허혈성 뇌졸중 실험 모델들에서 물리적장벽파괴술에 의한 역방향 혈관생성
Reverse arteriogenesis with mechanical barrier disruption in experimental models of ischemic stroke
- 주제(키워드) Arteriogenesis , Angiogenesis , Ischemic stroke , Erythropoietin , cranial burr hole , Mechanical barrier disruption
- 발행기관 아주대학교
- 지도교수 홍지만
- 발행년도 2020
- 학위수여년월 2020. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029710
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Stroke, which has the second highest mortality rate worldwide, is caused by bleeding or occlusion of the cerebral vasculature. Particularly, insufficient blood flow in the ischemic penumbra after injury leads to irreversible tissue damage as a result of cerebral autoregulation failure and impairment in metabolic compensation. Among the potential recovery strategies, such as neurogenesis, synaptogenesis, enhancement of neuronal and synaptic plasticity, and augmentation of angiogenesis, revascularization is of particular importance as it is necessary to revitalize the ischemic region. Mechanical barrier disruption (MBD) of the protective layers between the intracranial and extracranial arterial systems, which includes the formation of cranial burr holes and small disruptions in the meninges, is a minimally invasive indirect revascularization procedure. The use of cranial burr holes has been reported to be beneficial in certain progressive cerebrovascular occlusive disorders, such as Moyamoya disease (MMD). However, its use is still limited in clinical situations as there is no guarantee of revascularization or immediate vascular connection, and its exact mechanism is not fully understood. This study was aimed at investigating the feasibility and mechanism of mechanical barrier disruption (MBD) in experimental models of ischemic stroke. The study had four objectives. First, the feasibility of reverse arteriogenesis by MBD was confirmed on MMD-like animal models. To establish MMD-similar models with sustained hypoperfusion, the mild ischemia model rats was performed by ligating bilateral ICAs (bICAL) in SD. MBD procedures in mild ischemia models led to transient BBB breakdown in the intracranial environment as well as restoration of the damaged periosteum in the extracranial environment within 2 weeks. After this spontaneous healing process, transcranial vessels between intracranial and extracranial arterial systems, which were originally divided by the skull, were observed by time-of-flight MRA and vascular casting using India-Ink perfusion at 3 months. Intriguingly, the vessels were derived from external carotid artery (ECA). As a result, enhanced angiogenesis was observed within the MBD region only. Second, whether cerebral inflammation affects reverse arteriogenesis using the MBD procedure in mild ischemia models was determined as inflammation caused by MBD has the potential to induce angiogenesis. To induce systemic inflammation, mild ischemia models were intra-peritoneally injected with two concentrations of lipopolysaccharide (LPS) (Low dose, 0.1 mg/kg; high dose, 1 mg/kg). LPS injection for 3 days caused weight loss, which is a hallmark of infection in LPS-treated animals. High dose LPS significantly increased inflammatory cytokines and induced cerebral inflammation in the brain of mild ischemia models. With an increase in inflammatory cytokines after MBD, these cytokines exacerbated BBB permeability that was induced by MBD. As a result, they induced enhanced angiogenesis at 1 month. Third, whether combination therapy with an angiogenic booster and MBD could enhance reverse arteriogenesis was investigated in mild ischemia models. To enhance angiogenesis and arteriogenesis in the MBD regions, erythropoietin (EPO) was administrated as an angiogenic booster. The ipsilateral hemisphere with MBD showed a significant increase in vessels in the intracranial vasculature adjacent to the MBD region. The levels of pro-angiogenic and inflammatory factors with prominent markers of vessel permeability were also significantly increased. In the EPO-administered group, such elevations in inflammation were significantly mitigated. The ipsilateral hemisphere with MBD-EPO (vs. MBD-only) showed a significant increase in vessels and their maturation, with upregulation of transforming growth factor-β1 (Tgf-β1) and matrix metalloproteinase-2 (Mmp-2). These phenomena were completely blocked by minocycline (MIC) administration during in vivo and in vitro experiments. Finally, a new severe ischemia model with perfusion impairment in rats was established and then the impact of combination therapy on the reverse arteriogenesis was investigated in the severe ischemia model. A new severe ischemia model was constructed by introducing focal ischemia (occlusion time for 30 min) into a mild ischemia model (bICAL+tMCAO) to mimic the MMD patient with acute ischemic stroke. This model had both hypoperfusion states and an infarction. Next, the feasibility of reverse arteriogenesis by combination therapy was confirmed in the severe ischemia model. Although a vascular network between intracranial and extracranial tissues was generated, angiogenic effects of the combination therapy could not be seen under severe ischemic conditions. Compared to the mild ischemia model, the severe ischemia model showed a decrease in hypoxia related factors and an increase in ROS and inflammation, indicating that hypoxia and its transcriptional targets are inversely correlated with ROS and inflammation. In order to overcome the limitation of MBD-induced angiogenesis being suppressed by excessive ROS or inflammation, therapeutic hypothermia (HT) was performed before reperfusion. Early hypothermia in the severe ischemia model decreased ROS and inflammation and increased the expression of hypoxia inducible factor-1 α (HIF-1α), resulting in attenuation of the neurological deficits and tissue loss. In addition, this experiment confirmed the feasibility of reverse arteriogenesis in severe ischemic models. In conclusion, these results indicate that the MBD procedure at angiogenic routes can facilitate successful reverse-arteriogenesis in subjects with intracranial perfusion insufficiency, but not severe injury.
