Experimental Models of Oligodendrocyte Physiology and Ischemic White Matter Injury
- 주제(키워드) Oligodendrocyte , Ischemic White Matter Injury , Experimental model
- 주제(DDC) 570
- 발행기관 아주대학교 일반대학원
- 지도교수 Jun Young Choi
- 발행년도 2025
- 학위수여년월 2025. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000034719
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Oligodendrocytes (OLs) are glial cells found in the central nervous system (CNS), generating myelin sheaths to enable saltatory conduction to designated neurons. Research on the normal physiology and function of OLs, the OL lineage spanning from OL progenitor cells (OPCs) to myelinating OLs, and the pathophysiology of OLs in diseases where they are involved; ischemic brain diseases, neurodegenerative disease, multiple sclerosis, neuromyelitis optica spectrum disorders, etc.., has been a long-established subject in the field of neuroscience. Various in vitro and in vivo models to study OLs have been devised and are currently in use. This dissertation, which commenced as a hypothesis-driven research project to uncover the role of the OL lineage in ischemic brain diseases, aims to present a variety of experimental models and analyses to study OLs and ischemic white matter injury (IWMI), newly developed by the author. I have developed a simple, two-step differential centrifugation method to isolate OPCs from neonatal rodent cerebral cortices and have optimized media compositions to better support the distinct phases of the OL lineage during in vitro primary culture. This dissertation also presents a validation of the functional robustness of cryopreserved primary rodent OLs to be used in in vitro experiments, along with computational analyses to acquire and interpret data from in vitro experiments on OLs. Lastly, I present in vivo/in vitro disease models to recreate IWMI in experiment environments. In conclusion, the presented dissertation demonstrates strategies to facilitate research on OLs, a crucial component of the CNS.
more목차
Ⅰ. INTRODUCTION 1
1. Oligodendrocytes and the oligodendrocyte lineage 1
2. Experimental models of oligodendrocytes and related diseases 3
3. Development of experimental models for research on oligodendrocytes and ischemic white matter injury 4
Ⅱ. MATERIALS & METHODS 6
1. Animals 6
2. The E3 method for primary OL culture 6
3. Primary OL culture (shaking method) 10
4. Cryopreservation of primary OPCs (E3 method) 10
5. EdU proliferation assay 11
6. WST-8 cell viability and proliferation assay 11
7. Genetic manipulation 12
8. One-step myelinating OL/neuron co-culture 13
9. Oxygen-glucose deprivation (OGD) 13
10. Cell death assays 14
11. In vivo hypoxia 14
12. Spectral Confocal Reflectance (SCoRe) microscopy 15
13. Immunofluorescence 15
14. Immunoblotting 16
15. Image analyses 16
16. Statistical analysis 18
Ⅲ. RESULTS 19
Part I: Development of the E3(Easy, Efficient, Effective) method for primary oligodendrocyte culture from neonatal rodent brains. 19
1. The necessity for the de novo development of a primary OL culture method 19
2. Trials with density gradient centrifugation and the discovery of a two-step differential centrifugation to isolate OPCs 27
3. Optimization of OPC proliferation media formulae 31
4. Cell death during OL differentiation and the need for the passaging of crowded OPC colonies 36
5. Optimization of OL differentiation media formulae 46
6. Applicability of the E3 method to C57BL/6 mice 51
7. In vitro myelination of OLs cultured through the E3 method 53
Part II: Cryopreservation of primary OLs, and spatial/morphological analyses to evaluate in vitro OL function. 56
1. Cryopreservation of primary OLs cultured through the E3 method 56
2. In vitro functional validity of cryopreserved OLs 60
3. Poisson point process-based evaluation of the spatial distribution of OPCs 65
4. Sholl analysis and fractal dimensionality analysis to assess the morphological complexity of mature OLs 68
Part III: Adjunctive experimental methods for in vitro primary OLs. 71
1. Genetic manipulation of primary rat OPCs 71
2. Modification of the E3 method for adult rodent brains 75
3. One-step neuron-OL myelinating co-culture 77
4. The role of osmolarity in OPC proliferation and OL differentiation 81
Part IV: In vitro and in vivo experimental models of ischemic white matter injury. 83
1. In vitro oxygen-glucose deprivation (OGD) 83
2. Cell death and damage assays to evaluate OL injury after OGD 88
3. In vivo rodent model of neonatal hypoxic-ischemic encephalopathy (HIE) 91
4. Label-free detection of myelin and myelin damage 93
Ⅳ. DISCUSSION 95
Ⅴ. REFERENCES 106
