstrated diabetic changes in the stria vascularis, basilar membrane, and hair cells of the cochlea [14,15]. The stria vascularis, which produces endolymph in scala media, is unique to the inner ear because of its rich microvasculature, making it particularly susceptible to diabetic microangiopathy. A previous study showed that CA increases vascular endothelial growth factor (VEGF) expression, resulting in improved microvascular perfusion in DM rats [16]. The improvement efficacy of CA with respect to cochlear dysfunction and morphological damage of hair cells in our study might be related to the microvascular perfusion improvement of CA. Hyperglycemia is known to adversely affect from the peripheral nerve to the central nervous system (CNS) with microglial activation [17]. Also, hyperglycemia is associated with reduced neuronal size and reduced dendritic branching and spines in the brains [18]. Dendritic branch atrophy and spine loss have previously been associated with cognitive dysfunction [19]. The chronic activation of microglia may in turn cause neuronal damage through the release of potentially cytotoxic molecules such as proinflammatory cytokines, reactive oxygen intermediates, proteinases and complement proteins [20]. Recent studies have reported that microglia cells are also distributed in cochlear [21]. Therefore, suppression of microglia-mediated inflammation has been considered as an important strategy in neurodegenerative disease therapy. In previous reports, the pretreatment of CA significantly inhibits the microglial activation, and CA may be neuroprotective for proinflammatory factor-mediated neurodegenerative disorders [22]. The neuroprotection efficacy of CA may be related to the effect of CA on the damaged auditory nervous system in our study. We suggest that the various actions of CA affect the complex mechanism of diabetic sensorineural auditory dysfunctions. Finally, we wanted to investigate whether the hypoglycemia efficacy of CA improved diabetic sensorineural auditory dysfunction using zebrafish larvae treated with GLM, a known hypoglycemic agent. GLM-treated zebrafish larvae did not show any effect in lateral line neuromasts and otic hair cells. These data indicate that the ameliorative efficacy of CA in diabetic sensorineural auditory dysfunctions seems to be an independent action of CA, rather than an effect of CA on hypoglycemia. Taken together, our data suggest that CA affects both the peripheral and central auditory systems in DM mice. Ultimately, CA has beneficial effects for the management of diabetic sensorineural auditory dysfunctions. Further studies will be necessary to clarify the mechanism of CA efficacy. Also, the higher frequency evaluation in ABR and OAE to appropriate audible frequency of animal is needed in future study. Conflict of interest The authors have no conflicts of interest to declare.
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Acknowledgements This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF2015R1D1A1A09060469 and NRF-2015R1D1A1A01056702). References [1] M. Acar, Z. Aycan, B. Acar, U. Ertan, H.N. Peltek, R. Karasen, Audiologic evaluation in pediatric patients with type 1 diabetes mellitus, J. Pediatr Endocrinol Metab. 25 (2012) 503–508. [2] K.E. Bainbridge, H.J. Hoffman, C.C. Cowie, Diabetes and hearing impairment in the United States: audiometric evidence from the National Health and Nutrition Examination Survey, 1999 to 2004, Ann. Intern. Med. 149 (2008) 1–10. [3] National Diabetes Data Group, NIoDaDaKD, Diabetes in America, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, 1995. [4] H. Fukushima, S. Cureoglu, P.A. Schachern, M.M. Paparella, T. Harada, M.F. Oktay, Effects of type 2 diabetes mellitus on cochlear structure in humans, Arch. Otolaryngol. Head Neck Surg. 132 (9) (2006) 934–938. [5] H. Fukushima, S. Cureoglu, P.A. Schachern, M.M. Paparella, T. Harada, M.F. Oktay, Effects of type 2 diabetes mellitus on cochlear structure in humans, Arch. Otolaryngol. Head Neck Surg. 132 (2006) 934–938. [6] K. Makishima, K. Tanaka, Pathological changes of the inner ear and central auditory pathway in diabetics, Ann. Otol. Rhinol. Laryngol. 