전신성허혈과 알츠하이머 병의 동물 모델에서 혈뇌장벽손상의 병리적인 역할
Pathological roles of blood-brain barrier damage in animal models of stroke and alzheimer's disease
- 주제(키워드) Free radical , Transient forebrain ischemia , blood-brain barrier , iron , alzheimer , amyloid beta
- 발행기관 아주대학교
- 지도교수 곽병주
- 발행년도 2010
- 학위수여년월 2010. 8
- 학위명 박사
- 학과 및 전공 일반대학원 신경과학기술과정
- 실제URI http://www.dcollection.net/handler/ajou/000000010983
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Recent data from humans and animals models suggest that pathological blood-brain barrier (BBB) opening contributes to nerve cell damage and dysfunction. We examined the possibility and underlying mechanims that transient forebrain ischemia (TFI) would render the BBB permeable to blood cells possibly through dysfunction and degeneration of endothelial cells. Extravasation of blood cells into the hippocampal parenchyma was manifest by increased levels of Evans blue and IgG that were detectable at 8 h and peaked at 48 h after TFI. TdT-mediated dUDP nick end labeling (TUNEL) revealed marked degeneration of endothelial cells primarily in the CA1 and 2 area within 48 h after TFI. Mitochondrial free radicals were produced in the endothelial cells as well as the hippocampal neurons before cell death occurred. Iron was overloaded in the microvessel endothelium of the CA1 and 2 area as early as 30 min, was higher than the control until 4 h after reperfusion. Administration of deferoxamine (DFO), a selective iron chelator, or Neu2000, a potent antioxidant, blocked free radical production, degeneration of the endothelial cells, and extravasation of the blood cells into the brain. The present study suggests that iron overload and iron-mediated free radical production cause degeneration of the endothelial cells, opening of the BBB, and possibly degeneration of the CA1 pyramidal neurons after TFI. Evidence has accumulated suggesting that BBB opening and iron deposit are associated with the pathology of Alzheimer’s disease (AD). We examined role of amyloid beta (Ab) peptides underlying initial BBB opening and iron deposit in APP/PS1 (which coexpress mutated human presenilin 1 and amyloid-β precursor protein). BBB opening was barely detectable in APP/PS1 at 3 months of age but significantly elevated in 5-month-old APP/PS1 mice as shown by infiltration of IgG in the cortex and hippocampus. Double staining with Evans blue and a collagen type IV antibody revealed basal lamina disruption and blood extravasation primarily in the cortex. BBB opening was accompanied by reduced levels of tight junction proteins such as occludin and adherence junction protein β-catenin. Soluble Ab40 was detected approximately to 0.35 pg/g at 2 - 5 months of age. In contrast, soluble Ab42 was slightly detected at 3 months of age but both soluble and insoluble forms of Ab42 were increased up to 0.04 and 0.025 pg/g at 5 months of age, respectively. Intracortical administration of soluble Ab42, but not Ab40, caused BBB opening around the injection site, suggesting that soluble Ab42 mediates initial BBB opening in APP/PS1 mice. Iron deposit was observed exclusively in plaques and blood vessels in 5-month-old APP/PS1 mice and following intrahippocampal administration of Ab42. Administration of deferoxamine, a selective iron chelator, for 2 months reduced amyloid plaque burdens as well as iron deposit. Furthermore, amyloid plaque burdens in APP/PS1 mice were locally increased at 15 d following intracortical administration of FeCl2. The present findings suggest that soluble Ab42 enhances amyloid plaque burdens by inducing BBB opening and iron deposit at early stage of AD pathology.
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ACKNOWLEDGEMENTS------------------------------------------------------------------- i
ABSTRACT------------------------------------------------------------------------------------- ii
TABLE OF CONTENTS-------------------------------------------------------------------- iv
LIST OF ABBREVIATION----------------------------------------------------------------- vi
LIST OF FIGURES--------------------------------------------------------------------------- vii
LIST OF TABLES---------------------------------------------------------------------------- viii
I. Introduction----------------------------------------------------------------------------------- 1
Overview-------------------------------------------------------------------------------------- 1
A. BBB opening in transient forebrain ischemia-------------------------------- 4
B. BBB opening in Alzheimer's disease-------------------------------------------- 6
C. Purpose of study----------------------------------------------------------------------- 8
II. Material & Methods----------------------------------------------------------------------- 9
A. Materials--------------------------------------------------------------------------------- 9
1. Human brain------------------------------------------------------------------------ 9
2. Animals------------------------------------------------------------------------------- 9
3. Reagents------------------------------------------------------------------------------ 9
4. Antibodies---------------------------------------------------------------------------- 9
B. Methods----------------------------------------------------------------------------------- 10
1. Biochemical methods------------------------------------------------------------- 10
(A) Western Blot Assay-------------------------------------------------------- 10
(B) Immunohistochemistry------------------------------------------------------ 10
(C) Iron histochemistry--------------------------------------------------------- 11
(D) Zinc staining------------------------------------------------------------------ 11
(E) Thioflavin-S staining------------------------------------------------------- 11
(F) In situ detection of free radical generation------------------------- 12
(G) Analysis of BBB permeability------------------------------------------ 12
(H) In situ apoptosis assay---------------------------------------------------- 12
2. In vivo methods------------------------------------------------------------------- 13
(A) Transient forebrain ischemia and drug treatment--------------- 13
(B) Tissue preparation--------------------------------------------------------- 13
3. Statistical analysis----------------------------------------------------------------- 13
III. Results--------------------------------------------------------------------------------------- 14
A. BBB opening after transient forebrain ischemia------------------------ 14
1. Pattern of BBB opening after TFI---------------------------------------- 14
2. Dysfunction of cerebral endothelial cells after TFI------------------ 17
3. ROS production in the cerebral endothelial cells after TFI------- 22
4. Iron accumulation in the cerebral endothelial cells after TFI---- 24
5. The effect of deferoxamine and antioxidant on ROS production and degeneration after TFI---------------------------------------------------- 27
B. BBB opening in Alzheimer disease---------------------------------------- 33
1. Temporal pattern of BBB opening in APP/PS1 mice------------- 34
2. The effect of amyloid peptide on BBB opening------------------- 35
3. Alteration of tight junction proteins in Alzheimer disease------- 39
4. Iron colocalized with amyloid plaque in APP/PS1 mice--------- 45
IV.Discussion------------------------------------------------------------------------------------ 53
A. BBB opening after TFI------------------------------------------------------- 53
B. BBB opening in Alzheimer's disease-------------------------------------- 61
V. Summary and clonclusion---------------------------------------------------------------- 66
BIBLIOGRAPHY-------------------------------------------------------------------------- 68
