!     SOLUBLE EPOXIDE HYDROLASE INHIBITION IMPROVES DILATION IN PARENCHYMAL ARTERIOLES AND PREVENTS MEMORY IMPAIRMENT IN HYPERTENSIVE RATS WITH CHRONIC CEREBRAL HYPOPERFUSION.  By Nusrat Matin       A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of  Pharmacology and Toxicology- Doctor of Philosophy 2016     !ABSTRACT SOLUBLE EPOXIDE HYDROLASE INHIBITION IMPROVES DILATION IN PARENCHYMAL ARTERIOLES AND PREVENTS MEMORY IMPAIRMENT IN HYPERTENSIVE RATS WITH CHRONIC CEREBRAL HYPOPERFUSION.  By Nusrat Matin Parenchymal arterioles (PAs) control blood flow to the neurovascular unit that consists of the neurons, glia and capillaries. Epidemiological studies highlight the involvement of PA dysfunction as being a key driver of cerebral small vessel disease, a leading cause of vascular cognitive impairment (VCI). Despite this the structural and functional features of PAs in physiological and pathological models have been largely unexplored.   There are no therapeutic interventions to halt the progression of VCI, a spectrum of cognitive disorders with a cerebrovascular origin. Chronic cerebral hypoperfusion (CCH) and hypertension are major risk factors of VCI. Understanding the combined effects of these morbidities on PA structure and function could provide mechanistic insights into VCI and assist in designing therapies for the condition. In cerebral arteries epoxyeicosatrienoic acids (EETs) are potent dilators that modulate neurovascular coupling. Soluble epoxide hydrolase (sEH) rapidly metabolizes EETs to less active metabolites, thus inhibition of sEH is pharmacologically feasible means of enhancing the pleiotropic effects of EETs by increasing their half-life.   !The present dissertation describes studies to validate and study a clinically relevant model of VCI induced by bilateral common carotid artery stenosis (BCAS) in adult normotensive Wistar Kyoto (WKY) rats and in spontaneously hypertensive stroke prone rats (SHRSP). The working hypothesis was that impaired endothelium dependent dilation and remodeling in PAs after BCAS would accompany cognitive dysfunction in WKY rats (Chapter 2) and SHRSP (Chapter 3). Data are shown as mean ± SEM, Sham vs BCAS. Impaired memory and spatial learning abilities were observed in WKY rats with BCAS. BCAS impaired endothelial function in PAs from WKY rats, as evidenced by reduced carbachol mediated dilation (% dilation at 10-5M: 29.6 ± 8.1 vs -10.2 ± 7.4, p<0.05). In SHRSP with BCAS memory was impaired, however there was no difference in spatial learning abilities. BCAS also impaired endothelial function in PAs from SHRSP (% dilation at 10-5M: 17.5 ±5.3 vs -0.2 ± 2.9, p<0.05). I hypothesized that effects of CCH on cognitive function and PA dilation would be exacerbated in SHRSP. However, Sham SHRSP had impaired spatial learning abilities and PA dilation compared to Sham WKY rats and BCAS did not worsen these impairments.   Furthermore, I hypothesized that chronic administration of a sEH inhibitor in SHRSP with BCAS would prevent CCH induced cognitive impairment by preventing endothelium dysfunction, and arteriole remodeling in the PAs (Chapter 4). Inhibition of sEH improved memory function, increased mRNA markers of neuroprotection and improved PA dilatory function (% dilation at 10-5M, BCAS + vehicle vs BCAS + sEH inhibitor: 3.6 ± 2.4 vs 14.9 ± 3.0, p<0.05). These studies suggest that chronic inhibition of sEH prevents CCH induced memory impairment via neuronal and vascular effects.    !!iv            To Ammu-Abbu, for their unconditional love.           !!v ACKNOWLEDGEMENTS  My PhD endeavor would not have been complete without the support and friendship of some very wonderful people.   First and foremost, I am indebted to Dr. Anne Dorrance, my mentor extraordinaire. AnneÕs intelligence, resilience, and optimism have been a source of inspiration. Her patience and unwavering support has been instrumental during my growth as a PhD candidate. Without her guidance this dissertation would not have been possible.   I also wish to express the deepest appreciation for my committee members Dr. William Jackson, Dr. James Galligan, and Dr. Michelle Mazei Robison for their time and invaluable inputs. Their perspectives and scholarship really helped me to critically evaluate my work. I hope to emulate their dedication and passion in my scientific journey.   I am truly blessed to have met some magnificent people during the last five years. A special thanks goes to Nadine El-Ayache, who has been like a sister to me. I greatly cherish the honesty and strength of her friendship. I am also grateful for the time spent with Janice Diaz Otero and Patricia Perez; our ridiculous musings will always bring a smile to my face. I am also thankful to Paulo Pires, ex post-doc in the Dorrance lab, for showing me how to mount an artery and for his moral support during the learning !!vi process. This dissertation would not have been possible without Courtney Fisher, who I have had a chance to mentor the last few years. Her responsibility and enthusiasm towards our projects has helped me immensely. She has been a pleasure to work with and I will truly miss all her hilarious antics.    I also want to thank the faculty of the Department of Pharmacology and Toxicology. Their brilliance and hard work have inspired and intellectually challenged me. A special mention goes to the administrative personnel for their warmth and ready support. They were always accessible and I could always count on them to make my problems go away.  Lastly, I would like to thank my wonderful family for keeping me in their hearts the last five years. I am forever grateful to my parents for encouraging me to stay curious, to my sisters for teaching me survival skills, and to my niece and nephew for mistaking me as their ÔleaderÕ.         !!vii TABLE OF CONTENTS  LIST OF TABLES                                                                  xii LIST OF FIGURES                    xiii KEY TO ABBREVIATIONS                xv CHAPTER 1                   1 The cerebral circulation in health and vascular cognitive impairment: focus on parenchymal arterioles                 2 1-Overview                       2 1.1 - Cerebral arteries versus peripheral arteries                  3 1.2 - Anatomy of the cerebral vasculature                                                                        6 1.2.1 - Extracranial arteries                   6 1.2.2 - Circle of Willis, and intracranial arteries              7 1.2.3 - Pial arteries                 8 1.2.4 - Parenchymal arterioles                         9 1.2.5 - Capillaries                        11 1.2.6 - Neurovascular unit                        12 1.3 - Physiology of cerebral arteries              13 1.3.1 - Myogenic tone, and autoregulation in cerebral arteries                    14 1.3.2 - Nitric oxide                          18 1.3.3 - Prostaglandins                                                 19 1.3.4 - Endothelium-derived hyperpolarizing factor                                   20 1.4 - The effects of hypertension on the cerebral circulation                     23 1.4.1 - Impaired endothelium dependent dilation                        23 1.4.2 - Cerebral artery remodeling                        25 1.4.3 - Stroke prone spontaneously hypertensive rat                     27 1.5- Vascular cognitive impairment                        28 1.5.1 - Vascular cognitive impairment: current concepts and clinical  developments                           29 1.5.2 - Hypertension as a risk factor for VCI                       30 1.5.3 - Current therapies for VCI                        32 1.5.4 - VCI models of chronic cerebral hypoperfusion                     33 1.5.5 - Acetazolamide challenge              35 1.5.6 - Cognitive assessments                        36 1.6 - The pleiotropic effects of EETs                         39 1.6.1 - Arachidonic acid                         39 1.6.2 - The transitory life of EETs                        41 1.6.3 - Physiological effects of EETs                        42 1.6.4 - Soluble epoxide hydrolase inhibition                       45 !!viii 1.7 - Scope of this project                          47 1.7.1 - BCAS in WKY rats                        47 1.7.2 - BCAS in SHRSP                        48 1.7.3 - Chronic TPPU treatment of SHRSP with BCAS                      48 REFERENCES                           50  CHAPTER 2                  77 Bilateral common carotid artery stenosis in normotensive rats impairs endothelium dependent dilation of parenchymal arterioles                 78 2.1 - Abstract              78 2.2 - Introduction                 79 2.3 - Methods                 80 2.3.1 - Animals, and surgery                    80 2.3.2 - Novel objection recognition test            82 2.3.3 - Morris water maze               82 2.3.4 - Cerebral tissue perfusion              83 2.3.5 - Acetazolamide challenge              83 2.3.6 - PA isolation, and cannulation             84 2.3.7 - Assessment of the structural, and mechanical properties in PAs        85 2.3.8 - Cannulation, and assessment of the structural, and mechanical  properties of PComAs                86 2.3. 9 - Real time-polymerase chain reaction                  86 2.3.10 - Statistical analyses               87 2.3.11 - Chemicals, and reagents              87 2.4 - Results                  87 2.4.1 - BCAS impaired memory function, spatial learning abilities, and abolishes cerebrovascular reserve capacity            87 2.4.2 - BCAS impaired dilation in PAs            88 2.4.3 - Endothelium dependent dilation in PAs was independent of NO, and  prostaglandin                 88 2.4.4 - EETs-mediated dilation in PAs from Sham rats                 89        2.4.5 - BCAS did not induce remodeling the PAs            89 2.4.6 - PComAs from BCAS rats exhibited structural remodeling         89 2.4.7 - BCAS changed mRNA expression of key mediators in  EETs dilatory pathway                 90 2.5 - Discussion                 99 2.5.1 - BCAS impaired memory function, spatial learning abilities, and  abolished cerebrovascular reserve capacity            99 2.5.2 - BCAS attenuated endothelium-dependent dilation in the PAs       100 2.5.3 - BCAS impaired EETs-mediated dilation in the PAs                    101 2.5.4 - Heterogeneous remodeling of the PAs and PComAs after BCAS              103 2.6 Ð Limitations               104 REFERENCES               105  !!ix CHAPTER 3                     111 Carotid artery stenosis in hypertensive rats impairs dilatory pathways in parenchymal arterioles and posterior communicating arteries        112 3.1 - Abstract                112 3.2 - Introduction               113 3.3 - Methods                       114 3.3.1 - Animals, and surgery              114 3.3.2 - Measurement of cerebral perfusion           115 3.3.3 - Novel object recognition test            115 3.3.4 - Morris water maze             116 3.3.5 - Acetazolamide challenge            116 3.3.6 - PA isolation and cannulation            117 3.3.7 - Assessment of dilatory pathways in the PAs          117 3.3.8 - Cannulation, and assessment of dilatory pathways in the PComAs        118 3.3.9 - Assessment of the structural and mechanical properties of arteries     118 3.3.10 - Statistical analysis             119 3.3.11 - Chemical reagents             119 3.4 - Results                119 3.4.1 - 8 weeks after BCAS cerebral perfusion was restored, but CVR  was absent in both Sham and BCAS rats            119 3.4.2 - BCAS impaired memory function in SHRSP         120 3.4.3 - Altered dilatory signaling and remodeling in PAs after BCAS       120 3.4.4 - PComAs exhibit impaired dilation and outward remodeling after BCAS               122 3.5 - Discussion               133 3.5.1 - BCAS impaired memory function in SHRSP         133 3.5.2 - CVR was absent in SHRSP            134 3.5.3 - Dysfunctional dilatory pathways in PAs from BCAS rats        135 3.5.4 - Impaired myogenic tone, and dilation in PComAs from BCAS rats      137 3.5.5 - Differential remodeling in PComAs and PAs from BCAS rats      138 3.6 - Limitations               140 REFERENCES               141  CHAPTER 4                149 The effects of soluble epoxide hydrolase inhibition on parenchymal arterioles in a hypertensive model of cognitive impairment          150 4.1 - Abstract               150 4.2 - Introduction               151 4.3 - Methods              153 4.3.1 - Animals and surgery                                 153 4.3.2 - TPPU treatment             154 4.3.3 - Blood pressure measurement           154 !!x !4.3.4 - Novel objection recognition test           154 4.3.5 - Morris water maze             154 4.3.6 - Cerebral tissue perfusion and acetazolamide challenge        155 4.3.7 - PA isolation and cannulation            156 4.3.8 - Assessment of structural and mechanical properties in PAs       157 4.3.9 - High-performance liquid chromatography coupled with tandem  mass   spectrometry for TPPU measurement          157 4.3.10 - Real-time polymerase chain reaction                              158 4.3.11 - Statistical analyses             158 4.3.12 - Chemicals and reagents            159 4.4 - Results                          159 4.4.1 - Physiological parameters, and cerebrovascular reserve capacity  were unchanged in TPPU treated BCAS rats.         159 4.4.2 - TPPU prevented memory deficits in BCAS rats         159 4.4.3 - TPPU improved dilation in PAs from BCAS rats without affecting artery structure               160 4.4.4 - Chronic TPPU treatment enhanced PA dilation in Sham rats  and had no effect on memory            161 4.4.5 - Chronic administration of TPPU altered mRNA expression in  the brain and arteries from BCAS rats           161 4.5 - Discussion               171 4.5.1 - TPPU treatment prevented CCH-induced memory impairment  in BCAS rats                172 4.5.2 - TPPU treatment prevented endothelial dysfunction in PAs  from BCAS rats               173 4.5.3 - NO/prostaglandins, and EETs mediate dilation in PAs from  TPPU treated BCAS rats.                                                                                      174 4.5.4 - TPPU treatment had no effect on PA passive structure        176 4.5.5 - TPPU treatment had no effect on cerebrovascular reserve  capacity in BCAS rats              177 4.6 - Limitations               178 REFERENCES                         179  CHAPTER 5                186 Soluble epoxide hydrolase inhibition improves dilation in parenchymal arterioles and prevents memory impairment in hypertensive rats with chronic cerebral hypoperfusion                187 5.1 - General Conclusions                  187 5.2 - Considerations about the model used, and treatment regimens used       191 5.3 - Novel findings               193 5.3.1 - The effects of BCAS on WKY rats           194 5.3.2 - The effects of BCAS on SHRSP           194 5.3.3 - Sham WKY rats versus Sham SHRSP          195 5.3.4 - The effects of TPPU on SHRSP with BCAS, and on Sham SHRSP      196 !!xi 5.4 - Limitations               196 5.5 - Perspectives               198 REFERENCES               199                      !!xii LIST OF TABLES  Table 1: Significant key differences in dilatory pathways and structural properties between PAs from Sham WKY rats and Sham SHRSP.                  123                    !!xiii LIST OF FIGURES  Figure 1. 1: Major arteries and circle of Willis.                       6 Figure 1. 2: The cerebrovascular tree.                         9 Figure 1. 3:  PAs regulate neurovascular coupling.               11 Figure 1. 