염증 반응에 의한 도파민성 신경세포의 사멸과 Interleukin-4의 역할에 관한 연구
Studies on the Effect of Interleukin-4 on the Degeneration of Dopaminergic Neurons in the Substantia Nigra in vivo
- 주제(키워드) Interleukin 4 , Inflammation , microglia , leukocyte , dopaminergic neuron
- 발행기관 아주대학교
- 지도교수 진병관
- 발행년도 2006
- 학위수여년월 2006. 8
- 학위명 박사
- 학과 및 전공 일반대학원 신경과학기술과정
- 본문언어 영어
초록/요약
The present study showed that interleukin-4 was expressed in infiltrated T lymphocytes, and participated in the LPS-induced inflammatory processing and degeneration of dopaminergic neurons in the SN. In LPS-injected tissues, OX42-ip resident microglia (with ramified morphology) had activated morphology with large cell bodies and short processes, resulting in the expression of pro-inflammatory and/or cytotoxic factors, such as TNF-a IL-1b and iNOS. Previous studies determined that microglia undergo dramatic morphological changes from resting ramified cells to activated amoeboid microlgia. However, in the present study, it was thought that activated microglia (larger bodies and thicker processes) were died, but not changed amoeboid morphology, and amoeboid monocyte/macrophages were infiltrated into the SN, as expressing IL-1b and other inflammatory molecules. These infiltrated macrophages maybe participate to degeneration of dopaminergic neurons in the SN. Degeneration of TH-ip dopaminergic neurons paralleled with increasing of infiltration of macrophages, TH-ip dopaminergic neurons were started to die at 24 hr with damaged morphologies, such as small and shrunken cell bodies and short and dotted processes. And then most of TH-ip cells were disappeared from the SNpc, remaining some dotted processes at 3days and 7 days after intranigral injection of LPS. This neurotoxicity against dopaminergic neurons occurred in a time-dependent manner as determined by TH-ip cell counting. When quantified and expressed as a percentage of control values, treatment of LPS attenuated the number of TH-ip neurons by 26 - 72 % at 1 – 7 days, respectively compared with PBS treated control tissues. Neutralization of IL-4 effectively reduced LPS-induced degeneration of dopaminergic neurons, when quantified and expressed as the percentage of TH-ip neurons by 80 % compared with PBS-injected SN tissues. And also, in the LPS-injected tissue, OX42-ip cells were mostly presented as rounded morphology in the SN at 24hr, even at 1wk later. However, neutralization of IL-4 effectively reduced infiltration of rounded OX42-ip cells at 24hr, and only a few rounded OX42-ip cells were detected in the injected site at 1wk later. And also, neutralization of IL-4 effectively reduced ED1-ip phagocytes compared with LPS-injected tissues. When quantified and expressed as the percentage of ED1-ip cells on the ipsilateral SN, neutralization of IL-4 was found to reduce the number of ED1-ip cells in both SNpc and SNr by 43 % and 35 %, respectively, compared with LPS-injected SN tissues. Neutralization of IL-4 prevented the LPS-induced death of astrocytes and BBB disruption in the SN. These results suggest that interleukin-4 play important role in the LPS-mediated inflammatory process and degeneration of dopaminergic neurons by regulation of macrophage infiltration into the SN.
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ACKNOWLEGEMENTS i
ABSTRACT ii
TABLE OF CONTENTS iv
LIST OF FIGURES viii
LIST OF ABBREVIATION xi
I.INTRODUCTION 1
1.Origin of microglia 1
2.Characterization of microglia 3
3.Leukocyte infiltration into the CNS 3
3.1.Antigen presentation of dendritic cells in the CNS 4
3.2.Infiltration of lymphocytes into the CNS 5
3.3.Infiltration of granulocyte 6
3.4.Microglia and macrophage in the CNS 6
4.Anatomy and physiology of the blood-brain barrier 7
5.Adhesion of leukocytes to endothelial cells 9
6.Cytokines 11
7.Pro-inflammatory cytokines 12
8.Interkeukin-4 13
8.1.General Properties 13
8.2.Effect of IL-4 in the CNS 16
8.3.Interleukin-4 and adhesion molecules 17
9.Inflammation in the Parkinson’s disease 17
10.Parkinson’disease and peripheral immune cells 19
11.Specific aims of this study 21
II.MATERIALS AND METHODS 22
1.Stereotaxic injection of LPS 22
2.Tissue preparation and immunohistochemistry 23
3.FITC-labeled albumin leakage 24
4.Stereological cell counts 25
5.Immunofluorescence staining 26
