Table Of ContentUC San Diego
UC San Diego Electronic Theses and Dissertations
Title
Intravital Live cell Triggered Imaging System Reveals Monocyte Patrolling and Macrophage
Migration in Atherosclerosis
Permalink
https://escholarship.org/uc/item/68x8g3kx
Author
McArdle, Sara Ashley
Publication Date
2015
Supplemental Material
https://escholarship.org/uc/item/68x8g3kx#supplemental
Peer reviewed|Thesis/dissertation
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University of California
UNIVERSITY OF CALIFORNIA, SAN DIEGO
Intravital Live cell Triggered Imaging Reveals Monocyte Patrolling and
Macrophage Migration in Atherosclerosis
A dissertation submitted in partial satisfaction of the requirements for the degree
Doctor of Philosophy
in
Bioengineering
with a Specialization in Multi-Scale Biology
by
Sara Ashley McArdle
Committee in Charge:
Professor Klaus Ley, Chair
Professor Andrew McCulloch, Co-Chair
Professor Yury Miller
Professor Gina Sosinsky
Professor Shankar Subramaniam
2015
Copyright
Sara Ashley McArdle, 2015
All rights reserved.
Signature Page
The Dissertation of Sara Ashley McArdle is approved and it is acceptable in
quality and form for publication on microfilm and electronically:
Co-Chair
Chair
University of California, San Diego
2015
iii
Table of Contents
Signature Page ................................................................................................................... iii
Table of Contents ............................................................................................................... iv
List of Figures .................................................................................................................... ix
List of Tables .................................................................................................................... xii
List of Supplementary Files ............................................................................................. xiii
List of Abbreviations .........................................................................................................xv
Acknowledgements .......................................................................................................... xvi
Vita ................................................................................................................................. xviii
Abstract of the Dissertation ............................................................................................. xix
Chapter 1 Introduction ....................................................................................................1
1.1 Atherosclerosis ...........................................................................................1
1.2 Leukocytes in Atherosclerosis ...................................................................2
1.3 Scope of the thesis ......................................................................................6
Chapter 2 The Duffy Antigen/ Receptor for Chemokines Influences Atherosclerosis ..8
2.1 Background ................................................................................................8
2.2 Previous unpublished work in the lab ......................................................10
2.3 Methods ....................................................................................................15
2.3.1 Mice ....................................................................................................15
iv
2.3.2 Aorta Flow Cytometry ........................................................................15
2.3.3 Aorta RT-PCR ....................................................................................17
2.3.4 Flow Cytometry of Lymphoid Organs ................................................18
2.3.5 Flow Cytometry of Bone Marrow.......................................................20
2.3.6 Immunofluorescence ...........................................................................21
2.3.7 Statistics ..............................................................................................22
2.4 Results ......................................................................................................23
2.4.1 Gross anatomy ....................................................................................23
2.4.2 Aorta Flow Cytometry ........................................................................24
2.4.3 Aorta RT-PCR ....................................................................................24
2.4.4 Flow Cytometry of Lymphoid Organs ................................................27
2.4.5 Immunofluorescence ...........................................................................33
2.5 Discussion ................................................................................................37
2.5.1 Conclusion ..........................................................................................38
Chapter 3 lntravital Live Cell Triggered Imaging System ...........................................39
3.1 Intravital Imaging .....................................................................................39
3.2 Design Criteria .........................................................................................42
3.2.1 Excitation light penetration depth .......................................................42
3.2.2 Tissue Motion .....................................................................................43
v
3.2.3 Cardiac Triggering ..............................................................................44
3.3 Intravital Imaging Method (ILTIS) ..........................................................46
3.3.1 Surgery ................................................................................................46
3.3.2 Multiphoton Microscopy ....................................................................46
3.3.3 Triggered Imaging Method .................................................................48
3.3.4 Image Processing Algorithm...............................................................54
3.3.4.1 Frame Selection ............................................................................54
3.3.4.2 Image Registration .......................................................................55
3.4 Testing ILTIS ...........................................................................................58
3.4.1 Methods...............................................................................................58
3.4.1.1 Mice ..............................................................................................58
3.4.1.2 Movie Quality Quantification ......................................................59
3.4.1.3 Histology and Immunofluorescence of the RCA .........................59
3.4.1.4 Flow Cytometry............................................................................61
3.4.2 Results .................................................................................................61
