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Graduate School of Ajou University Department of Neuroscience and Technology Course 3. Theses(Master)

혈액에서 유입된 염증세포와 신호전달분자 Rac, SOCS에 의한 뇌염증반응의 조절에 관한 연구

DC Field Value Language
dc.contributor.advisor조은혜-
dc.contributor.author지경애-
dc.date.accessioned2019-10-21T06:45:59Z-
dc.date.available2019-10-21T06:45:59Z-
dc.date.issued2006-08-
dc.identifier.other1612-
dc.identifier.urihttps://dspace.ajou.ac.kr/handle/2018.oak/16363-
dc.description학위논문(박사)--아주대학교 일반대학원 :신경과학기술과정,2006. 8-
dc.description.tableofcontentsLIST OF CONTENTS ACKNOWLEDGEMENTS i ABSTRACT ii LIST OF CONTENTS v LIST OF FIGURES xii LIST OF TABLES xvi LIST OF ABBREVIATION xvii I. INTRODUCTION 1 1. Brain glial cells and their roles in brain inflammation 1 1.1. Microglia 1 1.2. Astrocytes 1 2. Brain inflammation and neuronal damage 2 3. Stimulators of brain inflammation 3 3.1. Interferon-gamma 3 3.2. Thrombin 4 3.3. Lipopolysaccharide (LPS) 5 4. Signaling molecules that mediate brain inflammation 6 4.1. Positive regulators: JAK/STAT pathway 6 4.2. Negative regulators: SOCS family 7 5. Roles of blood inflammatory cells in brain inflammation 8 6. Aims of this study 9 II. MATERIALS AND METHODS 11 1. Materials 11 2. in vitro Methods 12 2.1. Cell preparation 12 2.2. Generation of cells stably expressing RacN17 12 2.3. Western blot analysis 13 2.4. Reverse Transcription and Polymerase Chain Reaction (RT-PCR) 13 2.5. GST-PAK-PBD binding Assays 14 2.6. Transfection and luciferase assay 14 2.7. Immunoprecipitation 15 2.8. Released H2O2 assay 15 2.9. PGE2 and LTB4 assay 16 3. in vivo Methods 16 3.1. Stereotaxic injection of LPS or NMDA 16 3.2. Irradiation 17 3.3. Tissue preparation and Immunohistochemistry 18 3.4. Evaluation of blood-brain barrier (BBB) permeability 19 3.5. TUNEL assay 20 3.6. Stereological cell counts 21 3.7. Statistical analysis 21 III. RESULTS 22 Part A. Rac1 contributes to maximal activation of STAT1 and STAT3 in IFN-g-stimulated rat astrocytes 22 1. IFN-g stimulates the activation of Rac1 22 2. Dominant negative Rac1 reduces IFN-g-induced GAS promoter activity in rat astrocytes 24 3. The expression of IFN-g-responsive genes is attenuated in C6 cells expressing RacN17 27 4. RacN17 does not affect the expression level of IFN-g receptor alpha subunit 28 5. Rac1 plays a role in IFN-g-stimulated phosphorylations of STAT1 and STAT3 but not JAK1 31 6. Rac1 associates with IFN-g receptor alpha, STAT1, and STAT3 32 Part B. Thrombin induces expression of cytokine-induced SH2 protein in rat brain astrocytes: involvement of phospholipase A2, cyclooxygenase and lipoxygenase 39 1. Thrombin induces CIS expression in C6 astroglioma cells 39 2. Thrombin induces CIS expression via a STAT- and MAPK-independent pathway 41 3. Cytosolic phospholipase A2 , cyclooxygenase, and lipoxygenase are involved in thrombin-induced CIS expression 44 4. Reactive oxygen species mediates thrombin-induced CIS expression 46 5. PGE2 and LTB4 enhance thrombin-induced CIS expression 51 6. CIS reduces IFN-g-activated STAT and GAS promoter activity in C6 cells 51 Part C. Lipopolysaccharide induces reversible neuronal damage in the rat brain cortex: comparison to N-methyl d-aspartate (NMDA) -induced neuronal death 55 1. LPS induces transient disappearance of neuronal markers, NeuN and MAP-2 in the rat brain cortex 55 2. NeuN-negative regions coincide with OX-6-positive microglia detected regions 60 3. The expression of a neurogenesis marker, doublecortin, is not changed during the reappearance of NeuN-immunopositive cells 62 4. N-methyl d-aspartic acid (NMDA) induces neuronal degeneration in the cerebral cortex 63 5. TUNEL assay supports the possibility that LPS induced reversible neuronal damage but not neuronal death 66 Part D. Critical roles of neutrophil infiltration in inflammation and death of dopaminergic neurons in the substantia nigra: implications of Parkinson’s disease 68 1. Inflammatory stimulation causes a dramatic increase in the number of OX-42-ip cells in the SNpc but not the cortex 68 2. Expression of MPO, a neutrophil marker, by OX-42-ip cells in the SNpc following injection of LPS 70 3. Differential patterns of neutrophil infiltration in the SNpc and the cortex following LPS injection. 