80 (2) (1971) 218–228. [7] B.N. Hong, T.H. Yi, R. Park, S.Y. Kim, T.H. Kang, Coffee improves auditory neuropathy in diabetic mice, Neurosci. Lett. 441 (2008) 302–306. [8] K. Denman-Johnson, A. Forge, Establishment of hair bundle polarity and orientation in the developing vestibular system of the mouse, J. Neurocytol. 28 (1999) 821–835. [9] Y.H. Nam, B.N. Hong, I. Rodriguez, M.G. Ji, K. Kim, U.J. Kim, T.H. Kang, Synergistic potentials of coffee on injured pancreatic islets and insulin action via KATP channel blocking in zebrafish, J. Agric. Food Chem. 63 (23) (2015) 5612–5621. [10] A.J. King, The use of animal models in diabetes research, Br. J. Pharmacol. 166 (2012) 877–894. [11] B.N. Hong, T.H. Kang, Distinction between auditory electrophysiological responses in type 1 and type 2 diabetic animal models, Neurosci. Lett. 566 (2014) 309–314. [12] F. Ottaviani, N. Dozio, C.B. Neglia, S. Riccio, M. Scavini, Absence of otoacoustic emissions in insulin-dependent diabetic patients: is there evidence for diabetic cochleopathy? J. Diabetes Complicat. 16 (2002) 338–343. [13] K.N. Owens, D.E. Cunningham, G. MacDonald, E.W. Rubel, D.W. Raible, R. Pujol, Ultrastructural analysis of aminoglycoside-induced hair cell death in the zebrafish lateral line reveals an early mitochondrial response, J. Comp. Neurol. 502 (4) (2007) 522–543. [14] S. Nakae, M. Tachibana, The cochlea of the spontaneously diabetic mouse. II. Electron microscopic observations of non-obese diabetic mice, Arch. Otorhinolaryngol. 243 (5) (1986) 313–316. [15] E. Raynor, W.G. Robison, C.G. Garrett, W.T. McGuirt, H.C. Pillsbury, J. Prazma, Consumption of a high-galactose diet induces diabeticlike changes in the inner ear, Otolaryngol. Head Neck Surg. 113 (6) (1995) 748–754. [16] D. Bagdas, B. Cam Etoz, S. Inan Ozturkoglu, N. Cinkilic, M.O. Ozyigit, Z. Gul, N.I. Buyukcoskun, K. Ozluk, M.S. Gurun, Effects of systemic chlorogenic acid on random-pattern dorsal skin flap survival in diabetic rats, Biol. Pharm. Bull. 37 (3) (2014) 361–370. [17] R. Sonneville, H.M. den Hertog, F. Guiza, J. Gunst, I. Derese, P.J. Wouters, J.P. Brouland, A. Polito, F. Gray, F. Chretien, P. Charlier, D. Annane, T. Sharshar, G. Van den Berghe, I. Vanhorebeek, Impact of hyperglycemia on neuropathological alterations during critical illness, J. Clin. Endocrinol. Metab. 97 (6) (2012) 2113–2123. [18] J.I. Malone, S. Hanna, S. Saporta, R.F. Mervis, C.R. Park, L. Chong, D.M. Diamond, Hyperglycemia not hypoglycemia alters neuronal dendrites and impairs spatial memory, Pediatr. Diabetes 9 (6) (2008) 531–539. [19] R. Beeri, N. Le Novere, R. Mervis, Enhanced hemicholinium binding and attenuated dendrite branching in cognitively impaired acetylcholinesterase-transgenic mice, J. Neurochem. 69 (1997) 2441–2451. [20] S.T. Dheen, C. Kaur, E.A. Ling, Microglial activation and its implications in the brain diseases, Curr. Med. Chem. 14 (11) (2007) 1189–1197. [21] J.T. O’Malley, J.B. Nadol Jr., M.J. McKenna, Anti CD163+ Iba1+, and CD68+ cells in the adult human inner ear: normal distribution of an unappreciated class of macrophages/microglia and implications for inflammatory otopathology in humans, Otol. Neurotol. 37 (1) (2016) 99–108. [22] W. Shen, R. Qi, J. Zhang, Z. Wang, H. Wang, C. Hu, Y. Zhao, M. Bie, Y. Wang, Y. Fu, M. Chen, D. Lu, Chlorogenic acid inhibits LPS-induced microglial activation and improves survival of dopaminergic neurons, Brain Res. Bull. 88 (5) (2012) 487–494.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Chlorogenic acid rescues sensorineural auditory function in a diabetic animal model Bin Na Hong a,b , Youn Hee Nam a,c , Sang Ho Woo a,c , Tong Ho Kang a,c,∗ a b c
College of Life Sciences, Kyung Hee University, Gyeonggi 446-701, Republic of Korea Department of Audiology, Nambu University, Gwangju 506-706, Republic of Korea Graduate School of Biotechnology, Kyung Hee University, Gyeonggi 446-701, Republic of Korea
h i g h l i g h t s • CA may improve damaged peripheral and central auditory function in the DM mouse model. • CA may aid in the recovery of OHCs damage in the DM mice cochlea and otic hair cells damage in AIZL. • The efficacy of CA in diabetic sensorineural auditory dysfunctions seems to be an independent action of CA, rather than an hypoglycemiant effect.