4:  Autoregulation in cerebral arteries.                 17 Figure 1. 5: Endothelium-dependent dilatory pathways.           22 Figure 1. 6: The effects of hypertension on the cerebral circulation.          26 Figure 1. 7: Hypertension increases the risk of developing VCI.          32 Figure 1. 8: Cognitive assessments.              38 Figure 1. 9: Arachidonic acid signaling cascade.            40 Figure 1.10: EETs-mediated dilation.              43 Figure 1.11: Overarching hypothesis of the dissertation.           49 Figure 2. 1: Bilateral common carotid artery stenosis.            81 Figure 2. 2: BCAS impaired memory function and spatial learning abilities.         91 Figure 2.3: 8 weeks after BCAS cerebral perfusion was restored, however cerebrovascular reserve capacity was impaired.                92                                                       Figure 2.4: Impaired dilation in PAs from BCAS rats.            93 Figure 2.5: EETs - mediated dilation is impaired in PAs from Sham rats.         94 Figure 2.6: There was no difference in passive structure of PAs between  the two groups.                 96  Figure 2.7: Outward remodeling in the PComAs from BCAS rats.             97 Figure 2.8: Altered expression of markers involved in EETs-mediated  signaling in BCAS rats.                98 !!xiv  Figure 3. 1: Cerebral perfusion was restored in BCAS rats and cerebrovascular  reserve capacity was absent in both Sham and BCAS rats.            124  Figure 3.2: BCAS impaired memory function with no change in spatial learning  abilities.                                                                              125            Figure 3.3: Dilation was abolished in PAs from SHRSP with BCAS.                           126 Figure 3.4: Inhibition of CYP epoxygenase restored dilation in PAs from BCAS rats. 127 Figure 3.5: Hypotrophic remodeling in the PAs from BCAS rats.        129 Figure 3.6: Impaired myogenic tone generation, dilation, and remodeling  in the PComAs after BCAS.             131  Figure 4. 1: TPPU treatment did not change mean cerebral perfusion  or cerebrovascular reserve capacity 8 weeks after BCAS.            162  Figure 4. 2: TPPU treatment improved memory in BCAS rats.        163 Figure 4.3: Increased dilation in PAs from TPPU treated BCAS rats compared  to vehicle treated BCAS rats.             164  Figure 4. 4: Enhanced endothelium dependent dilation and altered dilatory  pathways in PAs from TPPU treated BCAS rats.           166  Figure 4. 5: TPPU enhanced PA dilation and had no effect on memory function  in Sham rats.                168  Figure 4. 6: Increased expression of mRNA levels in brains from TPPU  treated BCAS rats.                169  Figure 4. 7: No difference in mRNA expression of sEH and mediators of EETs signaling in MCAs and PAs from the BCAS groups.                    170  Figure 5. 1: Dilatory pathways assessed in parenchymal arterioles.       188 Figure 5. 2: Summary of novel findings.            193     !!xv KEY TO ABBREVIATIONS  AA : arachidonic acid  BBB: bloodÐbrain barrier  BCAS: bilateral common carotid artery stenosis BH4: tetrahydrobiopterine  BKCa: large conductance calcium activated potassium channels  CBF: cerebral blood flow CCH: chronic cerebral hypoperfusion COX: cyclooxygenase CVR: cerebrovascular reserve capacity  CYP: cytochrome P450  DAG: diacyl glycerol  DHET: dihydroxyeicosatrienoic acids  EDHF: endothelium-derived hyperpolarizing factor EET: epoxyeicosatrienoic acid  EGTA: ethylene glycol tetra acetic acid GSK101:(N-((1S)-1-{[4-((2S)-2-{[(2,4-Dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide  20-HETE: 20-hydroxyeicosatetranoic acid  IEL: internal elastic lamina IKCa: intermediate conductance calcium activated potassium channel !!xvi Indo: indomethacin IP3: inositol triphosphate L-NAME: N-nitro-L-arginine methyl ester MWM: Morris water maze MEP: myoendothelial projection  MCA: middle cerebral artery MS-PPOH: N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide NF-kB: nuclear factor kappa-light-chain-enhancer of activated B cells  NO: nitric oxide  NORT: novel object recognition test NOS: nitric oxide synthase NOS1: brain nitric oxide synthase NOS2: inducible nitric oxide synthase  NOS3: endothelial nitric oxide synthase NVC: neurovascular coupling PLA2: phospholipase A2  PA: parenchymal arterioles  PLC: phospholipase C  PSS: physiological salt solution PComA: posterior communicating arteries  PEG400: polyethylene glycol 400 PKC: protein kinase C  !!xvii ROS: reactive oxygen species  RyR: ryanodine receptor sEH: soluble epoxide hydrolase SHR: spontaneously hypertensive stroke-resistant rat  SHRSP: stroke prone spontaneously hypertensive rat  SKCa: small conductance calcium activated potassium channel  SNP: sodium nitroprusside SOD-3: superoxide dismutase-3 SR: sarcoplasmic reticulum  TPPU: 1-(1-propanoylpiperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl] urea TRP: transient receptor potential cation channel TRPC6: transient receptor potential cation channel subfamily canonical member 6 TRPM4: transient receptor potential cation channel subfamily melastatin member 4 TRPV4: transient receptor potential cation channel subfamily vanilloid member 4 UCP-2: uncoupling protein 2 VCI: vascular cognitive impairment WKY: Wistar Kyoto       !1            CHAPTER 1           !2 The cerebral circulation in health and vascular cognitive impairment: focus on parenchymal arterioles 1- Overview  !The cerebral circulation is exquisitely adapted to regulate blood flow to the brain, one of the most highly perfused organs in the body. Nitric oxide (NO), prostaglandins and endothelium-derived hyperpolarizing factor (EDHF) mediate dilation in cerebral arteries and the complex interplay between these signaling pathways is essential to brain homeostasis [56,77,81]. Chronic hypertension disrupts these signaling pathways and induces deleterious structural changes to the cerebral arteries, all of which can have a profound effect on brain function [207]. These pathological changes in the cerebral arteries may be intimately related to the development of vascular cognitive impairment (VCI).   Risk factors such as hypertension, and carotid stenosis lead to VCI and often appear as comorbidities [6,32,86,91]. Understanding the cerebrovascular effects of complicated etiologies has been one of the major challenges in finding reliable therapeutic options for controlling the advancement of VCI. This chapter summarizes the physiological characteristics of the cerebral vasculature, followed by a discussion of how changes in the vasculature could aide in the progression of VCI.   !3 1.1- Cerebral arteries versus peripheral arteries The high metabolic demand of neurons causes the brain to require 20% of the body's total oxygen consumption and 25% of total glucose utilization, despite the brain being only 2% of body weight. To meet this demand cerebral blood flow (CBF) is highly regulated and this involves multiple well-coordinated regulatory mechanisms unique to the cerebral circulation. Thus notable functional, and structural differences exist between the cerebral circulation and circulation in other organs [207]. In this section I will address some of those significant differences.  Cerebral arteries and arterioles consist of three concentric layers with the innermost layer, the tunic intima, consisting of a single layer of endothelial cells and a well-developed internal elastic lamina[139]. Next to the tunic intima is the tunica media, which contains mostly smooth muscle cells with very little elastin and some collagen fibers. The outermost layer, tunica adventitia, is very thin in cerebral vessels compared to peripheral arteries and consists of associated cells such as perivascular nerves, pericytes and astrocytic end-feet. Cerebral arteries, in contrast to peripheral arteries, lack an external elastic lamina [139]. Smooth muscle cells in cerebral arteries are arranged circularly, perpendicularly oriented to the blood flow, a possible adaptation to the high wall tension. In contrast, most peripheral arteries have oblique oriented smooth muscle cells around the long axis of the vessel[34].   Another significant difference between the peripheral and the cerebral circulation is the presence of the bloodÐbrain barrier (BBB). BBB exists at all levels of the !4 cerebrovascular tree except the circumventricular organs[275]. The BBB consists of specialized endothelial cells that lack fenestrations and have apical tight junction proteins. The endothelium provides structural properties of the BBB, while astrocytes, pericytes and the extracellular matrix contribute to its functional properties. BBB acts as a selective transport barrier and forces most molecular traffic to take a transcellular rather than paracellular route across the BBB, unlike peripheral endothelial cells [97]. With a combination of intracellular and extracellular enzymes the BBB is also a 'metabolic barrier', inactivating many neuroactive and toxic compounds. In this manner the BBB allows the entry of required nutrients, while excluding potentially harmful compounds and limiting access of many humoral stimuli to cerebral smooth muscle cells or the brain.   In the peripheral circulation the majority of vascular resistance comes from small arteries and arterioles, whereas in the cerebral circulation 50-60% of total cerebral vascular resistance lies in the large extracranial and intracranial arteries. In the large cerebral arteries intravascular pressure is approximately half of systemic blood pressure highlighting their critical role in determining cerebral perfusion [94]. Thus, a prominent feature of large cerebral arteries is the regulation of CBF and microvascular perfusion pressure; this is key to protecting downstream vessels during fluctuations in arterial pressure. Small arterioles and capillaries within the brain parenchyma contribute around 33% of total vascular resistance in the cerebral circulation[94]. This segmental arrangement of vascular resistance is modulated by interactions between multiple cell !5 types, including vascular cells, neurons, and astrocytes to enable optimal regulation of microvascular pressure and maintenance of homeostasis in the brain[65].   Another example of cerebral vasculaturesÕ vigilance to maintain homeostasis is the cerebral arteriesÕ response to hypercapnia and hypoxia. Compared to peripheral vascular beds (eg. renal, cutaneous, and skeletal muscle) hypercapnia and hypoxia are potent vasodilators in the cerebral circulation since the direct vasodilator effects are not offset by vasoconstrictor response via chemoreflex [101].   These differences between the cerebral and peripheral arteries means it is impossible to accurately extrapolate findings in the periphery to the brain. However, many more studies are conducted in peripheral arteries than in cerebral arteries. Therefore, data from peripheral arteries have been used in this dissertation for discussion when no similar data in cerebral arteries were available.    !6  Figure 1.1: Major arteries and circle of Willis. 1: Vertebral arteries; 2: basilar artery; circle of Willis- {3: internal carotid arteries; 4: posterior cerebral arteries; 5: posterior communicating arteries; 6: anterior communicating arteries; 7: anterior cerebral arteries}; 8: middle cerebral arteries. Figure courtesy of Daniel Bollman.  1.2- Anatomy of the cerebral vasculature 1.2.1- Extracranial arteries. Blood is supplied to the brain from two pairs of large arteries, the right and left internal carotid and the right and left vertebral arteries, and the latter merge to form the basilar artery (Figure 1.1). The internal carotid arteries supply !7 the cerebrum, and branches of the vertebral and basilar arteries supply blood for the cerebellum and brain stem. In healthy adults, flow in the internal carotid arteries represents 72% of the total CBF, while vertebral arteries supply the rest [273]. The basilar artery joins the circle of Willis, a complete anastomotic ring at the base of the brain.   1.2.2 - Circle of Willis, and intracranial arteries. The circle of Willis consists of the posterior communicating arteries (PComAs), posterior cerebral arteries, internal carotid arteries, anterior cerebral arteries and the anterior communicating artery (Figure 1.1). Three pairs of large arteries: the anterior, middle, and posterior cerebral arteries, branch from this anastomotic ring. These arteries run along the surface of the brain and perfuse the cerebral cortex. Amongst the arteries emanating from the circle of Willis, blood supply from the middle cerebral artery covers the largest area of the brain. The middle cerebral artery and its branches supply the posterior parietal, angular, occipital cortices and regions in the temporal lobe to name a few [12]. The areas supplied are critical for speaking, mood, judgment, and motor synchronization. Branches of the posterior cerebral artery supply blood to the midbrain, the hippocampus, as well as the parieto-occipital and temporal cortex. These regions govern attention, memory and visual information processing[99]. The anterior cerebral artery and its branches supply blood to the superior and inferior parietal cortex, orbitofrontal and frontopolar cortex, and the pericallosal region. These regions are important for memory, judgment, mood and motor movement processing [11].  !8 The large arteries of the circle of Willis are part of the cerebral collateral circulation that serves as the subsidiary network of arteries that restore CBF when principal conduits fail such as during carotid artery stenosis[187]. The size and patency of these primary collateral networks are risk factors for cerebral infarction in patients with severe stenosis of a carotid artery [187]. Ophthalmic arteries and leptomeningeal arteries serve as secondary collaterals and are only recruited if the severity of chronic hypoperfusion exceeds the primary collateralsÕ capacity to restore CBF.  Blood flow across the anterior communicating artery and reversal of flow in the proximal anterior cerebral artery provide collateral support to both hemispheres in the anterior portion of the circle of Willis. The proximal posterior cerebral arteries can supply collateral flow from the vertebrobasilar circulation, but only to the posterior circulation, whereas the PComAs can provide collateral support to both the anterior and posterior circulations [144].   1.2.3 - Pial arteries. The large cerebral arteries branch into the pial arteries on the surface of the brain within the piaÐarachnoid (also known as the leptomeninges). They branch into smaller arteries and arterioles on the brain surface and provide an abundance of anastomoses that serve as important collaterals if a surface artery is occluded (Figure 1.2).  Nerves from the peripheral nervous system innervate the adventitial layer of pial arteries. The density of this extrinsic innervation decreases as the pial arteries branch into penetrating arterioles and enter the VirchowÐRobin space, a continuation of the subarachnoid space containing cerebrospinal fluid.  !9 Figure 1.2: The cerebrovascular tree. Pial arteries and arterioles have extensive two-dimensional anastomoses that serve as collateral networks when a surface artery is occluded. In contrast, penetrating arterioles and PAs are predominantly branchless and thus control parenchymal perfusion. As PAs dive further into the parenchyma they become capillaries that have a tortuous three- dimensional network. Adapted from [183], with permission.  