6.TdT-mediated dUTP Nick-End Labeling (TUNEL) assay 27
7.RT-PCR Analysis 28
8.Western immunoblot analysis 29
9.Statistical analysis 30
III.RESULTS 31
Part A.LPS induces infiltration of IL-4 expressing T lymphocytes into the SN 31
1.Comparisons of immunohistohemistry between anti-OX42 and anti-Iba1 in LPS-injected SN tissues 31
2.Infiltration of Interleukin-4 expressing cells into the SN 32
3.T lymphocytes are infiltrated into the SN, expressing Interleukin-4 39
Part B.Resident microglia & Infiltrated macrophage 42
1.LPS induces infiltration of phagocytes/macrophages into the SN 42
2.Chemokine expression 46
3.LPS induces expression of iNOS and proinflammatory cytokines, such as IL-6, IL-1b and TNF-a in the resident microglia 48
4.IL-1b expression shift from resident microglia to infiltrated macrophages 51
5.LPS-mediated infiltration of macrophages maybe accompanied with degeneration of dopaminergic neurons 53
6.LPS induces damage of astrocytes and disruption of blood-brain barrier 56
Part C.Interleukin-4 participates in the infiltration of macrophages into the SN by regulation of adhesion molecules 61
1.Neutralization of IL-4 prevents the death of LPS-induced dopaminergic neurons in the SN 61
2.Neutralization of IL-4 blocks LPS-induced inflammatory processing in the SN 63
3.Neutralization of IL-4 reduces infiltration of ED1-ip phagocytes mediated by LPS in the SN 67
4.Neutralization of IL-4 prevents the death of LPS-induced astrocytes in the SN 70
5.Neutralization of IL-4 reduces LPS-induced ICAM-1 expression in the SN 73
IV.DISCUSSION 76
Part A.LPS induces infiltration of IL-4 expressing T lymphocytes into the SN 76
Part B.Resident microglia & Infiltrated macrophage 77
Part C.Interleukin-4 participates in the infiltration of macrophages into the SN by regulation of adhesion molecules 81
V.SUMMARY AND CONCLUSION 87
VI.BIBLIOGRAPHY 88
VII.국문 초록 113
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Figure 1.Schema showing the proposed differentiation process of microglial cells during development 2
Figure 2.Schematic drawing showing some features of the perivascular sastrocytic end feet, which form ‘rosette’-like structures on the brain capillary surface 8
Figure 3.OX42 immunohistochemistry 34
Figure 4.Iba1 immunohistochemistry 36
Figure 5.LPS induced infiltration of IL-4 expressing cells into the SN 38
Figure 6.Comparison of immunohistochemistry between OX-42 and Iba1 39
Figure 7. IL-4 expressing T lymphocytes infiltrated into the SN 42
Figure 8.Infiltration of phagocytes/macrophage into the SN 45
Figure 9.Infiltration of phagocytes/macrophage into the SN 46
Figure 10.RT-PCR analysis of LPS-induced mRNA expression of chemokines in the SN 48
Figure 11.RT-PCR analysis of LPS-induced mRNA expression of iNOS and pro-inflammatory cytokines in the SN 50
Figure 12.Co-localization of iNOS or IL-1b-immunoreactivity within activated resident microglia in the SN 51
Figure 13.IL-1b expression shift from resident microglia to infiltrated macrophages 53
Figure 14.Iba-ip rounded cells were differentiated into thick and ramified morphology 55
Figure 15. TH-immunohistochemistry showing degeneration of dopaminergic neurons by intranigral injection of LPS 56
Figure 16.GFAP-immunostaining shows disruption of astrocyte after intranigral injection of LPS 58
Figure 17.LPS induces disruption of Blood-Brain Barrier 59
Figure 18.Schematic drawing shows that LPS induces activation of resident microglia and infiltration of monocyte/macrophages into the SN 60
Figure 19.Neutralization of IL-4 prevents the death of LPS-induced dopaminergic neurons in the SN 62
Figure 20.Neutralization of IL-4 blocks LPS-induced inflammatory processing in the SN 64
Figure 21.Neutralization of IL-4 blocks LPS-induced inflammatory processing in the SN 65
Figure 22. Neutralization of IL-4 blocks LPS-induced inflammatory processing in the SN 66
Figure 23.Neutralization of IL-4 reduces LPS-induced infiltration of phagocytes into the SN 69
Figure 24.Neutralization of IL-4 prevents damage of GFAP-ip astrocytes 71
Figure 25.Neutralization of IL-4 prevents disruption of Blood-Brain Barrier72
Figure 26.Neutralization of IL-4 reduces LPS-induced ICAM-1 mRNA in the SN 74
Figure 27.Schematic drawing for immune response mediated by LPS in the SN 75