72 4. Neutrophil infiltration coincides with a loss of dopaminergic neurons in LPS-injected SNpc 77 5. Death of dopaminergic neurons in the LPS-injected SNpc is attenuated in leukopenic animals. 79 Part E. Brain inflammation is a symphony orchestrated by microglia and blood-derived inflammatory cells 85 1. LPS induces the differential behaviors of OX-42- and Iba-1-ip cells in the SNpc 85 2. Resident microglia die in the SNpc following LPS injection 87 3. Infiltrated neutrophils die following LPS injection 89 4. OX-42/Iba-1-double-ip cells reappear in the SNpc following LPS injection 89 IV. DISCUSSION 94 Part A. Rac1 contributes to maximal activation of STAT1 and STAT3 in IFN-g-stimulated rat astrocytes 94 Part B. Thrombin induces expression of cytokine-induced SH2 protein (CIS) in rat brain astrocytes: involvement of phospholipase A2, cyclooxygenase and lipoxygenase 98 Part C. Lipopolysaccharide (LPS) induces reversible neuronal damage in the rat brain cortex: comparison to N-methyl d-aspartate (NMDA)-induced neuronal death 102 Part D. Critical roles of neutrophil infiltration in inflammation and dopaminergic neuron death in the substantia nigra: implications for Parkinson’s disease 106 Part E. Brain inflammation is a symphony orchestrated by microglia and blood-derived inflammatory cells 110 V. SUMMARY AND CONCLUSION 113 VI. BIBILOGRAPHY 114 국문요약문 135 LIST OF FIGURES Figure 1. IFN-g induces activation of Rac1 23 Figure 2. Expression of RacN17 markedly attenuates GAS promoter activity in rat astrocytes 25 Figure 3. IFN-g-enhanced GAS luciferase activity and gene expression are attenuated in cells expressing RacN17 29 Figure 4. The expression level of IFN-g receptor is not changed by expression of RacN17 30 Figure 5. Expression of RacN17 attenuates the IFN-g-induced phosphorylation of STAT1 and 3, but not JAK1 33 Figure 6. Rac1 interacts with IFN-g receptor alpha, JAK, and STAT 36 Figure 7. Thrombin induces CIS expression in C6 cells 40 Figure 8. Thrombin induces CIS expression in primary cultured rat brain astrocytes 42 Figure 9. Thrombin induces CIS expression via a STAT- and MAPK-independent pathway 43 Figure 10. cPLA2 mediates thrombin-induced CIS expression 45 Figure 11. Cyclooxygenase (COX) and lipoxygenase (LO) are associated with thrombin-induced CIS expression 48 Figure 12. Thrombin induces ROS generation, and ROS scavengers reduce thrombin-induced CIS expression 49 Figure 13. Indomethacin and NDGA reduce thrombin-induced ROS generation 50 Figure 14. PGE2 and LTB4 enhance thrombin-induced CIS expression 52 Figure 15. CIS reduces IFN-g-activated GAS promoter activity and inhibits sustained phosphorylation of STAT1 and STAT3 53 Figure 16. LPS induces reversible neuronal damage in the cerebral cortex of rat brain in vivo 56 Figure 17. Higher dose (50 ug) of LPS also induces reversible neuronal damage in the cerebral cortex of rat brain 58 Figure 18. Neurotoxic mediators affect on LPS-induced neuronal recovery in the cerebral cortex of rat brain 61 Figure 19. LPS-induced neuronal recovery is not involved in neurogenesis 64 Figure 20. NMDA, which has direct neurotoxicity, induces the death of neuron in the cerebral cortex of rat brain in vivo 65 Figure 21. LPS does not induce neuronal death in the cerebral cortex in vivo 67 Figure 22. The number of OX-42-ip cells increases in LPS-injected SNpc but not in the cortex 69 Figure 23. MPO-ip cells appear in the LPS-injected SNpc and are colabeled with OX-42 antibody 71 Figure 24. In leukopenic rats, the number of MPO-ip cells in the SNpc are reduced following LPS injection 73 Figure 25. Differential distribution of MPO-ip cells in the SNpc and the cortex following LPS injection 75 Figure 26. BBB is easily disrupted in LPS-treated SNpc, compared withcortex 76 Figure 27. MPO-ip cells express iNOS 78 Figure 28. Infiltration of MPO-ip cells coincides with the loss of TH-ip neurons in LPS-treated-SNpc 80 Figure 29. LPS-induced loss of TH-ip neurons is attenuated in neutropenic rats 82 Figure 30. Differential behaviors of OX-42- or iba-1-ip cells at the indicated times in the SNpc following LPS injection 86 Figure 31. Disappearance of iba-1-ip cell at 12 h in the SNpc following LPS injection in vivo 88 Figure 32. Death of MPO-ip neutrophils in the SNpc following LPS injection 90 Figure 33.Heterogeneity of OX-42-ip cells at 1 or 3 d in the SNpc following LPS injection 91 Figure 34. Reappeared Iba-1ip cells are from not migration of adjacent microglia, but blood into the SNpc following LPS injection 92-