1.2.4 - Parenchymal arterioles. Penetrating arterioles branch off from pial arteries and dive, almost perpendicularly, into the brain parenchyma [231]. This right angle !10 bifurcation ensures a significant decrease in blood pressure [256] and protects the penetrating arterioles and capillaries from increased wall stress. As they enter the brain these arterioles are bathed by cerebrospinal fluid in the Virchow-Robin space. Beyond the Virchow-Robin space penetrating arterioles turn into parenchymal arterioles (PAs) [207]. PAs can have varying depths of penetration [216], some PAs are long enough to run through the whole cerebral cortex. In the rat brain PAs supply blood to discrete cortical regions that extend up to 350 !m in radius with limited collateral support from neighboring PAs. Thus, in contrast to occlusions in pial arteries or capillaries, occlusion of a single PA drastically reduces flow through downstream capillaries [183] and can cause cognitive deficits [231] (Figure 1.2).   PAs are extensively covered by astrocytic end-feet with just one layer of smooth muscle cells [159] (Figure 1.3). PAs are the primary regulators of neurovascular coupling (NVC) [64], a process by which local CBF is temporally and spatially matched to neuronal activity [65].  Astrocytes receive noradrenergic, serotonergic, cholinergic, and GABAergic inputs from subcortical neurons in the locus coreuleus, raphe nucleus, basal forebrain, or local cortical interneurons and play an integral role in modulating PA tone [29,92,251]. PAs response to neurotransmitters differs from larger cerebral arteries due to differences in population of post-synaptic receptors on the smooth muscle cells and the endothelium. For example, unlike the middle cerebral arteries that have "-adrenoceptors, #-adrenoceptors are present in PAs and norepinephrine thus induces vasodilation [42]. Similarly, in contrast to the marked contractile effect of serotonin in the large cerebral arteries, PAs dilate to this neurotransmitter [146]. !11  Figure 1.3:  PAs regulate neurovascular coupling. PAs dive into the parenchyma from pial arteries on the surface of the brain. Beyond the Virchow-Robin space the PAs lose their extrinsic innervation and become completely wrapped in astrocytic terminal processes called Ôend-feetÕ. Changes in Ca2+ levels in end-feet convey changes in neuronal activity to PAs and modulate arteriolar tone to match the metabolic needs of the surrounding parenchyma. From [46], with permission.  1.2.5 - Capillaries. Capillaries are a three-dimensional network of microvessels (3-10!m) with extensive anastomoses, that facilitate a major portion of the exchange of gas, metabolites, and heat (Figure 1.2) [216]. Depending on the location and metabolic demand of the brain region, the density of capillaries varies significantly with higher density in grey matter compared to white matter [134]. Approximately 90% of the capillaries in the rat brain are continuously perfused [80]. PA dilation increases the intravascular pressure gradient between the pre-capillary arteriole and post-capillary !12 venule and increases capillary flow. Flow from the capillaries eventually drains into the parenchymal venules that return blood to central sinus in the cortex.   Capillaries lack smooth muscle cells and consists of endothelial cells that are encased long, thin pericyte processes and astrocytic end-feet. Together with astrocytes, pericytes may play a role in sensing metabolic changes in the microenvironment milieu and relaying that information to PAs upstream to modulate blood flow [103].   1.2.6 - Neurovascular unit. NVC occurs in the neurovascular unit that is comprised of neurons, astrocytes and vascular cells (Figure 1.3). An increase in blood flow in response to increased neuronal activity is called functional hyperemia and is made possible by NVC. Dilatory stimuli released from the foci of increased neuronal activity need to be conducted to arteries upstream for functional NVC [53,110]. There has been debate about the primary site of CBF regulation during functional hyperemia. An in vitro study showed that contractile pericytes might have the ability to actively regulate capillary diameter [205]. However, their contribution to functional hyperemia in vivo is yet to be determined [64]. Sensory stimulation evokes rapid and significant dilation of PAs, but only a slow and passive dilation of pericyte-covered capillaries [103] suggesting that PAs regulate blood flow during functional hyperemia. Moreover, as opposed to the noncircumferential, longitudinal morphology of pericytes that surround capillaries, actins are present in a circumferential band-like fashion in PA smooth muscle cells. This arrangement of actins allows them to exert radial forces necessary to control changes in vessel diameter.  !13 Increased neuronal activity releases glutamate that binds to metabotrophic glutamate receptors in astrocytes and elevates intracellular Ca2+ [Ca2+]i levels [244]. Depending on the intensity of the stimulation both presynaptic and postsynaptic neuronal activity may modulate arteriolar changes during NVC [206]. A widely held view is that large conductance Ca2+ activated potassium (BKCa) channels in astrocytic end-feet release K+ ions in response to an increase in astrocytic Ca2+ levels [66,79]. This increases the K+ concentration in the perivascular space and activates inward rectifier K+ channels on the smooth muscle cells of adjacent PAs [66,148]. Study suggests that K+ concentration <20 mM induces membrane hyperpolarization, whereas K+ concentrations ! 20 mM could lead to vasoconstriction through depolarization of PA smooth muscle cells [66]. Many other vasoactive agents have been proposed to modulate NVC including vasodilators such as epoxyeicosatrienoic acids (EETs), prostaglandins, adenosine, oxygen, NO, and H+ [5,109].  Astrocytes can also release arachidonic acid (AA) can that can be converted to 20-hydroxyeicosatetranoic acids (20-HETEs) in smooth muscle cells of the PAs to induce vasoconstriction [174]. Constriction and dilation during NVC is dependent on multiple factors such as astrocytic Ca2+ levels [79], NO [164],  and oxygen levels in the surrounding tissue [85].  1.3 - Physiology of cerebral arteries While NVC enables variation in blood flow in the microcirculation to match the demands of the neurons and the glia, CBF has to remain constant even when there are fluctuations in systemic arterial pressure. Cerebral autoregulation maintains a constant CBF over a range of arterial pressure, and is a consequence of the ability of resistance !14 arteries to generate myogenic tone. Myogenic tone is the intrinsic property of smooth muscle cells to constrict in response to an increase in intravascular pressure [10], independent of neuronal, endothelial and circulating factors [161]. This section will briefly discuss autoregulation, generation of myogenic tone and endothelial control of tone.   1.3.1- Myogenic tone, and autoregulation in cerebral arteries. The mechanism involved in the mechanotransduction of pressure or stretch into sustained myogenic tone is complex and still not well understood. In recent years several lines of work implicated the activation of mechanosensors transient receptor potential cation channels (TRPs) as being critical in mediating pressure induced membrane depolarization in cerebral arteries [49,264]. In cerebral artery smooth muscle cells, an increase in intravascular pressure activates TRP subfamily canonical member 6 (TRPC6) channels directly [84] or indirectly via Gq/11 mechanosensors such as AT1 angiotensin II receptor[160]. Ca2+ influx through TRPC6 leads to Ca2+-induced Ca2+ release via inositol triphosphate receptors on the sarcoplasmic reticulum (SR) and is fundamental to the generation of tone. Increased  [Ca2+]i level activates TRP subfamily melastatin member 4 (TRPM4) channels and results in an influx of Na+ and depolarization of cerebral smooth muscle cell [15,84]. Depolarization activates voltage-gated Ca2+ channels, and Ca2+ influx through these channels results in pressure-induced vasconstriction and myogenic tone.  !15 PAs are exposed to lower pressure than pial arteries and thus PA smooth muscle cells generate tone in response to lower levels of intravascular pressure. In isolated PAs increasing intravascular pressure from 5mmHg to 40mmHg can depolarize smooth muscle cells from -60mV to -35mV. Depolarization results in Ca2+ influx via opening of L-type voltage-gated Ca2+ channels[181,188], activation of Ca2+-dependent molecular machinery responsible for actin-myosin cross-bridge cycling, smooth muscle cell contraction and generation of up to 40% myogenic tone [135,170]. PAs generate more tone compared with pial arteries and studies suggest that this can be attributed to lack of negative feedback from BKCa channel-mediated hyperpolarization under resting conditions [41]. PA tone sets the level of resting perfusion and enables PAs to dilate and constrict to match neuronal activity.  Myogenic tone is modulated by vasoactive factors released from the endothelium [22,35,151], perivascular nerves[92], as well changes in the cerebral microenvironment. For example, reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide can have potent dilatory effects on cerebral arteries and this reduces myogenic tone [140]. Alternatively, at high concentrations, superoxide also increase myogenic tone by inactivating NO[59], generating peroxynitrite[57], or by the generation of F2 isoprostanes from AAs[105]. Similar biphasic effect has also been reported with H2O2 [140].   Myogenic reactivity, defined as alterations in tone in response to changes in intravascular pressure, enables cerebral autoregulation [162]. Autoregulation is crucial !16 to maintain perfusion, to protect downstream arterioles, capillaries and the BBB from fluctuations in arterial pressure[136].  In healthy humans, autoregulation in large cerebral arteries is maintained when arterial pressure is between 50 and 150$mm$Hg[204] (Figure 1.4). Pressures below the lower limit of 50 mmHg can lead to cerebral hypoperfusion, ischemia and neuronal death. At pressures above the upper limit of 150 mmHg arteries exhibit forced dilation with complete loss of tone production, and rapid increase in wall tension. Pressure beyond the upper limit can induce hyperperfusion, and vasogenic edema[194].   These limits of cerebral autoregulation in cerebral arteries are not entirely fixed, and can be modulated by myogenic, metabolic, and neurogenic mechanisms [82,138,161]. Markedly impaired autoregulation as been reported in chronic hypertension, stroke and AlzheimerÕs disease[44,58,184]. In young hypertensive rats the autoregulatory range shifts to the right towards higher pressures [72]. The degree of autoregulatory dysfunction induced by hypertension correlates with the severity of periventricular white matter injury[86], suggesting that autoregulation disruption plays a significant role in driving cerebrovascular pathologies. The lower limit of autoregulation can be particularly important in models of carotid artery occlusion/stenosis. Under normal conditions a reduction in intravascular pressure after carotid artery occlusion/stenosis would lead arteries, downstream from the occlusion, to dilate and increase perfusion. However, hypertension increases the lower limit of autoregulation. Thus after carotid artery occlusion/stenosis the intravascular pressure may drop below the lower limit of autoregulation, this could cause large artery collapse that can worsen cerebral !17 hypoperfusion [72]. Following carotid artery occlusion, pressure measured in the carotid artery was above the lower limit for the normotensive controls whereas for SHR it remained below the lower limit. The authors suggested that this critical reduction in perfusion pressure in carotid arteries from SHR might be a major causative factor for the high mortality rate and extensive cerebral infarction following bilateral carotid artery occlusion in this model of hypertension [71].    Figure 1. 4: Autoregulation in cerebral arteries. During autoregulation CBF (blue line) remains constant as intravascular pressure increases from 50 to 150 mmHg. Myogenic reactivity is active within this range and reduces lumen diameter of the arteries (top red circles). Below the lower limit arteries fail to dilate to maintain CBF and !18 causes hypoperfusion, and arteries collapse causing ischemia.  Above the upper limit of autoregulation arteries exhibit force-mediated dilation that can lead to vasogenic edema. From [207], originally modified from [153] with permission.   1.3.2 - Nitric oxide. The discovery of NO, initially called endothelium-derived relaxing factor, was the most significant contribution to understanding of circulatory control[73,111,196]. NO synthase (NOS) [260] converts L-arginine to L-citrulline and releases NO as a by-product. The reaction takes place in the presence of several co-factors including the easily oxidized tetrahydrobiopterine (BH4) [141].  NOS exists as three major isoforms: endothelial NOS (NOS3), brain NOS (NOS1), and inducible NOS (NOS2) [260]. Activation of endothelial receptors or deformation of the endothelium by mechanical forces increases [Ca2+]i leading to the formation of Ca2+-calmodulin complex that activates NOS3. Alternatively, shear stress induced NOS3 phosphorylation leads to increased NO production mostly independent of changes in [Ca2+]i [143, 292]. NO diffuses from the endothelium to the vascular smooth muscle, where it stimulates soluble guanylyl cyclase to generate cyclic guanosine monophosphate. Cyclic guanosine monophosphate activates protein kinases resulting in reduced [Ca2+]i, decreased sensitivity of contractile apparatus to Ca2+, and K+ efflux via activation of  Ca2+ activated potassium (KCa) channels [13,222].   The physiological significance of NO is evident because infusion of inhibitors of NOS induces vasoconstriction in vascular beds, including the cerebral circulation [56,77,250]. Additionally, chronic NO inhibition increases systemic arterial blood pressure[218]. !19 These studies showed that NOS3 regulates basal tone in vascular beds and is critical for vascular homeostasis, thus its expression and activity needs to be tightly controlled. The [Ca2+]i level is an important regulator of NOS3, since Ca2+ activated calmodulin releases NOS3 bound to its regulatory binding partner calveolin-1, the major coat protein of calveolae [235]. Phosphorylation of NOS3 can increase or decrease enzyme activity depending on phosphorylation sites. NOS3 phosphorylation is a regulatory mechanism during chronic hypertension [143]. Along with phosphorylation, NOS3 activity can also be modulated by acetylation[121], and S-glutathionylation[31]. A critical determinant of NOS3 activity is the availability of BH4 [266]. Pathological states that limit the co-factor availability can lead to NOS3 uncoupling with reduced NO levels and elevated superoxide production [4,127,197]. Furthermore, NO itself modulates enzyme activity by its ability to form stable nitrosyl complexes with heme-containing NOS3 [3,128,168]. 1.3.3 - Prostaglandins. Cyclooxygenase (COX) enzymes break down AA to produce vasodilators like prostaglandin I2 (prostacylin or PGI2), prostaglandin E2, and prostaglandin D2. Three COX isoforms, COX-1, COX-2, and COX-3 are expressed in the neurons, glia, and cerebral vessels[132].  