a r t i c l e
i n f o
Article history: Received 26 August 2016 Received in revised form 24 November 2016 Accepted 12 January 2017 Available online 16 January 2017 Keywords: Diabetic mellitus Chlorogenic acid Sensorineural auditory function Mice
a b s t r a c t Recently, many studies have reported that sensorineural hearing impairment related to neurological disorders may be caused by diabetes mellitus. However, to date, only a small number of studies have investigated the treatment of sensorineural hearing impairment. In the present study, the effects of chlorogenic acid on diabetic auditory pathway impairment were evaluated by neuro-electrical physiological measurements and morphological investigations. We have shown that CA efficiently prevents the progression of auditory pathway dysfunction caused by DM using auditory brainstem responses and auditory middle latency responses in mice. Additionally, using transient-evoked otoacoustic emissions measurement and scanning electron microscope observation of hair cells in DM mice, we found that CA may aid in the recovery from outer hair cell and otic hair cell damage. In conclusion, CA has beneficial effects for the management of diabetic sensorineural auditory dysfunction. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Recently, many studies have reported that sensorineural hearing impairment is a neurological disorder possibly caused by diabetes mellitus (DM) [1]. Additionally, DM was shown to be an independent risk factor for hearing impairment in a large population-based dataset study [2]. DM causes neuropathy and microvascular damage, especially affecting the peripheral arteries and nerves, kidneys, and retinas [3]. The cochlea and auditory nerves are likewise at risk [4]. Previous studies have reported thickened vessels of the stria vascularis, atro-
Abbreviations: DM, diabetes mellitus; CA, chlorogenic acid; ABRs, auditory brainstem responses; AMLRs, auditory middle latency responses; TEOAE, transient evoked optoacoustic emission; SEM, scanning electron microscope; GLM, glimepiride; AIZL, alloxan induced zebrafish larvae. ∗ Corresponding author at: Graduate School of Biotechnology, Department of Oriental Medicine Biotechnology, Kyung Hee University, 1732, Deogyeong-daero, Giheung-gu,Yongin-si, Gyeonggi-do, 17104, Republic of Korea. E-mail address: [email protected] (T.H. Kang). http://dx.doi.org/10.1016/j.neulet.2017.01.030 0304-3940/© 2017 Elsevier B.V. All rights reserved.
phy of the stria vascularis, and loss of outer hair cells (OHCs) in the cochlea [5]. Auditory neural changes have also been demonstrated by histologic studies revealing auditory nerve demyelination and spiral ganglion loss [6]. But few studies on preventing or treating diabetic sensorineural hearing impairments have been reported. Accordingly, this study investigated the anti-diabetic sensorineural auditory dysfunction efficacy of chlorogenic acid (CA) administration in a DM mouse model and a zebrafish model of alloxan-induced neuromast damage. To demonstrate the effect of CA in diabetic sensorineural auditory dysfunction, we performed electrophysiological auditory functional studies in a DM mouse model. Neurological evaluation of the auditory brainstem responses (ABRs) served to assess the integrity of the peripheral auditory nerve and the lower part of the brain. Furthermore, assessment of the auditory middle latency responses (AMLRs) provided useful insight into the neurological function of the higher central auditory nervous system. We also performed transient evoked otoacoustic emissions (TEOAEs) testing to directly measure cochlear function and used scanning
B.N. Hong et al. / Neuroscience Letters 640 (2017) 64–69
electron microscope (SEM) observation to directly measure the outer hair cells in DM mice. We confirmed the ameliorative action of CA using live image observation of neuromasts and otic hair cells in a zebrafish larvae model of alloxan-induced neuromast damage. Finally, in a comparative experiment with hypoglycemic agents, we verified whether the efficacy of CA was related to its glucoselowering effects.
