Microvascular plasticity in the healthy and diseased mouse cortex by Patrick Reeson B.Sc., University of Calgary, 2005 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSPHY in the Division of Medical Sciences (Neuroscience) Patrick Reeson, 2018 University of Victoria All rights reserved. This dissertation may not be reproduced in whole or in part, by photocopy or other means, without the permission of the author. ii Supervisory Committee Microvascular plasticity in the healthy and diseased mouse cortex by Patrick Reeson B.Sc., University of Calgary, 2005 Supervisory Committee Dr. Craig E. Brown, Division of Medical Sciences Supervisor Dr. Patrick C. Nahirney, Division of Medical Sciences Departmental Member Dr. Bob Chow, Department of Biology Outside Member iii Abstract The brain relies on a properly functioning vasculature system to deliver oxygen and nutrients and remove metabolic waste. However as in all biological systems, the brain is sometimes challenged by small or large-scale failures in the vascular system, which threaten the neuronal networks they support. Cerebral capillaries are uniquely prone to spontaneous obstructions, randomly stopping flow on a moment to moment basis. While not surprising given that capillaries are narrow, low pressure tubes that pass relatively large and adherent cells and debris, the ultimate outcomes of these obstructions are unknown. The vascular response to these events could have profound effects on brain health, as these random events accumulate over time. Similarly, while much research has studied the neural and vascular responses to large vessel obstructions (ischemic stroke), how common comorbidities which also afflict the vasculature, like diabetes, alters vascular plasticity and in turn neuronal rewiring and functional recovery, is not understood. This dissertation furthers our understanding of how microvascular plasticity, in response to either small or larger interruptions to blood flow, affect brain health. In the first aim I examined the fates of cortical capillaries in the mouse somatosensory cortex to either spontaneous or experimentally induced obstructions. Using in vivo 2 photon imaging of cortical blood flow, I found that ~0.12% of cortical capillaries become obstructed each day. Tracking natural or microsphere induced obstructions in anesthetized or awake mice revealed that most capillaries recanalize. Remarkably, 30% of all obstructed capillaries failed to recanalize and were pruned by 21 days. This loss was not compensated for by any angiogenic sprouting in any imaging area. Using this iv information, I was able to predict capillary loss over time that closely matched experimental estimates. From a mechanistic perspective, endothelium specific genetic knockdown or pharmacological inhibition of VEGF-R2 signaling was a critical factor in promoting capillary re-canalization and mitigating subsequent pruning. Thus, this work reveals the incidence, mechanism and long-term outcome of capillary obstructions and contributes to our understanding of age related capillary rarefaction. In the second aim, I examined the vascular adaptions that accompany large scale disruptions to the cerebral blood flow in the form of ischemic stroke. Using a mouse model of type 1 diabetes, I revealed that ischemic stroke leads to an abnormal and persistent increase in Vascular Endothelial Growth Factor Receptor 2 (VEGF-R2) expression in peri-infarct vascular networks. Correlating with this, BBB permeability was markedly increased in diabetic mice which could not be prevented with insulin treatment after stroke. Imaging of capillary ultrastructure revealed that BBB permeability was associated with an increase in endothelial transcytosis rather than a loss of tight junctions. Pharmacological inhibition or endothelial-specific knockdown of VEGF-R2 after stroke attenuated BBB permeability, loss of synaptic structure in peri-infarct regions, and improved recovery of forepaw function. However, the beneficial effects of VEGF-R2 inhibition on stroke recovery were restricted to diabetic mice and appeared to worsen BBB permeability in non-diabetic mice. These results showed that aberrant VEGF signaling and BBB dysfunction after stroke plays a crucial role in limiting functional recovery in an experimental model of diabetes. Overall this dissertation demonstrates that the structure, integrity, and function of mature cerebrovascular networks undergoes substantial changes in the face of both small v and large scale vascular insults. Furthermore, I have revealed a critical role for endothelial VEGF-R2 signalling in mediating many of these vascular changes, which in turn, had important consequences for brain aging and the recovery of function after stroke. vi Table of Contents Supervisory Committee ...................................................................................................... ii Abstract .............................................................................................................................. iii Table of Contents ............................................................................................................... vi List of Abbreviations ......................................................................................................... ix List of Figures ..................................................................................................................... x Acknowledgments............................................................................................................. xii Dedication ........................................................................................................................ xiv Chapter 1 ............................................................................................................................. 1 1.0 Introduction ............................................................................................................. 1 1.1 On the paradox of cerebral blood flow ................................................................... 5 1.2 The anatomy of the cerebral blood supply .............................................................. 7 1.3 The structure of cerebral blood vessels ................................................................. 10 1.4 The Blood-Brain Barrier ....................................................................................... 12 1.4.1 Endothelial Tight Junctions .............................................................................. 14 1.4.2 BBB Endothelial transporters ........................................................................... 16 1.4.3 BBB Endothelial transcytosis ........................................................................... 16 1.4.4 Formation and Regulation of the BBB ............................................................. 17 1.5 The nature of blood flow in cortical capillaries .................................................... 20 1.6 The effects of shear stress on endothelial cell function ........................................ 23 1.7 VEFG-R2 is a master regulator of Endothelial cell function ................................ 24 1.8 Vascular plasticity in the developing and adult brain ........................................... 28 1.9 Vascular obstruction in the healthy brain ............................................................. 30 1.10 Capillary loss with aging ...................................................................................... 31 1.11 Stroke .................................................................................................................... 33 1.12 Stroke in diabetics ................................................................................................. 38 1.13 Rationale and aims ................................................................................................ 44 Bibliography ..................................................................................................................... 47 Chapter 2 VEGF signaling regulates the fate of obstructed capillaries in mouse cortex . 72 2.1 Abstract ....................................................................................................................... 72 2.2 Introduction ................................................................................................................. 73 2.3 Results ......................................................................................................................... 74 2.3.1 Superficial and lower order cortical capillaries are prone to obstruction ................ 74 2.3.2 Fates of obstructed cortical capillaries ..................................................................... 79 2.3.3 Impact of capillary pruning on local blood flow ..................................................... 92 2.3.2 Lower capillary density in aged mice is predicted by obstruction and pruning rates ......................................................................................................................................... 100 2.3.3 VEGF-R2 signaling dictates capillary recanalization ............................................ 106 2.4 Discussion ................................................................................................................. 115 2.4.1 Microsphere Model of obstruction......................................................................... 116 2.4.2 Vascular remodelling in the mature brain .............................................................