목차

Ⅰ. Introduction 1
1. Ischemic stroke and secondary immune response 1
2. Loss of neural connections after stroke 3
3. Current treatment for acute ischemic stroke patients 4
3.1 Tissue plasminogen activator (tPA) 4
3.2 Endovascular thrombectomy 4
4. Therapeutic approaches to target post-stroke inflammation 5
5. Augmentation of the intrinsic capacity of neural plasticity 7
6. Aim of the study 8
II. Methods and materials 9
1. Animals 9
2. Photothrombotic ischemic stroke 10
3. Intracortical injection of adeno-associated virus (AAV) 11
4. Behavior test 11
4.1 Pellet retrieval test 11
4.2 Ladder walk test 13
4.3 Cylinder test 13
4.4 mNSS scoring 13
5. Tissue preparation and immuno-histochemistry 14
6. Image acquisition and analysis 15
7. Single cell dissociation and fluorescent activated cell sorting (FACS) cell sorting 16
8. RNA extraction and cDNA synthesis 17
9. Cytokine PCR array and string analysis 18
10. Semi and Real-time PCR 19
11. Tissue protein extraction and western blot analysis 20
12. In vivo phagocytosis analysis and 3D image reconstruction 21
13. Primary bone marrow-derived macrophage culture 22
14. Primary microglia culture 22
15. In vitro macrophage-microglia interaction model 23
16. Arginase activity assay 24
17. Botulinum toxin injection 24
18. Intra-cortical BDA analysis 25
19. MATLAB analysis 26
20. Statistical analysis 28
III. Results 29
Chapter A. Establishment of photothrombotic ischemic stroke model and behavioral test batteries to study therapeutic approaches for ischemic stroke 29
1. photothrombotic ischemic stroke mice model 29
2. Validation of behavioral test batteries for evaluation of functional recovery after stroke 32
Chapter B. Modulation of post-stroke inflammation targeting Arginase-1 (Arg1) following ischemic stroke 35
3. Assessment of time-dependent expression of Arg1 following photothrombotic ischemic stroke 35
4. Characterization of Arg1 expressional source after ischemic stroke 37
5. Specification of cellular source of arg1 expression using reporter animals 39
6. Validation of conditional knockout of (cKO) arg1 following ischemic stroke 41
7. Behavioral analysis for evaluation of functional recovery after ischemic stroke in Arg1 cKO animals 44
8. Histological analysis of immune cell activity and glial scar formation 47
9. Fibrotic scar formation after ischemic stroke in Arg1 cKO animals 49
10. Peri-neuronal net formation after ischemic stroke 52
11. Excitatory synapses in peri-infarct area 54
12. In vivo microglial synaptic elimination and phagocytic activity 56
13. Phagocytic marker expression in peri-infarct microglia 60
14. Microglial Cytokine profiles following deletion of Arg1 in infiltrating macrophages 62
15. Macrophage-microglial interactions under hypoxic induction of Arg1 66
16. Proposed model for role of Arg1 expressing infiltrating macrophages following stroke 68
Chapter C. Enhancement of neural plasticity and activity dependent strategies for treatment of ischemic stroke 70
17. Development of intra-cortical Biotinylated Dextran Amine (BDA) axonal tracing system 70
18. Tissue processing and data acquisition of BDA-labeled axon 72
19. Machine learning algorithm for pattern classification 74
20. Conversion of BDA axon signals to the pixelated axon density map 76
21. Comparison of axonal sprouting following stroke using machine learning algorithm 78
22. Classifier accuracy-based statistical analysis 80
23. Validation of knock-out of PTEN assessing down-stream target of PTEN: P-S6 kinase 82
24. Assessment of functional recovery after PTEN KO in ischemic stroke model 84
25. Evaluation of intra-cortical axonal plasticity in PTEN KO after stroke 87
26. Combinational therapy of PTEN KO with Constraint-induced movement therapy (CIMT) using Botulinum toxin A 89
IV. Discussion 92
V. Summary and conclusion 100
References 101
국문요약 116