2. Materials and methods All of the experimental procedures described herein were performed in accordance with the Principles of Laboratory Animal Care (NIH publication, #80-23, revised 1996) and the Animal Care and Use Guidelines of Nambu University, Republic of Korea. Seven-week-old male Lepr (+/+)C57BL/KsJ (dbdb) mice and Lepr(+/−)C57BL/KsJ mice (dbh) as the appropriate control were purchased from Jung-Ang Lab Animal (Seoul, Republic of Korea). The mice were housed individually at a 12-h light/dark cycle with food and water ad libitum. Normal hearing mice were divided into four groups (n = 10/group) as follows: eight-week-old adult male Lepr(+/−)C57BL/KsJ (dbh) littermates (Nor), Lepr (+/+)C57BL/KsJ (dbdb) mice (DM), and Lepr (+/+)C57BL/KsJ (dbdb) mice treated with 10 mg/kg (C10) and 20 mg/kg (C20) CA. CA treatments were performed once daily for 8 weeks beginning in mice that were 8 weeks old. Body weights and blood glucose levels were evaluated in 16week-old mice of four groups (n = 10/group) using a method described by Hong and Kang [7]. At 16 weeks, the ABR, AMLR, and TEOAE measurements of four groups (n = 10/group) were measured on anesthetized mice after i.m. administration of xylazine 0.43 mg/kg (Bayer Korea) and ketamine 4.57 mg/kg (Yuhan Co., Korea) using a method described by Hong and Kang [7]. Peripheral auditory functions were assessed through measurements of ABRs and AMLRs using GSI Audera (Viasys Healthcare Inc., USA) and cochlear function was assessed with TEOAEs using ILO analyzer (Otodynamics Co. Ltd., UK). After the auditory tests, all mice of four groups (n = 10/group) were sacrificed and the cochleae were prepared for scanning electron microscopy (SEM) measurements, as described previously [8]. The hair cells of the organ of Corti were observed using a Hitachi S-500 SEM (Tokyo, Japan). Adult zebrafish or larvae maintenance and embryo production were performed as described previously [9]. The zebrafish larvae were divided into four groups (n = 10/group) as follows: no treated zebrafish larvae (Nor), alloxan-induced zebrafish larvae (AIZL) (DM), AIZL treated with 10 M glimepiride (GLM), and AIZL treated with 10 M CA (CA). Five days post-fertilization (dpf), the zebrafish larvae of DM, GLM, and CA groups were treated with 100 M alloxan for 15 min, and then placed in 0.03% sea salt solution. After 6 h, the zebrafish larvae of GLM or CA groups, respectively, were treated with 10 M glimepiride (GLM) or 10 M CA for 12 h. Following treatment, the zebrafish larvae of four groups (n = 10/group) were stained with 0.1% YO-PRO for 30 min to identify apoptotic cells and the morphologies of the lateral line neuromasts and otic hair cells were analyzed under a fluorescence microscope using a method described [9]. Data were analyzed using the Prism 5 Statistical Software package (GraphPad, San Diego, CA, USA). All data are expressed as mean ± standard error of the mean. Statistical comparisons between all groups were performed using a one-way ANOVA test with a Dunnett post hoc test. P values of <0.05, 0.01, and 0.001 were considered significant.