Vasodilatory prostaglandins increase cyclic AMP levels and protein kinase A activity in vascular smooth muscle cells. Activation of protein kinase A opens K+ channels to induce hyperpolarization, activates sarco/endoplasmic reticulum Ca2+-ATPase to reduced [Ca2+]i  and leads to closure of voltage-gated Ca2+ channels leading to vasodilation[203]. Interestingly prostaglandin E2, thought to modulate NVC during functional hyperemia [276], constricts PAs from !20 both rat and mouse by activation of E-prostanoid receptors on smooth muscle cells[40]. In contrast, PGI2 released from the endothelium in response to muscarinic agonists and various other stimuli [18,169], dilates PAs[40] by acting on GS-coupled I prostanoid receptors on smooth muscle cells [63,237].   1.3.4 - Endothelium-derived hyperpolarizing factor. EDHF, detected in the presence of NOS and COX inhibition, refers to a chemically diverse group of endothelium-derived compounds that activate Ca2+ activated potassium (KCa) channels in the endothelium and/or the smooth muscle cells leading to hyperpolarization and dilation[81] (Figure 1.5). In cerebral arterioles, EDHFs like H2O2 and EETs elevate endothelial [Ca2+]i that leads to activation of KCa channels[113,158,262]. EDHF increases [Ca2+]i  by Ca2+ release from intracellular stores coupled with an influx of extracellular Ca2+. The influx of extracellular Ca2+ through TRP subfamily vanilloid member 4 (TRPV4) generates elementary Ca2+ currents called Ca2+ sparklets. Ca2+ sparklets can activate IP3 receptors in the endoplasmic reticulum (ER) and further increase [Ca2+]i. In pressurized middle cerebral arteries an increase in [Ca2+]i  from 130-160 nM to 340nM activates KCa channels [154]. Activation of intermediate conductance KCa (IKCa) channels and small conductance KCa (SKCa) channels leads to K+ efflux[50,238]. An increase in extracellular K+ from 4 mM to approximately 12 mM activates inwardly rectifying K+ channels or Na+- K+-ATPase on smooth muscle cells resulting in hyperpolarization of the smooth muscle cells [50].   Alternatively, hyperpolarization can spread from endothelial cells to smooth muscle cells !21 by direct electrotonic transfer through gap junctions in myoendothelial projections (MEPs). MEPs are endothelial cell protrusions abut smooth muscle cells [17,30] (Figure 1.5).  Gap junction proteins are present at some MEPs and studies show that 18"-glycyrrhetinic acid, an inhibitor of gap junction communication, induces significant inhibition of dilation in the rat mesenteric artery[87,95]. In rat mesenteric arteries the number of MEPs increased as the artery size decreased suggesting an increasing importance of their functional role in very small arterioles[225]. This finding supports the argument that EDHF mediated dilation is more prominent in smaller resistance arteries and arterioles compared to larger conduit arteries[60,107,232].  One or more EDHF may exist in the same artery and the exact contribution of an EDHF may vary between vascular beds, strains and pathological conditions. Sheer stress, and activation of various endothelial receptors such as the muscarinic or bradykinin receptors, induce formation of EDHF [23,67,76,212]. In rat PAs EDHF mediates conducted dilation that is dilation several millimeters upstream and/or downstream from the foci at which it was stimulated[106]. In the cerebral circulation, conducted dilation is particularly important for coordinating vascular resistance to enable functional hyperemia. In peripheral arterioles an inhibitor of cytochrome P-450 (CYP) epoxygenase that produces EETs, and KCa blockers suppress conducted vasodilation to muscarinic receptor activation [104,265].     !22     Figure 1.5: Endothelium-dependent dilatory pathways. NO and prostaglandin produced in endothelial cells (EC) diffuses through the internal elastic lamina (IEL) to cause relaxation of underlying smooth muscle cells. EC can also release EDHF, a consortium of substances that act on smooth muscle cells to induce hyperpolarization and relaxation. Additionally, hyperpolarization in EC can spread to underlying smooth muscle cell (SMC) via myoendothelial gap junctions (GJ).       !23 1.4 - The effects of hypertension on the cerebral circulation Chronic and sustained elevations in systolic arterial pressure lead to detrimental changes in the structure and function of cerebral arteries. This section will review some of these changes relevant to the studies presented in the dissertation and discuss the main characteristics of the stroke prone spontaneously hypertensive rat (SHRSP).   1.4.1- Impaired endothelium dependent dilation. Reduced levels of endothelium-derived NO and impaired endothelium dependent dilation have been reported in patients with essential hypertension compared to normotensive subjects [199,200]. Reduced dilation to acetylcholine was also reported in normotensive subjects with a family history of essential hypertension implying that impaired endothelial response was not simply a consequence of high blood pressure but may be complicit in the progression of hypertension [227]. Similar impairments in endothelium-dependent dilation are observed in cerebral arteries from spontaneously hypertensive stroke-resistant rats (SHR), a genetic model of essential hypertension, that express reduced levels of NOS3 compared to normotensive Wistar Kyoto (WKY) rats [269].   Reduced NO production and/or reduced NO bioavailability impairs endothelium dependent dilation. Impaired transport of the NOS3 substrate L-arginine in patients with essential hypertension has been proposed to mediate reduced forearm blood flow to acetylcholine by reducing NO production [227]. Reduced NO bioavailability can be an outcome of elevated ROS levels. Under normal conditions superoxide dismutase (SOD) converts ROS like superoxide to oxygen or H2O2 and H2O2 further degrades into O2 and !24 H2O. In arteries from hypertensive models elevated ROS levels exhaust antioxidant defenses like SOD [202,208]. Thus the ROS reacts with NO and generate peroxynitrite, thus reducing NO bioavailability [202,208,247] (Figure 1.6). Many enzymatic systems produce ROS; these include nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, CYPs and COXs. ROS from one source can increase ROS production in other enzyme systems and lead to sustained oxidative stress [293].  For example, oxidation of BH4 causes NOS3 uncoupling which leads to a rise in production of peroxynitrite, another ROS.   EDHF is upregulated after a variety of pathologic conditions such as ischemiaÐreperfusion, traumatic injury, congestive heart failure, and coronary artery disease as a protective mechanism that compensates for insufficient NO mediated dilation[17]. However in hypertensive models, this compensatory increase in EDHF dilation has not been reported. Impaired EDHF dilation observed in mesenteric arteries from SHRSP [242] was attributed to impaired function of IKCa channels and SKCa channels [78]. Similarily, renal arteries from aged SHR rats exhibited a CYP epoxygenase dependent loss of EDHF mediated dilation compared to age-matched WKY rats [19]. Interestingly, EDHF-mediated relaxation was increased in renal artery rings from young SHR compared to age-matched WKY rats [19], suggesting an age dependent attenuation of EDHF in SHR. Studies demonstrate that anti-hypertensive therapies significantly improved EDHF dilation in mesenteric arteries in SHR [88,123,193].  !25  Figure 1.6: The effects of hypertension on the cerebral circulation.  Hypertension disrupts critical regulatory mechanisms of the cerebral circulation; impaired dilation and remodelling can lead to loss of cerebral autoregulation. Hypertension increases the incidence of stroke, and chronic cerebral hypoperfusion. It also impairs neurovascular coupling and consequently increases the risk of developing VCI; blood-brain barrier, BBB; CBF, cerebral blood flow; and ROS, reactive oxygen species. From [61], with permission.   1.4.2 - Cerebral artery remodeling. Hypertension induces structural alterations of the vascular wall known as artery remodeling. An increase in lumen diameter is called outward remodeling while a decrease in lumen diameter is termed inward remodeling. !26 Hypertrophic remodeling occurs when the artery wall cross-sectional area increases [207]. Eutrophic remodeling occurs where there is no change in the wall cross-sectional area and hypotrophic remodeling describes reduced wall cross-sectional area. Changes in artery structure can be the result of rearrangement of pre-existing tissues in the wall, growth/ atrophy in the arteriolar wall or both.  Cerebral arteries from SHRSP [8,208,210] respond to increased intravascular pressure with inward remodeling, thereby increasing vascular resistance and reducing intravascular pressure in downstream arterioles and capillaries. This adaptation is thought to prevent ruptures and hemorrhages in downstream arterioles and capillaries [98]. While this remodeling process is initially a protective one, long-term this process becomes maladaptive, disrupts autoregulation, and causes dysregulation of local blood flow [157,224] (Figure1.6). Thus hypertension associated remodeling of cerebral arteries and arterioles could potentially lead to chronic hypoperfusion in the microcirculation and lead to increased risk of VCI.   PAs from 18-week-old SHRSP exhibit inward remodeling compared to PAs from their normotensive controls, evidenced by reduced passive lumen diameter with an increase in wall thickness and wall-to-lumen ratio [209]. PAs from SHRSP have increased distensibility, which is usually seen as the arteriolar diameter decreases [245]. Increased distensibility in cerebral arterioles from both genetic and non-genetic experimental models of hypertension could be associated with thickening of the internal elastic lamina. This change in distensibility is attributed to the ratio of non-distensible (basement membrane) to distensible components (endothelium, internal elastic lamina, smooth muscle cells) in the arteriolar wall, orientation of wall components with respect !27 to vascular circumference as well as interconnections among the various components [7].   Segments of the cerebrovascular tree remodel differently in response to intravascular pressure, circulating factors, neural stimuli and genetic factors[7,9]. Reducing arterial pressure with anti-hypertensive drugs prevents inward remodeling in PAs from 18-week old SHRSP [209]. However it is possible to reverse hypertensive artery remodeling independent of blood pressure as delineated by attenuated inward remodeling in PAs as well as in middle cerebral arteries from SHRSP treated with mineralocorticoid receptor blocker [209,220]. Pressure-independent remodeling during chronic hypertension is a complex multifactorial process mediated by vessel wall growth, apoptosis, low-grade inflammation, and vascular fibrosis with numerous modulators [21].   1.4.3 - Stroke prone spontaneously hypertensive rat. SHRSP have been used extensively as a model of essential hypertension and cerebrovascular disease in humans. In 1963, SHR were developed by brother-sister-breeding of WKY rats[191]. Successive breeding of SHR produced a sub-strain with higher blood pressure than that in SHR and a spontaneous stroke phenotype. In SHRSP blood pressure increases dramatically between 6 to 12 weeks of age and begins to plateau at 16 weeks. Extensive end-organ damage in SHRSP such as malignant nephrosclerosis and cardiac hypertrophy strongly resembles pathologies observed in uncontrolled hypertension in patients[189]. Increased sympathetic nerve activity [133], and hyperactive renin-!28 angiotensin-aldosterone and endothelin system [129,226] have been linked to organ damage and stroke in SHRSP [179].   Compared to WKY rats, CBF, angiogenic factors, and microvessel density is reduced in the cortex of SHRSP[69,118,270]. Furthermore, in SHRSP detrimental remodeling in cerebral arterioles, and enhanced BBB permeability leads to fibrinoid necrosis of arterioles, lacunar or silent infarcts and micro-hemorrhages (Figure1.6) [131,177,178]. These pathological changes in cerebral arterioles from SHRSP are also observed in patients with cerebral small vessel disease [177]. Symptomatic similarities are also observed between SHRSP and patients with VCI. Behavioral abnormalities in SHRSP such as hyperactivity and disturbed circadian rhythms are similar to the delirium state observed in patients with dementia[167]. Impaired learning assessed using a passive avoidance task accompanied central cholinergic dysfunction in SHRSP [246], and this type of neurological impairment is frequently seen in patients with VCI and AlzheimerÕs disease [37,257]. These cerebrovascular pathologies along with white matter lesions and cognitive impairment [100,145] make SHRSP an excellent model for VCI.   1.5 - Vascular cognitive impairment VCI is a heterogeneous group of cognitive impairments due to cerebrovascular pathologies and is the second most common cause of dementia after AlzheimerÕs disease, accounting for 20% of cases of dementia.. According to World Health Organization 2016, an estimated 47.5 million people suffer from dementia, and the !29 number is expected to almost triple by 2050 to 135.5 million. Social and economic costs of dementia measured by direct medical costs, direct social costs and the costs of informal care are staggering; in 2010, global cost of dementia was estimated to be US$ 604 billion. This section will discuss major risk factors and clinical developments in the VCI field, characteristics of the chronic cerebral hypoperfusion (CCH) models of VCI and some of the cognitive tests used in animal models.    1.5.1- Vascular cognitive impairment: current concepts and clinical developments. VCI is intended to be an umbrella term that includes patients with cognitive deficits ranging from full-blown dementia to milder forms of cognitive loss [89]. The cognitive profile of VCI patients is characterized by memory loss, mental confusion and, more often, deficits in executive function [229].   Vascular risk factors such as hypertension and diabetes, increased age, lifestyle habits such as smoking, and genetic factors can increase the risk of developing VCI[44]. Distinguishing between genetic and vascular risk factors of VCI is difficult due to the presence of co-morbidities, and the frequent overlap with AlzheimerÕs disease and other neurodegenerative diseases [86].   Hypertension increases the risk of both macro and micro-bleeds and ischemic stroke [86,91] (Figure 1.7). One third of patients with stroke exhibit symptoms of VCI within 3 months of the ischemic event [86]. Microinfarcts can occur in brain regions critical for cognition and can lead to strategic-infarct dementia; this is usually only discovered during diagnostic imaging. Small infarcts can occur in multiple brain regions, and result !30 in multi-infarct dementia.  VCI risk factors can also lead to hypoperfusive ischemic cerebral injury resulting from cardiovascular and circulatory disorders. Cardiac arrest, arrhythmias, cardiac failure, or hypotension can interrupt blood flow to the brain and impair cognition transiently or permanently; this emphasizes the importance of constant blood flow to the brain [122,155,241]. Moreover, carotid stenosis associated with CCH, can lead to the development of VCI even in the absence of ischemic lesions [6,32,119,156].   