3.4.2.1 The external carotid artery is a useful model system of the aorta 61
3.4.2.2 Cardiac Triggering Improves Movie Quality ...............................65
3.4.2.3 2D Imaging ...................................................................................67
3.4.2.4 3D imaging ...................................................................................67
vi
3.4.2.5 Intravital imaging accurately images cells ...................................69
3.5 Discussion ................................................................................................71
3.5.1 Conclusion ..........................................................................................73
3.5.2 Acknowledgements .............................................................................73
Chapter 4 Monocyte Patrolling in Atherosclerotic Arteries revealed by ILTIS...........74
4.1 Introduction ..............................................................................................74
4.2 Methods ....................................................................................................75
4.2.1 Mice ....................................................................................................75
4.2.2 Characterization of Blood GFP+ cells ................................................76
4.2.3 Intravital Imaging of GFP+ cells in the blood. ...................................76
4.2.4 Cell Tracking ......................................................................................76
4.3 Results ......................................................................................................78
4.3.1 Characterization of Blood GFP+ cells ................................................78
4.3.2 Characterization of monocytes patrolling the atherosclerotic
endothelium. 79
4.3.3 Hotspot Patrolling ...............................................................................82
4.4 Discussion ................................................................................................84
4.4.1 Conclusion ..........................................................................................87
4.4.2 Acknowledgements .............................................................................87
Chapter 5 Macrophages and DCs in the atherosclerotic artery wall ............................88
vii
5.1 Introduction ..............................................................................................88
5.2 Methods ....................................................................................................89
5.2.1 Mice ....................................................................................................89
5.2.2 Aorta Flow Cytometry ........................................................................90
5.2.3 Whole Mount Immunofluorescence ...................................................93
5.2.4 3D Intravital Imaging of Cells in the plaques .....................................93
5.2.5 Cell Motion Analysis ..........................................................................95
5.3 Results ......................................................................................................96
5.3.1 Characterizing the visible cells in the plaque .....................................96
5.3.2 Whole Mount Artery Imaging ..........................................................106
5.3.3 3D multicolor imaging of macrophages in atherosclerotic plaques. .109
5.4 Discussion ..............................................................................................112
5.4.1 Conclusion ........................................................................................114
5.4.2 Acknowledgements ...........................................................................114
Appendix 1 .......................................................................................................................115
Appendix 2 .......................................................................................................................117
References ........................................................................................................................120
viii
List of Figures
-/- -/- -/-
Figure 2-1| Apoe Darc mice have larger plaques than Apoe mice..............................11
-/-
Figure 2-2| Lipid and glucose levels are similar between Apoe and
-/- -/-
Apoe Darc mice. ............................................................................................................12
Figure 2-3| EC- vs. RBC- DARC expression differentially regulates progression of
atherosclerosis. ...................................................................................................................14
-/- -/- -/-
Figure 2-4| Gross anatomical differences between Apoe and Apoe Darc mice. .........23
-/- -/- -/-
Figure 2-5| Cell composition of aortas of Apoe and Apoe Darc mice........................25
-/- -/- -/-
Figure 2-6| Gene expression in the aorta of Apoe and Apoe Darc mice. ....................26
Figure 2-7| Gating scheme for flow cytometry analysis of myeloid cells in the blood of
-/- -/- -/-
Apoe and Apoe Darc mice. .........................................................................................28
Figure 2-8| Gating scheme for flow cytometry analysis of myeloid cells in the spleen of
-/- -/- -/-
Apoe and Apoe Darc mice. .........................................................................................29
Figure 2-9| Gating scheme for flow cytometry analysis of lymphoid cells in the blood,
-/- -/- -/-
spleen, and para-aortic lymph nodes of Apoe and Apoe Darc mice. ..........................30
Figure 2-10| Increased T cells and decreased monocytes in the in the blood of
-/- -/-
Apoe Darc mice fed WD for 12 weeks. ........................................................................31
-/- -/-
Figure 2-11| Apoe Darc mice have a higher frequency of CD4+ T cells in the spleen
and para-aortic lymph nodes. .............................................................................................32
-/- -/-
Figure 2-12| Apoe Darc mice have more MDPs in their bone marrow than
-/-
Apoe mice. .......................................................................................................................34
Figure 2-13| DARC is present on some vasa vasorum in aortic roots. ..............................35
Figure 2-14| CCL5 localization in aortic roots. .................................................................36
Figure 3-1| Correlation between CD11c surface expression and YFP expression in
YFP
Cd11c mice in live CD45+ leukocytes the blood, spleen, and aorta via flow
cytometry. ..........................................................................................................................41
Figure 3-2| Atherosclerotic plaque in the external carotid artery. .....................................43
Figure 3-3| Mirror and filter combination used in all intravital imaging experiments. .....48
ix
