MPTP로 유도된 파킨슨병 동물 모델에서 에틸 피루브산의 도파민성 신경세포사멸 억제 효과에 대한 연구
Study on the Neuroprotective Effects of Ethyl pyurvate in MPTP-induced Mouse PD Models
- 주제(키워드) Parkinson`s disease , MPTP , Ethyl pyruvate , glia , ROS , Neurotrophic factors
- 발행기관 아주대학교
- 지도교수 진병관, 백은주
- 발행년도 2012
- 학위수여년월 2012. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 파일정보 microsoft world
- 실제URI http://www.dcollection.net/handler/ajou/000000012408
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The present study examined whether ethyl pyruvate (EP) promotes the survival of nigrostriatal dopaminergic (DA) neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease. MPTP induced degeneration of nigrostriatal DA neurons and glial activation as visualized by tyrosine hydroxylase, macrophage antigen complex-1, and/or glial fibrillary acidic protein immunoreactivity. Western blotting and immunohistochemistry showed activation of microglial NADPH oxidase and astroglial myeloperoxidase (MPO), and subsequent reactive oxygen species (ROS)/reactive nitrogen species (RNS) production and oxidative DNA damage in the MPTP-treated substantia nigra. Additional immunohistochemical staining also showed that MPTP induced glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) in the substantia nigra (SN). Interestingly, treatment with EP prevented degeneration of nigrostriatal DA neurons, increased striatal dopamine levels, and improved motor function. This neuroprotection afforded by EP was associated with the suppression of astroglial MPO expression, NADPH oxidase- and/or inducible nitric oxide synthase-derived ROS/RNS production by activated microglia. In parallel, treatment of EP significantly increased GDNF and CNTF in the MPTP-treated SN. Also, EP was found to protect DA neurons from 1-methyl-4-phenyl-pyridinium (MPP+) neurotoxicity in co-cultures of mesencephalic neurons and glia, but not in neuron-enriched mesencephalic cultures devoid of glia. The present findings show that EP may inhibit oxidative stress and increase levels of neurotrophic factors in glia, suggesting that EP may have therapeutic value in the treatment of aspects of Parkinson’s disease related to glia activation.
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ABSTRACT i
TABLE OF CONTENTS iii
LIST OF FIGURES ⅳ
ABBREVIATION vii
I. INTRODUCTION 1
A. Parkinson's disease 2
1. Characteristics of MPTP-induced mouse PD model 5
2. Role of glia in PD 8
3. Oxidative stress and PD 10
4. Neurotrophic factors and PD 12
B. Ethyl pyruvate 13
C. Purpose of present study 14
II. MATERIALS AND METHODS 16
A. Materials 16
1. Animals and treatment∙16
B. methods 16
1. Immunohistochemistry 16
2. Detection of dying neurons (Fluorojade-C staining) 17
3. Double-immunofluorescence Staining 18
4. Stereological cell counting 19
5. Densitometric analyses 20
6. Rotarod test 20
7. HPLC analyses 21
8. GC-MASS analyses 21
9. Determination of O2- and O2--derived oxidants 22
10. Immunoprecipitation 23
11. Western Blotting 23
12. Neuron-Enriched Mesencephalic Cultures 24
13. Mesencephalic Microglia Cultures 25
14. Co-cultures of Mesencephalic Microglia and Neurons 25
15. Measurement of Dopamine Uptake 26
16. Assays for intracellular ATP and ROS levels 26
17. Statistical analysis 27
III. RESULTS 28
Part A. Ethyl pyruvate (EP) protects nigrostriatal dopaminergic neuron from MPTP
neurotoxicity 28
1. MPTP induces nigrostriatal DA neurodegeneration 28
2. MPTP induces glial activation in SN and STR. 30
3. EP protects nigrostriatal DA neurons from MPTP-induced neurotoxicity
in vivo. 33
4. EP increases striatal dopamine levels and improves motor behavior in
MPTP mice. 39
5. EP does not prevent MPP+-induced neurotoxicity in neuron-enriched mesencephalic cultures. 41
Part B. EP decreased oxidative stress increased by MPTP neurotoxicity 45
1. EP inhibits microglial activation in the SN in vivo 45
2. EP attenuates MPTP-induced reactive oxygen species (ROS) production
and oxidative damage via microglial NADPH oxidase 48
3. EP attenuates iNOS expression and nitrosative damage 53
4. EP protects mesencephalic DA neurons from microglia-derived
neurotoxicity 56
5. EP inhibits astrocytes activation in the SN in vivo∙ 59
6. EP inhibits myeloperoxydase (MPO) expression in astrocytes of the
MPTP-treated SN in vivo 61
7. The expression of a variety of neurotrophic factors in the SN in MPTP-induced PD model 64
8. Pyruvate (Py) protects DA neuronal death in MPTP neurotoxicity 71
IV. DISCUSSION 73
V. CONCLUSION 82
REFERENCES 83
국문요약 100