Risk factors also induce profound changes in the small arteries and arterioles in the brain. Endothelial dysfunction [90,96,213,261] in cerebral small arteries and PA can drive loss of smooth muscle cells. Endothelial dysfunction also leads to atherosclerosis with a reduction in lumen diameter, and is a prominent feature of cerebral small vessel disease that can account for up to 45% of VCI cases [86,198]. Hypertension promotes these pathological changes in the small arteries and PAs supplying the deep gray nuclei, and the white matter. This leads to white matter lesions (leukoariaosis) (Figure 1.7) [198,239] that are associated with a decrease in episodic memory and executive function and may be one of the key pathologies driving hypertension associated cognitive decline[176].  1.5.2 - Hypertension as a risk factor for VCI. In humans, hypertension is defined as systolic arterial pressure %140 mmHg and/or diastolic arterial pressure % 90 mmHg. In the United States, around 70% of people who are 60 years and older have hypertension and 15 to 18 million people from this age group are expected to develop VCI by 2050 !31 [83]. In a 40-year longitudinal study, mid-life systolic arterial pressure co-existed with cardiovascular risk factors and potentiated the risk of developing VCI [223]. Uncontrolled mid-life hypertension was also associated with late-life AlzheimerÕs disease and has been shown to exacerbate cognitive symptoms in AlzheimerÕs patients[108,234]. However, the influence of late-life hypertension on cognition is still unclear; some studies have reported a positive association, and others have reported a U-shaped relationship between arterial pressure and cognitive function[74]. Indeed, studies show that with increased age low systolic arterial pressure was associated with poorer cognitive status than that observed in individuals with normal or high systolic arterial pressure. The studies suggested that the maintenance of optimal systolic arterial pressure in older adults might be crucial for optimal cognitive functions[74].  Hypertensive patients have accelerated brain aging evidenced by brain atrophy with reductions in brain grey matter and increased sulca and ventricle size [215]. Hypertension associated brain atrophy was observed in the prefrontal cortex, the hippocampus, and the inferior temporal cortex. Furthermore, hypertension has been shown to exacerbate age-dependent detrimental changes in the brain [74].   !32  Figure 1.7: Hypertension increases the risk of developing VCI. Hypertension causes atherosclerosis of major extracranial and intracranial arteries and is a leading cause of stroke. Hypertension also increases the incidence of microbleeds and macrobleeds and is a leading risk factor for the progression of cerebral small vessel disease. Lacunar infarcts (silent infarcts), leukoaraiosis, and microinfarcts are characteristic of cerebral small vessel disease. From[61], with permission.   1.5.3 - Current therapies for VCI. Currently there are no FDA approved treatments for VCI[20]. Anti-hypertensive treatments have been useful in preventing the onset of post-stroke dementia[86]. However, in middle-aged and young elderly subjects anti-hypertensive trials have not shown consistent improvement in cognitive outcome [86,166,267]. In 2013, the European Society of Hypertension and of the European Society of Cardiology acknowledged that the current evidence for improved cognition !33 with antihypertensive therapy is weak and needs more thorough investigation [152]. Similar to the trials with anti-hypertensive drugs, antioxidants, anti-inflammatory drugs or drugs aiming to increase CBF have also shown inconsistent results[20]. Clinical trials involving cholinergic stimulants, vasodilators, and platelet aggregation inhibitors are currently underway for the treatment of VCI (www.clinicaltrials.gov).   Increasing evidence suggests that the ÔtreatmentÕ for VCI may just be reducing the severity of risk factors using a multi-pronged approach. Pre-hypertensive and hypertensive adults who were on antihypertensive drugs along with diet and aerobic exercise over a 4-month period had better cognitive outcome. The study subjects had improved executive function, memory, and psychomotor speed associated with reduced systolic and diastolic arterial pressure[236]. Rigorous control of risk factors like cholesterol, blood pressure, and fasting glucose along with lifestyle changes can reduce risk of developing VCI[166,217,252]. Physical and mental activity, social engagement, and a diet rich in antioxidants or polyunsaturated fatty acids are promising strategies to prevent the onset of VCI [44,152]. However, these conclusions are based on observational studies and have not been confirmed by large-scale randomized clinical trials of risk factor modification [44].         1.5.4 - VCI models of chronic cerebral hypoperfusion. The lack of suitable animal models for VCI have prevented mechanistic insights into disease progression and hindered the development of treatments [86]. The most widely accepted model for VCI is CCH induced by interrupting blood flow through the common carotid arteries. There !34 are several variations of the CCH models; the most widely accepted is a complete occlusion of both common carotid arteries. However, these bilateral common carotid artery occlusion models have substantial mortality rates in rats (50Ð60%) [230]. Many have attempted to increase survival rates by doing a staggered occlusion where common carotid arteries are occluded separately with a 1-week interval and by the use of microcoils to gradually reduce the diameter of common carotid arteries [142,230].   Bai et al reported a novel manner of inducing stenosis using a blunted needle as guide to reduce the common carotid artery lumen diameter [142]. This study compared cognitive deficits, hippocampal damage and white matter lesions between rats with bilateral common carotid artery stenosis group and rats with bilateral common carotid artery occlusion. 4 weeks after the surgeries there were no differences in cognitive impairment or the extent of brain damage between the groups. However the stenosis model dramatically increased survival rate post surgery. In my experience the stenosis model is the preferred model of inducing CCH in animals with a compromised cerebral circulation such as in the SHRSP.    A sudden drop in blood supply to brain regions can cause stroke, while a moderate but persistent reduction in regional CBF may compromise different aspects of cognitive functions and contribute to the development and progression of VCI [86]. Carotid artery occlusion in rats leads to a large reduction in CBF in cortical, white mater areas, as well as the hippocampus [190,195,248,249]. After 1 week of carotid artery occlusion CBF slowly begins to recover, and by 2-3 months post-occlusion blood flow is only slightly !35 reduced compared to controls [190,195]. With time neuropathology and cognitive deficits worsen despite the apparent restoration of CBF. Reduced blood flow induces neuronal energy failure with rapid depletion of ATP leading to dysfunction of energy dependent ion pumps, neuronal depolarization and toxic build-up of reactive oxygen species[62]. Increased levels of reactive astrocytes and microglia activation after carotid occlusion in rats have also been reported [1,201,228]. Bilateral common carotid occlusion models exhibit white mater damage, oxidative stress, inflammation and cognitive deficits, all pathological characteristics observed in patients with VCI [45,112,150].   The collateral circulation is critical for restoration of CBF and regulator of the severity of brain damage induced by CCH [130]. In a bilateral common carotid artery occlusion model PComA integrity influences the severity of white matter lesions [130]. Underdeveloped PComAs in Wistar rats were associated with larger reductions in CBF after carotid artery occlusion and worse white matter injury when these rats were compared to Sprague Dawley rats. Outward remodeling of the collateral arteries such as the basilar artery, the posterior cerebral artery, and the PComA was observed 15 weeks - 6 months after the onset of bilateral carotid artery occlusion [33]. It is possible outward remodeling progresses with time and then reaches a plateau, however such an investigation has not been carried out.    1.5.5 - Acetazolamide challenge. Acetazolamide, a carbonic anhydrase inhibitor, causes carbonic acidosis by reducing extracellular and intracellular pH to induce dilation !36 [281,289, 290]. It is used to assess cerebrovascular reserve capacity [281] that is a frequently used tool in clinical practice [286,291]. Impaired CVR in patients is associated with cognitive dysfunction and is predicative of increased risk of ischemic events in patients with carotid artery stenosis [278,283,284]. Impaired response to acetazolamide has been reported immediately post-occlusion in WKY rats with common carotid artery occlusion and this persisted for 4 weeks [289]. Prolonged chronic cerebral hypoperfusion could increase oxidative metabolism in the brain [277], enhance constrictor activity [279,285], and impair cerebrovascular reserve capacity.  Similar impairment in CVR in response to acetazolamide was reported in patients with severe hypertension and in healthy volunteers with increased age [280,288]. Age dependent reductions in CVR were also observed in mice after unilateral internal carotid occlusion [282]. Reduced cerebral perfusion pressure in the hypertensive or the aging brain can induce autoregulatory vasodilation and prevent further dilation of cerebral arteries to acetazolamide [287].  1.5.6 - Cognitive assessments. The Morris water maze (MWM) testing is based on the concept that the animal must learn to use distal cues (black cross and blue star in Figure 1.8 A) to navigate to a hidden platform after being placed in the swimming tank from different, and random locations. Apart from spatial learning MWM can be used to assess reference memory, and long-term memory that has been consolidated and can be retrieved. This relatively simple test does not employ external reinforcements such as food rewards nor does it require extensive pre-training. Additionally, a water maze reduces the chance of rats using a non-spatial strategy such as odor to find the hidden !37 platform [124]. MWM is useful test for studying the cognitive effects of lesions in the hippocampus, the prefrontal cortex, the neostriatum, and even the cerebellum [39].   Control procedures such as cued learning are essential during MWM to ensure that the spatial deficit is indeed present. Cued learning is where proximal cues (for example, a red flag attached to the hidden platform) are used to find the hidden platform. It requires the same basic abilities as spatial learning such as intact eyesight, swimming ability, learning to swim away from the wall, learning to climb on the platform) and the same motivation (escape from water) [254].  Thus, if the animal cannot perform the cued task, it is likely that it lacks the capacity to learn due to impaired sensorimotor abilities. Excessive thigmotaxis or swimming along the wall, excessive jump-offs, swim overs and/or deflections are other ways of determining if sensorimotor interference is occurring. It might also be necessary to measure stress and anxiety levels of the groups being compared to minimize any confounding stress components, associated with forced swimming during MWM.  Another test frequently used to assess cognitive function is the novel object recognition test (NORT). NORT uses the ratÕs innate exploratory behavior in the absence of externally applied reinforcements to test memory by evaluating differences in the exploration time of novel and familiar objects[52] (Figure 1.8B). NORT, like MWM, requires no external motivation, reward, or punishment and can be completed in a relatively short time. There is a need for habituation to the room and the apparatus in which the test is carried out. The novel exploration quotient or the time spent exploring !38 the novel object to the total exploration time is influenced by the interval between time spent with the novel object, and time spent with the sample object (retention time) as well as the time allowed for rats to explore the sample during the familiarization phase. NORT paradigm can be used to assess both hippocampal and cortical lesions [36], highlighting that different brain regions have different roles in the consolidation of memory [2,93].   Figure 1.8: Cognitive assessments. (A) Morris water maze can be used to assess spatial learning abilities. Latency time or time required to locate the hidden platform (grey box) is longer for normal rats as opposed to cognitively impaired rats. (B) Novel !39 object recognition test is used to evaluate memory. During the familiarization phase the rat is allowed to explore two similar sample objects (blue circles in the left box). Then after a time delay known as the retention period, the rat is placed inside the same testing apparatus for the test phase. During the test phase novel object (orange circle) replaces one of the sample objects and a rat with memory deficits spends a smaller percent of time exploring the novel object compared to a normal rat.   1.6 - The pleotropic effects of EETs  Various studies have established the dilatory, anti-inflammatory and angiogenic properties of EETs. In recent years there is great interest in elevating EETs levels by inhibition of the metabolizing enzyme soluble epoxide hydrolase (sEH) as treatment for cardiovascular diseases such as hypertension and stroke [117]. This dissertation proposes that inhibition of sEH can have beneficial effects in patients at risk of developing VCI. This section will outline the production, metabolism, and physiological roles of EETs, and highlight the significance of sEH inhibition.  1.6.1 - Arachidonic acid. AA, a 20-carbon polyunsaturated fatty acid, is a basic constituent of cell membranes and the main precursor of EETs (Figure 1.9). Phospholipase A2 (PLA2) hydrolyzes phospholipids in the cell membrane to release AA. While PLA2 pathway is predominant, AA can be also be liberated through a set of reactions initiated by phospholipases C and D [221]. Activation of PLA2 is tightly controlled and can be activated by pro-inflammatory cytokines, growth factors, hypoxia and elevated [Ca2+]i levels [16,137,165].  !40  In cells, AA readily undergoes oxygenation by COX, lipoxygenases, or CYP monooxygenases (Figure 1.9). These pathways result in the formation of prostanoids, leukotrienes, lipoxins, HETEs, and EETs that have diverse biological functions.  HETEs and EETs, monooxygenized metabolic products of AA are produced by CYP monooxygenases.  CYPs are multifunctional enzymes located in the ER that are involved in the conversion of cholesterol, steroids, bile acids, vitamins, and xenobiotics. CYPs also generate a considerable amount of ROS [26].    Figure 1.9: Arachidonic acid signaling cascade. Cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP) pathways metabolize free arachidonic acid (AA). Cytochrome P450 (CYP) pathways comprise of two enzymatic pathways catalyzed by the hydroxylases and the epoxygenases. The CYP hydroxylase (CYP4 A !41 and CYP4F) convert arachidonic acid into hydroxyeicosatetraenoic acids (HETEs). The CYP epoxygenase generate four regioisomeric epoxyeicosatrienoic acids (EETs), by epoxidation of AA olefin bonds. Soluble epoxide hydrolase (sEH) metabolize epoxyeicosatrienoic acid (EET) into corresponding DHETs (dihydroxyeicosatrienoic acids) regioisomers.  