소음에 의한 와우 손상의 약리학적 치료
Pharmacological Management of Noise-induced Cochlear Injury
- 주제(키워드) Noise , Hearing loss , Reactive oxygen species , NADPH oxidase , Mitochondria , Cysteinyl leukotriene , Pravastatin , Methylene blue , Montelukast
- 발행기관 아주대학교
- 지도교수 박상면
- 발행년도 2014
- 학위수여년월 2014. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000016195
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Noise-induced hearing loss (NIHL) has become one of the most common occupational disease in both developing and developed countries and is also the major contributing factor of age-related hearing loss. NIHL is uniquely preventable sensorineural hearing loss and researchers have investigated additional preventive strategy based on the discovered pathogenesis of NIHL. The pathogenetic mechanisms of NIHL are considered as multifactorial such as glutamate excitotoxicity, ischemia-reperfusion injury, ATP depletion and reactive oxygen species (ROS) formation. Among them, I determined that blockade of ROS formation is one of the most efficient targets of prevention using an animal model of NIHL. Firstly I reported that noise exposure induced superoxide formation through the activation of NADPH oxidase complex and inhibition of complex formation by pravastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which is a rate-limiting enzyme of cholesterol synthesis, before noise exposure protected against cochlear injury in BALB/c mice. Noise exposure produced both compound threshold shift (CTS) and permanent threshold shift (PTS) over 40 dB at 16 and 32 kHz. Pretreatment with pravastatin (25 mg/kg) for 5 days significantly decreased both CTS and PTS. Pravastatin also reduced hair cell death after noise exposure in the cochlea which was identified by surface preparation and scanning electron microscopy (SEM). It also reduced the formation of noise-induced 4-hydroxynonenal (4-HNE), a byproduct of lipid peroxidation. Activation of Rac1, one of the subunits of the NADPH oxidase complex was inhibited by the administration of pravastatin due to its pleiotropic effect. Secondly, I also showed that methylene blue (MB) pretreatment for 4 consecutive days significantly decreased both CTS and PTS after exposure to intense broad band noise for 3 h. MB reduced outer hair cell (OHC) death in the cochlea that was identified by surface preparation and SEM, and it also reduced ROS and RNS formation after noise exposure. MB significantly protected against rotenone- and antimycin A-induced cell death, and also reversed ATP generation in vitro. Furthermore, MB effectively attenuated noise-induced impairment of complex IV activity. MB also increased the neurotrophin-3 (NT-3) level which could affect synaptic connections between hair cells and spiral ganglion neurons in the noise-exposed cochlea. MB promoted the conservation of both efferent and afferent nerve terminals on the OHC and inner hair cells. Finally, I found the expression of cysteinyl leukotriene type 1 receptor (CysLT1) were increased at 3 days after noise exposure, but not in control mice. Enhanced CysLT1 expression was mainly occurred in the spiral ligament and the organ of Corti. Expression pattern of upstream enzyme 5-lipoxygenase was similar to CysLT1. Consistent with these results, elevated cysteinyl leukotrienes (CysLTs) concentration was also accompanied, as compared to control mice. Posttreatment of leukotriene receptor antagonist (LTRA), montelukast (10 mg/kg) for 4 consecutive days significantly decreased not CTS but PTS. Montelukast also reduced hair cell death after noise exposure in the cochlea which was identified by surface preparation and SEM. Taken together, pharmacological management using pravastatin, MB and montelukast will be helpful for providing novel strategies for the prevention of NIHL and other hearing loss-related diseases possibly related to oxidative stress or leukotriene signaling.
more목차
Acknowledgement i
ABSTRACT iii
TABLE OF CONTENTS vi
LIST OF FIGURES x
LIST OF TABLES xii
I. INTRODUCTION 1
A. Noise-induced hearing loss 1
1. Definition 1
2. Characteristics 1
3. Pathophysiology 2
B. Preventive methods 6
1. Hearing protective devices 6
2. Pharmacological treatment 6
C. Pravastatin 7
D. Methylene blue 8
E. Cysteinyl leukotriene receptor antagonist 9
II. MATERIALS AND METHODS 11
A. Experimental groups 11
1. Pravastatin experiment 11
2. Methylene blue experiment 11
3. Cysteinyl leukotriene receptor antagonist experiment 12
B. Experimental procedures 12
1. Noise exposure 12
2. Auditory Brainstem Response (ABR) analysis 13
3. Immunohistochemistry 13
4. Surface preparation 14
5. Electron microscopy 16
6. Tissue homogenization and Western blotting 17
7. Measurement of serum cholesterol content 17
8. Rac1 activity assay 17
9. Cell culture and viability 18
10. Intracellular ATP assay 19
11. Mitochondrial complex IV activity assay 19
12. Total RNA extraction and RT-PCR 20
13. Determination of cysteinyl leukotriene concentration in cochlear homogenates 21
14. Gene expression profiling and data analysis 21
15. Statistical analysis 23
III. RESULTS 25
A. Pravastatin attenuates noise-induced cochlear injury 25
1. Administration of pravastatin before noise exposure attenuated noise-induced threshold shift 25
2. Pravastatin pretreatment reduced hair cell loss after acoustic overstimulation 26
3. Pravastatin pretreatment attenuated reactive oxygen species (ROS) generation in the cochlea after acoustic overstimulation 32
4. Protective effect of pravastatin is associated with decreased Rac1 activity in vivo 33
B. Methylene blue alleviates oxidative stress and preserves synaptic structures 37
1. Administration of methylene blue before noise exposure attenuated noise-induced threshold shift 37
2. MB pretreatment reduced hair cell loss after acoustic overstimulation 38
3. MB pretreatment attenuated both ROS and RNS generation in the cochlea after acoustic overstimulation 42
4. MB treatment selectively ameliorated impaired mitochondrial electron transport chain (ETC) in inner ear cell line and in vivo 46
5. NT-3 was upregulated in noise-exposed cochlea by MB pretreatment 48
6. Improved mitochondrial function and upregulation of NT-3 by MB protected cochlear efferent and afferent synaptic structures from acoustic overexposure 51
C. Cysteinyl leukotriene signaling affects noise exposed cochleae 54
1. Leukotriene receptor gene expression in normal cochlear tissue of mice 54
2. Induction of LT signaling in the noise exposed mouse cochlea 54
3. Temporal and spatial expression pattern of CysLT1 receptor after noise exposure 58
4. Noise exposure increased concentration of cochlear CysLTs 58
5. Blockade of CysLT signaling using montelukast attenuated noise induced threshold shift 61
6. Montelukast reduced hair cell loss after acoustic overstimulation 64
7. Montelukast affected gene expression patterns after acoustic overstimulation 67
8. Inhibition of MMP-3 after noise exposure attenuated cochlear injury 67
IV. Discussion 73
V. Conclusion 87
References 88
국문요약 99