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3. Results To investigate the anti-diabetes efficacy of CA, we evaluated body weights and blood glucose levels in 16-week-old mice (Fig. 1). The DM group exhibited significantly increased body weights and blood glucose levels compared with the Normal group. However, the body weights in the C20 group showed significantly decreased compared with DM group. Mice in the CA treatment groups exhibited decreased blood glucose levels compared with DM group. These results demonstrate that CA improved the hyperglycemia observed in the diabetic mouse model. To detect any improvement mediated by CA in peripheral auditory function damaged by DM, hearing threshold and latency tests were performed using ABRs. Hearing thresholds or latencies in response to clicks, 4-kHz TBs and 8-kHz TBs in the DM group were significantly higher or delayed compared with the Normal group, respectively. Hearing thresholds or latencies of the CA treatment groups decreased significantly compared to the DM group (Fig. 2AF). These data indicate that CA may improve damaged peripheral auditory function in the DM mouse model. In order to investigate any protective effects of CA against abnormalities in central auditory functions, AMLR amplitudes and latencies were measured. Pa latencies and the Na-Pa amplitudes of the AMLRs in the DM group were significantly delayed and lower compared with the Normal groups. However, in the CA treatment groups, Pa latencies decreased and Na-Pa amplitudes increased compared with DM group to an extent similar to that observed in the Normal group. These data indicate that the central auditory pathway may become affected in diabetic mice over time, whereas CA may suppress central auditory pathway dysfunction in DM mice models (Fig. 3A, B). To assess the efficacy of CA against the normality of cochlear function and morphological differences in the DM mouse model, TEOAE tests and SEM measurements were performed. TEOAE SNRs of the DM group were significantly lower compared with the Normal group. The results of the morphological analysis of hair cells in the organ of Corti performed using SEM were similar to the physiological findings. Damage to the stereocilia of OHCs occurred in the DM mice. In the CA treatment groups, TEOAE SNRs were increased significantly in a dose-dependent manner excluding 4 kHz TB stimulus (Fig. 4A, B, C). Also, the morphologic results of OHCs in mice treated with CA were similar to those of the Normal mice (Fig. 4D). The concordance between these morphological and physiological data suggests that CA may aid in the recovery of OHC damage in the cochlea. In order to morphologically confirm the efficacy of CA, lateral line neuromasts and otic hair cells were observed in AIZL using live images obtained with a fluorescence microscope. In AIZL, damage to lateral line neuromasts and otic hair cells was observed. However, in the CA-treated zebrafish, number of trunk neuromasts increased significantly compared with the number in DM zebrafish (Fig. 5A, B). The number of otic hair cells also increased and the increased distance between each hair cell decreased significantly compared with DM zebrafish (Fig. 5C–E). We confirmed the ameliorative action of CA on alloxan induced damage to neuromast and otic hair cells. In order to determine whether the effect of the CA was mediated through a glucose-lowering effect, a comparative experiment using zebrafish larvae treated with GLM, a hypoglycemic agent, was performed. GLM-treated zebrafish larvae showed no significant difference compare with animals in the alloxan-treated group with respect to the number of trunk neuromasts and the number of otic hair cells or the increased distance between hair cells.
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Body weight
weight (g)
60 40
*** ***
20 0 Normal
DM
C10
C20
blood glucose level (mg/dl)
Blood glucose level
80
400 300 200
* **
100 0 Normal
DM
C10
C20
Fig. 1. Body weights and glucose levels in diabetic mice. Body weights and blood glucose levels were measured in the following groups: dbh mice (Normal), db/db mice (DM), db/db mice treated with chlorogenic acid 10 mg/kg (C10), and db/db mice treated with chlorogenic acid 20 mg/kg (C20). Data are shown as means ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate significant differences from the DM group.
Fig. 2. ABR hearing thresholds in response to stimulation with clicks, 4-kHz TBs, or 8-kHz TBs were measured (A,C,E). Wave I and IV absolute latencies of ABRs after stimulation with either clicks, 4-kHz TBs, or 8-kHz TBs were measured (B,D,F). Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate significant differences from the DM group.
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Fig. 3. Pa latencies (A) and Na-Pa amplitudes (B) of the AMLRs were measured. Data are expressed as mean ± SEM. * p < 0.05 and ** p < 0.01 indicate significant differences from the DM group.
Fig. 4. Ratios of the signal intensity to noise for TEOAEs in the analyzed response to 2-kHz TBs, 3-kHz TBs and 4-kHz TBs were measured (A,B,C). Results of SEM in Normal, DM, and C20 animals at 16 weeks of age (D). Data are expressed as mean ± SEM. *p < 0.05, ** p < 0.01 and *** p < 0.001 indicate a significant difference from the DM group.
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Fig. 5. Number of trunk neuromasts (A), live images of lateral line neuromasts (B), number of otic hair cells (C), the increased distance between hair cells (D), and live images of otic hair cells (E) in zebrafish larvae. Data are expressed as mean ± SEM. * p < 0.05 and *** p < 0.001 indicate a significant difference from the DM group. ## p < 0.01 and. ### p < 0.001 indicate a significant difference from the NOR group.