1.6.2 - The transitory life of EETs. CYP epoxygenases metabolize polyunsaturated fatty acids such as AA, eicosapentanoic acid and docosahexaenoic acid. These enzymes convert AA to EETs, and in the vasculature they are members primarily of the CYP2C and CYP2J classes [116]. In the brain, they are expressed in neurons, astrocytes [114], as well as in endothelial and vascular smooth muscle cells. CYP epoxygenase expression is increased in smaller resistance-sized arteries and arterioles suggesting an increasing functional role of the enzyme with reducing artery diameter [115]. There are four different regioisomers of EETs: 5,6-, 8,9-, 11,12-, and 14,15-EET. While each CYP epoxygenase isozyme can produce all four EET regioisomers, the predominant products vary depending on the isozyme and the tissue. 14,15-EETs and 11,12-EETs account for 67Ð80% of the total EETs produced by rat CYP epoxygenases [27].   EETs have close structural similarity to AA and can be incorporated into phospholipids in various tissues [25,125,126,268]. Agonists stimulation or shear stress can release these preformed stores of EETs in endothelial cells by activating phospholipid hydrolysis to induce dilation [263]. EETs are primarily metabolized by sEH, but can also !42 undergo chain elongation, !-oxidation or "-oxidation when tissue sEH activity is low or inhibited [240]. sEH converts EETs into the corresponding dihydroxyeicosatrienoic acids (DHETs) by hydrolysis of the epoxide group.   14,15-DHET activates peroxisome proliferator-activated receptor-", a nuclear hormone receptor with known effects on lipid metabolism, atherosclerosis, and vascular inflammation [54,219]. While some studies reported that DHET regioisomers lack vasoactive properties [28,214] others suggested that 11,12-DHET could act as potent vasodilators [55,192]. These differences in reports could be attributed to the differences in species and vascular beds studied. Compared to SHRs, SHRSP exhibited similar levels of EETs and significantly lower plasma DHETs levels, suggesting that the decreased amount of DHETs produced in the SHRSP may be associated with impaired vascular functions and increased stroke susceptibility [38].   1.6.3 - Physiological effects of EETs. EETs-mediated hyperpolarizations are not blocked by NOS or COX inhibition, but are sensitive to CYP epoxygenase inhibitors, KCa channel inhibitors and EETs antagonists [14,75,149,255,262]. In mesenteric resistance arteries EETs-mediated dilation is partially impaired by charybdotoxin and apamin, blockers of SKCa and IKCa respectively [48]. EETs activate TRPV4 in the endothelium [68] and this increases Ca2+ sparklets that activates endothelial SKCa channel and IKCa channel and induces endothelial cell hyperpolarization. Increase in extracellular K+ levels, due to SKCa and IKCa dependent K+ efflux, activates inward rectifying K+ channels and/or ouabain-sensitive Na+-K+-ATPase (Figure 1.10) [51]. Alternatively, EETs induced !43 hyperpolarization of the endothelial cell can be transferred to the smooth muscle cell via gap junctions [211].   Figure 1.10: EETs-mediated dilation. Receptor activation or sheer stress induces production of EETs from arachidonic acid. EETs released from the endothelial cell (EC) activation promote Ca2+ influx through TRPV4. Ca2+ sparks (yellow dot) can lead to Ca2+ induced Ca2+ release (orange dot) via IP3 receptors in the endoplasmic reticulum (ER) or via ryanodine receptors (RyR) in the sarcoplasmic reticulum (SR). This creates a microdomain of high intracellular Ca2+ causes activation of small conductance (SKCa) and intermediate conductance (IKCa) KCa channels (purple) and causes K+ efflux. Increase in extracellular K+ increases activity of Na+-K+-ATPase or inward rectifying K+ channel and leads to hyperpolarization of smooth muscle cell (SMC). In the SMCs Ca2+  !44 Figure 1.10 (contÕd)  sparks activates large conductance (BKCa) KCa channel and induces hyperpolarization. EETs can also directly activate BKCa channels.  Alternatively, EETs induced endothelial hyperpolarization can spread to smooth muscle cells through gap junctions in the myoendothelial projection that provide tight electrical continuity.   EETs produced in the endothelium can diffuse to the smooth muscle cells, where they can activate TRPV4. In cerebral smooth muscle cells EETs increased the generation of Ca2+ sparklets through TRPV4 [47] creating a microdomain of high [Ca2+]i [163]. These sparklets activate RyRs in the SR, initiates Ca2+ induced Ca2+ release to produce Ca2+ sparks that opens BKCa channels in cerebral smooth muscle cells and induce hyperpolarization (Figure 1.10) [47,68]. EETs can also directly activate BKCa channels possibly via a GS-coupled pathway [24]. Several studies show that EETs increase cyclic AMP levels in the smooth muscle cells [43,186] and labs have theorized the existence of a G protein coupled EETs receptor. While potent EETs agonists and antagonists have been discovered [272], to date an EETs receptor has not been identified.   EETs modulate NVC, with administration of EET antagonist diminishing, and sEH inhibitors prolonging functional hyperemia in rat cerebral cortex [147]. Glutamate release from neurons can activate astrocyte metabotrophic glutamate receptors to induce production of EETs that increase the activity of BKCa channel on the end-foot [102]. K+ efflux from BKCa channels increases perivascular K+ that activates inward rectifier K+ channel on the PA smooth muscle cells, and induces hyperpolarization to !45 increase blood flow in cerebral arterioles [147]. Alternatively, EETs derived from astrocytes can activate BKCa channels on PA smooth muscles to induce dilation [23].   Astrocyte derived EETs can also influence mitogenesis and morphogenesis of cerebral capillary endothelial cells, resulting in angiogenesis [175]. EETs also exhibit anti-inflammatory properties in various models. They have been shown to inhibit pro-inflammatory transcriptional factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). NF-kB decreases endothelial vascular cell adhesion molecule-1 expression and inhibits mononuclear cell adhesion [185]. In vascular endothelial cells, addition of physiologically relevant concentrations of EETs or overexpression of CYP2J2 increases tissue plasminogen activator expression and fibrinolytic activity. Overexpression of CYP2J2 has also been shown to protect against hypoxia-reoxygenation injury by reducing levels of lipid peroxidation and extracellular superoxide [271].  Thus, EETs play a critical role in maintaining vascular homeostasis. 1.6.4 - Soluble epoxide hydrolase inhibition. In the brain, sEH is highly expressed in neuronal cell bodies, astrocytes and oligodendrocytes [243]. sEH is also present in endothelial cells and smooth muscle cells of arterioles and in red blood cells [115,243]. In mammals the 125-kDa dimer protein is found mostly in the cytosol and also in the peroxisomes of some organs. The C-terminus contains the hydrolase activity, and the smaller N-terminus is a phosphatase and the two domains may be inhibited independently [173].  !46 Inhibition of sEH likely increases EET concentrations in plasma and tissues moderately since other routes such as beta-oxidation can metabolize EETs. This limits target-related side effects even with large doses of sEH inhibitors [173]. Amides, carbamates and urea-based sEH inhibitors have nanomolar Ki values and form stable transition states during inhibition. Between ureas, carbamates and amides, the urea pharmacophore seems to be the most potent inhibitor. 1,3-disubstituted ureas such as 1-(1-propanoylpiperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl] urea (TPPU) inhibit the C-terminal hydrolase activity of the sEH enzyme without changing phosphatase activity of the N-terminus [171,172]. The sEH inhibitor TPPU shows high potency (human IC50 0.9 nM; rat 5 nM), and can be stably formulated in the drinking water, and water concentrations correlate well with the resulting plasma and tissue levels of the drug. TPPU is absorbed effectively when administered orally and is metabolically stable with a half-life of more than 24 hours after a single oral dose [171].  Pharmacological inhibition of sEH is suggested to be a potential therapeutic intervention for various cardiovascular diseases [117,259,274]. sEH inhibition and the resulting increase in EETs reduces hypertension in many models, especially in Ang II-dependent hypertension, possibly as a result of reduced vascular resistance and increased renal sodium excretion[120,274]. Interestingly, sEH inhibition does not reduce arterial pressure in SHRSP. This resistance to the anti-hypertensive effects of sEH inhibitors could be linked to the polymorphisms in the sEH gene (Ephx2) in SHRSP.  Ephx2 polymorphism in SHRSP encodes for lower expression and activity of sEH than in SHR [38]. It is plausible that increased risk for target organ injury and reduced sEH function in !47 SHRSP is mediated by effects of the phosphatase domain of the enzyme[182]. This counterintuitive finding in SHRSP suggests that the physiological and pathophysiological functions of sEH are more complex than is currently understood. Activity of sEH was increased in the brain of VCI patients with higher sEH immunoreactivity near arterioles with microinfarcts, suggesting a role for the enzyme in the progression of cognitive decline [180]. Furthermore, sEH inhibitors reduced infarct size and neurodeficit score in SHRSP via vascular and neuronal protective mechanisms[233]. Thus it is conceivable inhibition of sEH could alleviate cognitive impairment in VCI models.  1.7 - Scope of this project Based on the gaps in the literature and the rationale provided in the introduction, the central hypothesis of this research was that impaired endothelium dependent dilation and remodeling in PAs from BCAS rats would contribute to cognitive dysfunction and the effects of BCAS will be exacerbated in SHRSP. I also hypothesized that chronic pharmacological inhibition of sEH would improve endothelium dependent dilation and remodeling in PAs from SHRSP after BCAS with a concurrent improvement in cognitive functions. The hypothesis was tested by the following experimental protocols:  1.7.1 - BCAS in WKY rats. As mentioned previously, BCAS is a well-established model of VCI. This study predicted that BCAS would impair memory and spatial learning abilities in WKY rats. It was expected that cerebrovascular reserve capacity assessed using acetazolamide challenge would be diminished in the BCAS rats accompanied by !48 impaired endothelium dependent dilation in the PAs.  Impaired PA dilation would be due to a reduction in EHDF dilation, since studies show EDHF is the predominant pathway for arteriole dilation. Both passive structures of PA and PComAs were assessed to determine the extent and pattern of vessel remodeling after CCH.   1.7.2 - BCAS in SHRSP. SHRSP are a model of cerebrovascular pathologies, thus after CCH induced by BCAS it is anticipated that memory and spatial learning abilities in SHRSP would be clearly evident, and exacerbated in comparison to WKY rats with BCAS. Dilation is impaired in large cerebral arteries from SHRSP and thus BCAS would induce drastic impairment in cerebrovascular reserve capacity and impair dilation in the PAs and PComAs. Inward remodeling is well documented in cerebral arteries from SHRSP. The effects of CCH on inwardly remodeling arteries would lead to a diminished outward remodeling in the PAs and the PComAs in SHRSP with BCAS compared to WKY rats with BCAS.   1.7.3 - Chronic TPPU treatment of SHRSP with BCAS. This study was designed to investigate the effects of chronic sEH inhibition on cognitive function, cerebrovascular reserve capacity and PA dilations in SHRSP with BCAS. I hypothesized that TPPU treatment would prevent CCH-induced memory deficits and spatial learning abilities in SHRSP with BCAS and that cerebrovascular reserve would be improved. Endothelium dependent dilation in PAs from TPPU treated rats would be increased via improved EDHF mediated dilation. Detrimental remodeling in PAs from BCAS rats would be attenuated in the TPPU treatment group. !49   Figure 1.11: Overarching hypothesis of the dissertation. CCH induced by BCAS will impair cognitive abilities, cerebrovascular reserve capacity, PA dilation and induce remodeling (Aim 1). These changes in cognitive abilities and vascular functions after BCAS will be exaggerated in SHRSP, a genetic model of malignant hypertension and cerebrovascular disease (Aim 2). Inhibition of sEH will alleviate BCAS induced vascular dysfunctions and improve cognitive performance of SHRSP after CCH (Aim 3).    !50            REFERENCES           !51 REFERENCES  1. Abraham H, Lazar G. 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Reduced blood flow through the common carotid arteries induced by bilateral carotid artery stenosis (BCAS) is a physiologically relevant model of chronic cerebral hypoperfusion. We hypothesized that BCAS in 20-week-old Wistar Kyoto (WKY) rats would impair cognitive function and lead to reduced endothelium dependent dilation and outward remodeling in the parenchymal arterioles (PAs). After 8 weeks of BCAS, both short-term memory and spatial discrimination abilities were impaired. In-vivo assessment of cerebrovascular reserve capacity showed a severe impairment after BCAS. PA endothelial function and structure were assessed by pressure myography. BCAS impaired endothelial function in PAs, as evidenced by reduced dilation to carbachol. Addition of nitric oxide synthase and cyclooxygenase inhibitors did not change carbachol-mediated dilation in either group. Blocking the production of epoxyeicosatrienoic acid (EETs) that acts as an endothelium-derived hyperpolarizing factor (EDHF), abolished dilation in PAs from Sham rats, but had no effect in PAs from BCAS rats. Expression of transient receptor potential cation channel subfamily vanilloid member 4 (TRPV4), downstream target of EETs, was decreased and maximal dilation to a TRPV4 agonist was attenuated after BCAS. Together these data suggest that EETs-mediated dilation is impaired in PAs !79 after BCAS. Thus impaired endothelium dependent dilation in the PAs may be one of the contributing factors to the cognitive impairment observed after BCAS.  2.2 - Introduction Chronic cerebral hypoperfusion has been implicated in dementias ranging from vascular cognitive impairment [19] to AlzheimerÕs disease [1,12]. Animal models of chronic cerebral hypoperfusion exhibit neuronal loss, white matter lesions, and increased levels of reactive astrocytes [10,27,28]. Few studies have investigated the effect of chronic cerebral hypoperfusion on parenchymal arterioles (PAs) [26,45]. However, none have reported changes in endothelial function in PAs or studied the effects of prolonged hypoperfusion.    PAs arise from the pial arteries, perfuse the parenchyma and eventually branch into the capillaries. These capillaries are in intimate contact with neurons, astrocytes and pericytes and together these cell types form the neurovascular unit. Maintaining a constant and controlled flow of blood to the brain parenchyma is important for its homeostasis. PAs, act as a bottleneck to parenchymal perfusion, and are responsible for modulating nutrient and oxygen delivery to the neurovascular unit [35]. Despite studies, which suggest that PA dysfunction can cause and exacerbate cerebrovascular disorders, a knowledge gap exists regarding endothelial function in these arterioles during chronic cerebral hypoperfusion.   !80 Endothelium dependent dilation is mediated by nitric oxide (NO), prostacyclin and endothelium-derived hyperpolarizing factors (EDHF). EDHF is more prominent in smaller resistance arteries and arterioles compared to larger conduit arteries [4,15,44]. In this study we sought to determine the effect of chronic cerebral hypoperfusion on EDHF mediated dilation in the PAs in normotensive Wistar Kyoto (WKY) rats with bilateral common carotid artery stenosis (BCAS). We investigated the contribution of epoxyeicosatrienoic acids (EETs), that act as an EDHF [5], to dilation after cognitive dysfunction was established. EETs are important regulators of parenchymal blood flow and neurovascular coupling [22] and studies suggest that cognitive impairment in humans is linked to dysfunctional EETs metabolism [32]. For comparative analysis of the effect of BCAS on arterioles and the major collateral artery between the posterior and anterior cerebral circulation, we also studied structural and mechanical changes in the PAs as well as posterior communicating arteries (PComAs) and evaluated cerebrovascular reserve capacity, a critical prognostic factor for cognitive impairment [17]. The aim of this study was to evaluate changes in the PAs and collaterals such as the PComAs in a model of cognitive impairment to understand the mechanism by which cerebral perfusion and brain functions are linked.   2.3 - Methods 2.3.1 - Animals, and surgery. 20-week-old male WKY rats, purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN), were randomized into two groups: one group underwent Sham surgery and the other group had bilateral common carotid stenosis (BCAS). Rats were anesthetized using isoflurane and were placed in a supine position !81 and the skin over the common carotid arteries was shaved and cleaned with alcohol. An incision was made in the skin, the right common carotid artery was separated from adherent connective tissue and a 27-gauge blunt needle was placed next to the vessel. Two 6-0 silk sutures were used to firmly tie the carotid artery and the needle together. After the ties were in place, the needle was carefully removed [28]. In this manner the needle was used as a guide to induce partial occlusion or stenosis of the artery. The procedure was then repeated on the left common carotid artery (Figure 2.1). Ketoprofen (5mg/kg/day) and Combi-pen 48 (22,000 units/day) were administered subcutaneously three and two days post surgery, respectively.   Figure 2.1: Bilateral common carotid artery stenosis. Diameters of common carotid arteries (CCAs) were reduced using a blunted 27-gauge needle as a guide.   The stenosis surgery had 100% survival rate and rats were monitored until they were completely awake and moving around freely. The rats were singly housed for the duration of the experimental period. The experimental protocol was approved by the Michigan State University Institutional Animal Care & Use Committee and was in !82 accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals (2011).  2.3.2 - Novel object recognition test. An open box with opaque walls was utilized for the novel object recognition test [23]. Rats were acclimatized to the box for 15 minutes/day for three days. On the test day, during the familiarization phase the rats were removed from the box after they explored two identical objects for 30 seconds. Ninety minutes after the end of the familiarization phase, during the acquisition phase, a novel object of similar size and texture replaced one of the familiar objects in the box. The time spent exploring the novel object was recorded along with the total exploration time. For both the familiarization and acquisition phase, exploration took place when the rat pawed at, sniffed or whisked with its snout at the object from a distance of under !1 cm [23]. The positions of the familiar and novel objects were alternated to prevent bias for a particular location. After each trial, 10% alcohol was used to clean the objects and the box to remove any olfactory cues. Novel exploration quotient was measured as a ratio of the time spent exploring the novel object to the total exploration time [31].   2.3.3 - Morris water maze. A circular tank filled with water (30¡C), was positioned in a room with external cues visible to the swimming rat. During the training phase, rats were placed in the tank from all four possible directions (north, south, west and east), and they learned to escape by locating a platform located 1 cm above the water level.  The test phase started 4 weeks after BCAS. The test was carried out in trials of 2 from all four directions per session and was performed 5 weeks after the surgery, for three !83 weeks. The protocol for the test was the same as training with the exception that the platform was hidden 1 inch below the surface of water colored opaque with non-toxic tempera paint. To evaluate the ratÕs spatial learning ability, latency (time required to reach the platform) was measured [48].   2.3.4 - Cerebral tissue perfusion. A scanning laser Doppler (PeriScan PIM 3, Perimed, Stockholm, Sweden) was used to measure cerebral tissue perfusion, while the rat was under isoflurane anesthesia. The scanning laser Doppler was positioned !18 cm above the exposed and cleaned skull, and cerebral tissue perfusion was recorded. The wavelength of the laser light was 670Ð690 nm with a penetration depth of 0.5 - 1 mm. A total of 4 consecutive scans were performed immediately after BCAS, and 8 weeks after BCAS. Mean perfusion was analyzed using the LDPIwin 3.1 software (Perimed). Results were expressed as mean perfusion units.   2.3.5 - Acetazolamide challenge. Changes in cerebral perfusion following administration of acetazolamide, a potent cerebral vasodilator, were used to estimate cerebrovascular reserve capacity [25]. Cerebrovascular reserve capacity was calculated as the percentage increase in cerebral tissue perfusion after acetazolamide relative to baseline perfusion [25]. Rats were anesthetized with isoflurane and placed on a heated platform in a supine position while acetazolamide (0.2mg/g) was injected into the tail vein. Scanning laser Doppler was used to assess cerebral tissue perfusion as described above. Perfusion was measured every five minutes for thirty minutes.   !84 2.3.6 - PA isolation, and cannulation. After 8 weeks of BCAS, rats were anesthetized using isoflurane. After thoracotomy, blood was collected by cardiac puncture, the heart was removed and the rat was decapitated. The brain was placed in ice-cold Ca2+-free physiological saline solution (PSS, in mM: NaCl 140, KCl 5, MgCl2¥7H2O 1, HEPES 10, Dextrose 10) for isolation of the PAs. To dissect out the PAs, a section of brain tissue containing the middle cerebral artery was removed and placed in Ca2+-free PSS at 4¡C, with 10% bovine serum albumin. PAs branching from the middle cerebral artery were carefully dissected and transferred to a cannulation chamber using a Wiretrol II positive displacement pipette (Drummond, PA). PAs were cannulated between two glass micropipettes (<40 !m) bent to a 45! angle and mounted on a small, 3-axis micromanipulators (MT-XYZ, Newport, Irvine, CA) such that the tips of the pipettes could be adjusted in three dimensions [6]. Cannulated PAs were bathed in warm (37¡C) PSS containing 1.8mM Ca2+, and pressurized at 60 mmHg until spontaneous myogenic tone developed. The perfusion chamber was positioned on the stage of an inverted microscope (Leica DMIL, Wetzlar, Germany) and was measured using a 20x objective (Leitz Wetzlar objective, numerical aperture: 0.3). PA outer and lumen diameters were constantly tracked and recorded using MyoView 2.0 software (Danish Myo Technology, Aarhus, Denmark). To assess endothelium dependent dilation, PAs were incubated with increasing concentrations of the muscarinic receptor agonist carbachol (1nM to 100µM) in the bath. Carbachol dilates cerebral vessels via production of NO, cyclooxygenase (COX) metabolites and EDHF [8]. To assess the role played by EDHF in the carbachol-induced dilation, PAs were incubated with the inhibitor of NO synthase (NOS), N(G)-!85 nitro-L-arginine methyl ester (L-NAME, 100 !M) and the COX inhibitor indomethacin (Indo, 10 !M) for 30 minutes, prior to pressurization and development of myogenic tone and prior to carrying out the concentration-response protocols. To evaluate the contribution of EETs in endothelium-dependent dilation, PAs were incubated in a similar fashion with cytochrome (CYP)-450 epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH; 10 !M). GSK1016790A, a transient receptor potential cation channel subfamily vanilloid member 4 (TRPV4) agonist, was used to assess downstream mediators of EETs signaling. Endothelium-independent dilation was studied by incubating PAs with sodium nitroprusside (SNP: 1nM to 100 !M). Only one concentration-response experiment was performed on each cannulated PA. Baseline tone was calculated using the following formula: [1 & (active external diameter/passive external diameter)] * 100. % Dilation was calculated using the formula: ((external diameter at drug concentration Ðbaseline external diameter)/(passive external diameter-baseline external diameter))*100.  2.3.7 - Assessment of the structural, and mechanical properties in PAs. After the end of the vasoreactivity studies the PAs were bathed in Ca2+-free PSS containing 2mM ethylene glycol tetra acetic acid (EGTA) + 100!M SNP to assess the passive structure of the vessels as described previously [3]. A CCD camera (Hitachi Kokusai Electric Inc., Japan), with number of effective pixels 768 (H) x494 (V), was connected to a video dimension analyzer (Living Systems Instrumentation, Burlington, VT), a final magnification of x1100 was used. The video dimension analyzer operates on the relative optical density changes of wall structures at the chosen level of a pre-selected !86 scan line. It was calibrated using a stage micrometer according to the manufacturerÕs protocol. Lumen diameter and wall thickness was measured after 5 minutes at each pressure step and intraluminal pressure was increased from 3 to 180 mmHg in 20 mmHg increments. Outer diameter was calculated as lumen diameter + left wall thickness and right wall thickness. The wall-to-lumen ratio and circumferential wall stress were calculated as described previously, as was the passive distensibility [3].   2.3.8 - Cannulation, and assessment of the structural, and mechanical properties of PComAs. The PComA was carefully dissected from the brain and transferred to a pressure myograph chamber. A branchless segment of the PComA was cannulated between two glass micropipettes as previously described [42]. The structural and mechanical properties of PComAs were assessed and calculated as described for the PAs.   2.3.9 - Real time-polymerase chain reaction. Total mRNA was isolated from the brain region around the middle cerebral arteries from which the PAs were dissected and a Qiagen RNeasy lipid tissue kit (Qiagen Sciences) was used to extract RNA. Total mRNA was also extracted obtained from the dissected middle cerebral arteries and the PAs that were used for the vasoreactivity studies. The vessel samples were homogenized in Trizol reagent (Life Technologies, Gaithersburg, MD) and total RNA was extracted from the tissue according to the manufacturer's suggested protocol. Total mRNA collected from the tissues was reverse-transcribed using a qScript cDNA Synthesis Kit (Quanta Biosciences, Gaithesburg, MD).  PCR was then performed using Taqman primer and !87 probe sets in a 7,500 real time PCR system (Applied Biosystem, Foster City, CA). Fold changes in expression from Sham group were calculated using the 2-''CT method [29] with #-2-microglobulin used as endogenous control[41].  2.3.10 - Statistical analyses. Novel object recognition test, myogenic tone and resting lumen diameter data were analyzed by StudentÕs t-test or a non-parametric alternative if the data were not normally distributed. Cerebrovascular reserve capacity, dilation, passive and mechanical properties were analyzed by two-way ANOVA followed by Sidak correction for multiple comparisons, or a non-parametric alternative. Analyses were performed using GraphPad Prizm 6.0 (La Jolla, CA, USA).  2.3.11 - Chemicals, and reagents. MS-PPOH was purchased from Cayman Chemical (Ann Arbor, MI, USA). Acetazolamide was purchased from X-Gen (Big Flats, NY, USA). All other chemicals and reagents were purchased from Sigma-Aldrich (Saint Louis, MO, USA).  2.4 - Results 2.4.1 - BCAS impaired memory function, spatial learning abilities, and abolished cerebrovascular reserve capacity. 8 weeks of BCAS reduced the novel exploration quotient (Figure 2.2A). BCAS rats also showed reduced spatial discrimination abilities as evidenced by increased time to find the platform in the Morris water maze test (Figure 2.2B). While Sham rats showed significant improvement in learning abilities at the 3rd week, BCAS rats did not.   !88 A scanning laser Doppler system was used to measure cerebral tissue perfusion immediately after surgery and then after 8 weeks of BCAS. While perfusion fell immediately in BCAS rats compared to Sham rats (Figure 2.3A), there was no difference in baseline mean perfusion between Sham and BCAS rats at the end of 8 weeks (Figure 2.3B). Acetazolamide, a potent carbonic anhydrase inhibitor, was used to measure cerebrovascular reserve capacity. The initial dilatory response between Sham and BCAS did not differ, however tissue perfusion in BCAS rats was not sustained and perfusion rapidly diminished in this group (Figure 2.3C).   2.4.2 - BCAS impaired dilation in PAs. There was no difference in myogenic tone generation between PAs from Sham and BCAS rats (Figure 2.4A). Endothelium dependent dilation, assessed using carbachol, was impaired in PAs from BCAS rats compared to Sham rats (Figure 2.4B). Carbachol induced negligible dilation in PAs from BCAS rats. Endothelium-independent dilation to the NO-donor SNP was unaltered between PAs from Sham and BCAS rats at the lower concentrations, however at the highest concentration dilation in PAs from BCAS rats was impaired (Figure 2.4C). EC50 of SNP did not differ between the groups.   2.4.3 - Endothelium dependent dilation in PAs was independent of NO, and prostaglandin. Different inhibitors were added to the bath before the development of myogenic tone to assess the contribution of EDHF and the NO/prostaglandin pathways to carbachol dilation in the PAs. Myogenic tone was unaltered with addition of the inhibitors (Figure 2.5A). Residual dilation after inhibition of NOS and COX is considered !89 to be EDHF mediated [5]. In PAs from Sham rats, carbachol-mediated dilation was unaltered in the presence of L-NAME and Indo (Figure 2.5B). There was very little dilation in PAs from BCAS rats with or without the inhibitors (Figure 2.5C).   2.4.4 - EETs-mediated dilation in PAs from Sham rats. MS-PPOH (10-5M), an inhibitor of CYP450 epoxygenases, blocked EETs production. Dilation of PAs from Sham rats was abolished when EETs production was inhibited (Figure 2.5D). There was no change in dilation in PAs from BCAS rats with inhibition of the CYP450 epoxygenase (Figure 2.5E). In cerebral arteries, EETs-mediated dilation involve activation of TRPV4 [13]. Therefore, we also assessed the effects of BCAS on dilation induced by the TRPV4 agonist, GSK1016790A.  We found that dilation to GSK1016790A was significantly impaired in PAs from BCAS rats (Figure 2.5F).   