dc.language.isoeng-
dc.publisherThe Graduate School, Ajou University-
dc.rights아주대학교 논문은 저작권에 의해 보호받습니다.-
dc.title혈액에서 유입된 염증세포와 신호전달분자 Rac, SOCS에 의한 뇌염증반응의 조절에 관한 연구-
dc.title.alternativeKyung-Ae Ji-
dc.typeThesis-
dc.contributor.affiliation아주대학교 일반대학원-
dc.contributor.alternativeNameKyung-Ae Ji-
dc.contributor.department일반대학원 신경과학기술과정-
dc.date.awarded2006. 8-
dc.description.degreeMaster-
dc.identifier.localId565539-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000001612-
dc.subject.keywordbrain inflammation-
dc.description.alternativeAbstractBrain inflammation is a process of host defense against infection of injury. During this process, excessive inflammation could be harmful to surrounding tissue and consequently aggravate brain injury. Thus, to understand how brain inflammation occurs and how its duration and extent are controlled is important to limit neuronal damage in the brain. The first part of this thesis showed that activation of Rac1, a small G-protein, is involved in IFN-g-signaling in astrocytes. Compared to levels in control cells, IFN-g-induced GAS promoter activity and expressions of several IFN-g-responsive genes were markedly reduced in both cells expressing RacN17, a well-characterized Rac1 negative mutant. Thus, Rac1 may contribute to maximal activation of IFN-g responsive inflammatory signaling in rat astrocytes. The second part showed that thrombin, an inflammatory stimulator, increased expression of full name of cytokine-induced SH2 protein (CIS), one of SOCS family proteins. Since CIS reduced IFN-g-induced GAS-luciferase activity and tyrosine phosphorylation of STAT1 and STAT3, I concluded that thrombin could control duration and extent of inflammation by inducing expression of negative regulators of inflammation as well as pro-inflammatory mediators. In the third part, I showed regional differences in the extent of brain inflammation and neuronal damage in the substantia nigra pars compacta (SNpc) and the cortex, and underlying mechanisms that cause these differences. Microinjection of lipopolysaccharide (LPS) induced transient inflammatory responses and reversible neuronal damage in the cortex, but relatively severe and long-lasting inflammation and neuronal death in the SNpc. I found that the differential extent of neutrophil infiltration in these two areas could cause differential extent of inflammation. In the SNpc, cells labeled with two markers of microglia, OX-42 and ionized calcium binding adaptor molecule (Iba-1) showed different behaviors: The number of OX-42-ip cells increased at 12 h after LPS injection while the number of Iba-1-ip cells were dead. Since the OX-42 antibody detects CD11b that is expressed in neutrophils as well as in microglia, infiltration of neutrophils was detected using myeloperoxidase (MPO) as a specific marker of neutrophils. At 12 h following LPS injection, there was a dramatic increase in the number of MPO-ip neutrophils in the SNpc while relatively sparsely in the cortex. The MPO-ip cells were co-labeled with OX-42 and these cells expressed inducible nitric oxide synthase (iNOS). In agreement with these results, in leukopenic rats, MPO-ip cells were not detected for up to 3 days after LPS injection, and the loss of dopaminergic neurons were significantly attenuated. These results indicate that the extent of infiltrated neutrophil could determine the severity of inflammation in the cortex and the SNpc, and this event could be linked to the severity of neuronal damage in these two brain areas.-
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