more목차
Ⅰ. INTRODUCTION １
A. Ischemic stroke １
1. Clinical significance of stroke １
2. Pathophysiology of ischemic stroke １
3. Current treatment of ischemic stroke ２
B. Moyamoya disease and syndrome ４
1. Epidemiology and clinical symptoms of MMD ４
2. Pathophysiology of MMD ５
3. Current treatment of MMD ６
C. Mechanical barrier disruption (MBD) ８
D. Crosstalk of focal inflammation and regional angiogenesis １０
E. Neovascularization １１
1. Vasculogenesis, angiogenesis, and arteriogenesis １１
2. Post-stroke angiogenesis １２
F. Erythropoietin as angiogenic boosters １４
G. Aims of this study １５
Ⅱ. MATERIALS AND METHODS １６
A. Ethics statement １６
B. Animal and general procedures １６
C. Animal models １６
D. Animal groups and drug administration １７
E. Mechnical barrier disruption (MBD) １８
F. Therapeutic hypothermia (TH) １８
G. Behavioral test １８
H. Measurement of hematocrit １９
I. In vivo brain imaging １９
1. Measurement of cerebral blood flow １９
2. Time-of-flight (TOF) magnetic resonance angiography (MRA) １９
J. Post-mortem microangiography ２０
1. Black India ink ２０
2. Blue dye injection ２０
K. Blood-brain barrier (BBB) leakage analysis ２１
L. TTC staining ２１
M. Immunohistochemistry ２１
N. Cresyl violet staining ２２
O. Quantitative real time polymerase chain reaction (qRT-PCR) ２２
P. Western blot ２３
Q. In vitro experimental design ２４
R. Tube formation of HUVECs ２５
S. Wound healing of hAoSMCs ２５
T. Statistical analysis ２６
Ⅲ. RESULTS ３１
PART A. Reverse arteriogenesis by MBD procedure in the mild ischemic rat model. ３１
1. Establishment of mild ischemia model ３１
2. MBD modulates extracranial and intracranial environments ３３
3. MBD induce reverse arteriogenesis from extracranium ３６
PART 2. The effect of cerebral inflammation on reverse arteriogenesis by MBD procedure in mild ischemic rat model ３９
1. The effect of systemic inflammation by LPS treatment in the mild ischemic rat model ３９
2. The effect of systemic inflammation in mild ischemic rat model with MBD procedure. ４４
PART 3. The effect of erythropoietin pretreatment on reverse arteriogenesis by MBD procedure in mild ischemic rat model ５０
1. EPO pretreatment enhance angiogenesis and arteriogenesis in MBD region ５０
2. EPO pretreatment mitigated inflammation in MBD region ５８
3. EPO stimulated Angiogenesis and arteriogenesis by regulating of TGF-β1 and MMP-2 ６４
4. Minocycline suppress the ability of EPO to induce vascular maturation ７０
PART 4. The feasibility of MBD procedure in severe ischemic rat model ７４
1. Establishment of severe ischemia model ７４
2. Futility of combination therapy in severe ischemia model ８２
3. Comparison of mild and severe ischemia model ８６
4. Challenge for reverse arteriogenesis in severe ischemia model ９２
Ⅳ. DISCUSSION ９９
1. MBD-induced reverse arteriogenesis ９９
2. Extracranial and intracranial angiogenic modulation by MBD procedure １００
3. Intracranial milieu modulation １０２
1) Inflammation modulation. １０２
2) Vessel maturation. １０４
4. Reverse arteriogenesis in severe ischemia model １０５
5. Intracranial and extracranial milieu modulation by combination therapy １０６
Ⅴ. CONCLUSION １１０
REFERENCES １１１
CONTRIBUTIONS １２６
국문요약 １２７