4. Discussion This comprehensive neuro-electrophysiological and morphological study demonstrates for the first time that CA ameliorates the sensorineural auditory dysfunction caused by DM in the db/db mouse model. The db/db mouse was used as spontaneous type 2 diabetic animal model. It becomes hyperphagic and hyperinsulinemic, insulin resistant early in life (within 2 weeks of age), then develops obesity at the age of 3–4 weeks [10]. To investigate the therapeutic actions of CA in diabetic sensorineural auditory dysfunction, auditory functions have been assessed by ABRs, AMLRs, and TEOAEs. Increased ABR or AMLR latencies in diabetic mice have been interpreted as evidence of neuropathy, central and/or peripheral, as the basis of auditory dysfunction [11]. The availability of TEOAEs to directly investigate
cochlear function, particularly outer hair cell function, has also suggested possible impairment in this organ [12]. CA resulted in auditory function improvement in hair cells of the cochlear and peripheral and central auditory pathways damaged by DM. The reduced hearing thresholds in ABRs of the CA treated group are an exemplary result showing an improvement in peripheral hearing function. Additionally, the decreased latencies of the CA treated group in ABRs demonstrated that the auditory nervous system damage caused by DM was suppressed. Also, the decreased latencies and increased amplitudes in AMLRs of the CA treated group indicated an improvement in the auditory central nervous system. In order to morphologically confirm the efficacy of CA, lateral line neuromasts and otic hair cells were observed in AIZL using live images. Alloxan is a cytotoxic agent that causes pancreatic -cell
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necrosis, which in turn decreases -cell mass and blocks insulin secretion to induce diabetes [10]. Physiologically, the lateral line neuromasts of zebrafish are a system of sense organs similar to human peripheral nerves. Also, otic hair cells of zebrafish are very similar to inner ear hair cells [13]. In this study, the observation of lateral line neuromasts in zebrafish might be support data of peripheral auditory nerves in mice. Unlike the results of TEOAE on CA efficacy in mice, there was no significant CA improvement effect on otic hair cell numbers in zebrafish. Morphological results of SEM showed that OHC damaged by DM improved by CA in mice. Likewise, otic hair cells damaged by DM seems to have improved morphologically by CA in zebrafish. Future studies will require morphological observation of otic hair cells rather than otic hair cell numbers in zebrafish. Evidence for the pathophysiological mechanisms by which diabetes may influence sensorineural hearing impairment continues to evolve. Numerous studies have demonstrated diabetic changes in the stria vascularis, basilar membrane, and hair cells of the cochlea [14,15]. The stria vascularis, which produces endolymph in scala media, is unique to the inner ear because of its rich microvasculature, making it particularly susceptible to diabetic microangiopathy. A previous study showed that CA increases vascular endothelial growth factor (VEGF) expression, resulting in improved microvascular perfusion in DM rats [16]. The improvement efficacy of CA with respect to cochlear dysfunction and morphological damage of hair cells in our study might be related to the microvascular perfusion improvement of CA. Hyperglycemia is known to adversely affect from the peripheral nerve to the central nervous system (CNS) with microglial activation [17]. Also, hyperglycemia is associated with reduced neuronal size and reduced dendritic branching and spines in the brains [18]. Dendritic branch atrophy and spine loss have previously been associated with cognitive dysfunction [19]. The chronic activation of microglia may in turn cause neuronal damage through the release of potentially cytotoxic molecules such as proinflammatory cytokines, reactive oxygen intermediates, proteinases and complement proteins [20]. Recent studies have reported that microglia cells are also distributed in cochlear [21]. Therefore, suppression of microglia-mediated inflammation has been considered as an important strategy in neurodegenerative disease therapy. In previous reports, the pretreatment of CA significantly inhibits the microglial activation, and CA may be neuroprotective for proinflammatory factor-mediated neurodegenerative disorders [22]. The neuroprotection efficacy of CA may be related to the effect of CA on the damaged auditory nervous system in our study. We suggest that the various actions of CA affect the complex mechanism of diabetic sensorineural auditory dysfunctions. Finally, we wanted to investigate whether the hypoglycemia efficacy of CA improved diabetic sensorineural auditory dysfunction using zebrafish larvae treated with GLM, a known hypoglycemic agent. GLM-treated zebrafish larvae did not show any effect in lateral line neuromasts and otic hair cells. These data indicate that the ameliorative efficacy of CA in diabetic sensorineural auditory dysfunctions seems to be an independent action of CA, rather than an effect of CA on hypoglycemia. Taken together, our data suggest that CA affects both the peripheral and central auditory systems in DM mice. Ultimately, CA has beneficial effects for the management of diabetic sensorineural auditory dysfunctions. Further studies will be necessary to clarify the mechanism of CA efficacy. Also, the higher frequency evaluation in ABR and OAE to appropriate audible frequency of animal is needed in future study. Conflict of interest The authors have no conflicts of interest to declare.
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