2.4.5 - BCAS did not induce remodeling the PAs. There was no difference in PA lumen diameter between BCAS and Sham rats (Figure 2.6A). There was also no difference in wall-to-lumen ratio between the groups (Figure 2.6B) or any changes in any of the biomechanical parameters between the groups (not shown).   2.4.6 - PComAs from BCAS rats exhibited structural remodeling. Passive structure and mechanics of the PComA was carried out for a comparative analysis of the effect of BCAS on arteries and arterioles. Lumen diameter was increased (Figure 2.7A) and the wall-to-lumen ratio was reduced in PComAs from BCAS rats (Figure 2.7B) compared to Sham rats. These changes were accompanied by increased wall stress in PComAs !90 from BCAS rats (Figure 2.7C). There was no change in distensibility (Figure 2.7D) between the groups.  2.4.7 - BCAS changed mRNA expression of key mediators in EETs dilatory pathway. We assessed the mRNA expression of TRPV4 which has been suggested to be the downstream regulator of EETs-mediated dilation[13]. mRNA expression of TRPV4 were reduced in the cerebral arteries and arterioles from BCAS rats (Figure 2.8A). We also measured the mRNA levels of soluble epoxide hydrolase, the enzyme that metabolizes EETs. Soluble epoxide hydrolase mRNA expression was unchanged in the middle cerebral arteries and arterioles between the groups (Figure 2.8B). However, mRNA for this enzyme was up-regulated in the brain region surrounding the PAs from BCAS rats (Figure 2.8C). Similarly, expression levels of the mRNA for CYP epoxygenase 2C11 also increased in the brains from WKY rats with BCAS (Figure 2.8D). !91 Figure 2.2: BCAS impaired memory function, and spatial learning abilities. (A) BCAS rat spend a smaller portion of the time exploring the novel object as depicted by reduced novel exploration quotient. (B) In the 3rd week of MWM, BCAS rats showed a significantly increased time to find the hidden platform, where Sham rats found the hidden platform in a significantly shorter time compared to 2nd week after the training. Each data point is mean ± SEM. * is data different from Sham rats and # is data different from Sham rats in the 2nd week; *, # p<0.05. !92  Figure 2.3: 8 weeks after BCAS cerebral perfusion was restored, however cerebrovascular reserve capacity was impaired. (A) Immediately after BCAS, mean perfusion in BCAS rats was reduced (B) however, 8 weeks after the surgery there was no different in cerebral perfusion between Sham rats and BCAS rats. (C) Cerebrovascular reserve capacity, measured during carbonic anhydrous inhibitor acetazolamide, quickly diminished in BCAS rats; *p<0.05, different from Sham rats. !93 Figure 2.4: Impaired dilation in PAs from BCAS rats. (A) Myogenic tone generation in the PAs was not affected by BCAS. PAs with at least 20 percent tone was used for further studies. (B) Endothelium dependent dilation assessed using carbachol was abolished in PAs from BCAS rats. (C) At the highest concentration of SNP, dilation in PAs from BCAS rats was reduced; *p<0.05, different from Sham rats. !94  Figure 2.5: EETs - mediated dilation was impaired in PAs from Sham rats. (A) No difference in myogenic tone generation with addition of the inhibitors. (B) Dilation to carbachol remained unaltered in the presence of L-NAME and Indo in PAs from Sham rats. (C) Inhibition of production of NO and prostaglandins had no effect on dilation in    !95 Figure 2.5 (contÕd)  PAs from BCAS rats. (D) Inhibition of EETs production with MS-PPOH abolished dilation in PAs from Sham rats. (E) Incubating with MS-PPOH had no effect on dilation in PAs from BCAS rats. (F) Dilation to TRPV4 agonist GSK 1016790A was reduced in PAs from BCAS rats; n= 4 to 6, *p<0.05, different from Sham rats.  !96  Figure 2.6: There was no difference in passive structure of PAs between the two groups. (A) Lumen diameters of the PAs were measured after the PAs were equilibrated in Ca2+ free PSS with EGTA and SNP. There was no difference between the lumen diameter of PAs from Sham rats and BCAS rats. (B) Wall to lumen ratio was not different between the two groups.   !97  Figure 2.7. Outward remodeling in the PComAs from BCAS rats . (A) Data from pressure myograph shows the lumen diameter increased in the PComAs from BCAS rats. (B) PComAs from BCAS rats also had reduced wall to lumen ratio. (C) Wall stress was increased in PComAs from WKY rats with BCAS. (D) There was no change in distensibility between the PComAs from the two groups; *p<0.05, different from Sham rats.  !98  Figure 2.8: Altered expression of markers involved in EETs-mediated signaling in BCAS rats. (A) mRNA levels of TRPV4 was reduced in middle cerebral arteries (MCAs) and PAs from BCAS rats. (B) There was no change in mRNA levels of soluble epoxide hydrolase (sEH) in MCAs and PAs after between the groups. (C) However, there was increased expression of sEH in brain region around the PAs from BCAS rats. (D) Increased expression of sEH was coupled with increased expression of Cyp2C11 in the brain from BCAS rats; *p <0.05, different from brains from Sham rats.    !99 2.5 - Discussion The novel finding of this study is that impaired dilation in the PAs is associated with the cognitive impairment resulting from chronic cerebral hypoperfusion. Although tissue perfusion was restored 8 weeks after the stenosis, reserve dilation was impaired and PA endothelium-dependent dilation was essentially abolished. This impairment appears to be the result of reduced EETs-mediated dilation. Together, these results provide evidence that dysfunction in the EETs dilatory pathway in PAs may be associated with the cognitive impairment observed with chronic hypoperfusion.   2.5.1 - BCAS impaired memory function, spatial learning abilities, and abolished cerebrovascular reserve capacity. After 8 weeks of chronic cerebral hypoperfusion, spatial discrimination and learning assessed using Morris water maze was impaired in BCAS rats. Carotid artery occlusion causes a similar impaired short-term memory and learning deficits, evident just 2 weeks after chronic cerebral hypoperfusion [28,33]. In the current study, BCAS rats also demonstrated impaired memory suggesting impaired function frontal-subcortical brain regions [14,23].  In models of chronic cerebral hypoperfusion vascular adaptations restore resting cerebral tissue perfusion to normal levels several weeks after the initial insult [16] as is the case in the current study. However despite the restoration of cerebral perfusion the cerebrovascular reserve capacity was impaired in the BCAS rats 7 weeks after the stenosis. Insufficient collateral supply after chronic cerebral hypoperfusion causes !100 dilation of the arterial circulation and reduced response to administered vasodilator. Acetazolamide induces dilation by extracellular and intracellular acidosis and is frequently used to assess cerebrovascular reserve capacity [18], an indicator of the severity of cognitive impairment [30].  The initial response to acetazolamide in the BCAS rats was not different from Sham rats but the cerebral vasculature appears to have lost its ability to maintain the dilatory response. Prolonged chronic cerebral hypoperfusion may have exaggerated the steal phenomenon, with an increase in flow velocity in the outward remodeled arteries in the circle of Willis and a flow reduction in the pial arteries and the arterioles measured with the scanning laser Doppler. Alternatively increased oxidative metabolism in the brain after chronic hypoperfusion [2] could enhance constrictor activity [11,40] and impair cerebrovascular reserve capacity. Impaired response to acetazolamide has been observed immediately post-occlusion in WKY rats with common carotid artery occlusion and this persisted for 4 weeks [46].   2.5.2 - BCAS attenuated endothelium-dependent dilation in the PAs. Normalization of resting cerebral tissue perfusion with persistent cognitive impairment hinted at dysfunction at the level of parenchymal perfusion. In our study endothelium-dependent dilation to carbachol was abolished and the response to the highest concentration of the endothelium-independent dilator SNP was reduced in PAs from BCAS rats. As there have been no other studies assessing the temporal effects of chronic cerebral hypoperfusion on the endothelium of the PAs, it is difficult to surmise whether this impairment occurs during the acute or chronic phase of BCAS. Studies in the literature !101 suggest this may be an acute response. In vivo assessment of the PAs 30 minutes after occlusion of carotid arteries showed a reduction in their resting diameter [26], suggesting an increase in resting tone generation. Regardless of the onset, impaired dilation could play a role in the development of the cognitive deficits observed in our model. Cognitive impairment is observed after occlusion in a single PA [35]. This may be due to functional changes in PAs, which may alter homeostasis in the neurovascular unit. Impaired dilation in a single PA may worsen the severity of hypoperfusion in the brain parenchyma due to lack of collateral flow between neighboring PAs [34].   We observed no difference in myogenic tone in the PAs between the groups. This is in contrast to the results obtained in a study using 4 weeks of unilateral common carotid occlusion in 14 week old WKY rats where myogenic tone in the PAs was reduced [45]. The duration, and severity of the hypoperfusion, as well as the age of the WKY rats used in this study may have negated this compensatory decrease in myogenic tone.   2.5.3 - BCAS impaired EETs-mediated dilation in the PAs. We observed NO and prostaglandin independent dilation in the PAs from Sham rats. This suggests that EDHF, a dilator pathway that is more prominent in smaller resistance arteries and arterioles compared to larger conduit arteries [4,15,44], is a major contributor of endothelium-dependent dilation in PAs from 28-week-old WKY rats.  CYP450 epoxygenase pathways metabolize arachidonic acid to produce EETs [43]. EETs act on TRPV4 to induce Ca2+ flux that causes NO synthase and cyclooxygenase independent dilation by membrane hyperpolarization [13]. In PAs from Sham rats !102 dilation was abolished by MS-PPOH, an inhibitor of CYP450 epoxygenases, primarily CYP2C11 enzymes [22]. The lack of dilation in PAs from the BCAS group and attenuated dilation with the inhibition of EETs in PAs from Sham group suggests that BCAS impairs EETs-mediated dilation. Coupled with the observed of impaired dilation to the TRPV4 agonist GSK1016790A, reduced expression of TRPV4 mRNA, and impaired dilation in PAs from BCAS rats suggests that chronic cerebral hypoperfusion causes impaired EETs induced TRPV4 activity. We also observed increased levels of soluble epoxide hydrolase in the brain region around the PAs from BCAS rats, suggesting that after prolonged hypoperfusion there might be increased degradation of EETs. This increased activity of soluble epoxide hydrolase may also have caused a compensatory increase in expression of CYP2C11 mRNA, the epoxygenase that produces EETs in the brain.   Evidence suggests that cognitive impairment in humans is linked to dysfunctional EETs metabolism [32]. In our model of cognitive impairment, despite restoration of cerebral perfusion [36,37], impaired PA dilation suggest that the arterioles may be less responsive to vasodilatory agents released from astrocytes and may exhibit impaired neurovascular coupling. Inhibition of EETs production reduces coupling of blood flow to neural activation [39], suggesting that impaired EETs-mediation dilation after BCAS may worsen cognitive outcomes. Disruption of the spatial and temporal relationship between neural activity and cerebral blow flow is observed during AlzheimerÕs disease and after ischemic stroke [20], a major risk factor for vascular cognitive impairment.   !103 2.5.4 - Heterogenous remodeling of the PAs and PComAs after BCAS. Structural changes in the PAs after 8 weeks of BCAS were not observed. However, arteries in the vetebrobasilar circulation have increased lumen diameters [7,16] suggesting that in chronic cerebral hypoperfuson models remodeling depends on the artery type studied. Following the stenosis, redistribution of flow results in increased intravascular shear forces. As a result, in our study PComAs from BCAS rats exhibited an increase in lumen diameter of approximately 14% accompanied by reduced wall-to-lumen ratio and increased wall stress. This remodeling is essential for restoration of cerebral perfusion. Lack of a fully formed PComA in gerbils exacerbates the effects of chronic hypoperfusion [16], whereas in rats a complete circle of Willis allows for restoration of perfusion [7,10].   Normalization of cerebral perfusion, observed in the current study, may be associated with outward remodeling of collaterals such as the PComA. However, one of the characteristic outcomes of reducing blood flow through the carotid arteries is sustained impairment in cognitive function [9,47], suggesting that these adaptations after BCAS are not enough to prevent cognitive impairment. Cognitive deficits after BCAS may be an outcome of a synchronized dysfunction in the cerebral microcirculation and the neurons. However, there is a growing body of evidence that cerebrovascular dysfunction may precede measurable cognitive deficits [21,24,38]. Our study emphasizes the contribution of impaired PA dilation that may worsen cognitive end points. Investigating drugable targets that restore the dilatory properties of PAs may allow us to intervene even before cognitive impairment is observed. Therapeutic agents !104 that enhance EETs-mediated dilatory pathway may be a credible option for reducing microcirculatory dysfunction and improving associated cognitive functions during vascular cognitive impairment.   2.6 - Limitations. A few limitations and caveats of our study should be acknowledged. First, since we used a scanning laser Doppler flowmetry, we did not perform quantitative measurements of cerebral blood flow. Even though it measures perfusion in arbitrary units (perfusion units), calibration of the laser detector is not changed between subjects. Thus, it still allows for comparative analyses between experimental groups. Second, we did not assess anxiety levels in the rats and that could affect the tests used to evaluate cognitive deficits. Third, we did not measure the contribution of other mediators in the EDHF consortium, since MS-PPOH abolished dilation in the Sham rats. Fourth, we only studied PAs from male 28 weeks old WKY rats and cannot confirm that similar findings can be expected in female rats or in rats of a different age.  Finally, the conclusions of this study are supported by associative evidence rather than direct links. 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Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 1: 848-858, 2006.           !111            CHAPTER 3           !112 Carotid artery stenosis in hypertensive rats impairs dilatory pathways in parenchymal arterioles and posterior communicating arteries  3.1- Abstract   Hypertension is a leading risk factor for vascular cognitive impairment. Hypertension is strongly correlated with carotid artery stenosis in patients. In normotensive rats chronic cerebral hypoperfusion induced by bilateral common carotid artery stenosis (BCAS) leads to cognitive impairments that were associated with impaired endothelium dependent dilation in parenchymal arterioles (PAs). The aim of this study was to assess changes in function and structure of PAs from 28-week-old adult male stroke prone spontaneously hypertensive rats 8 weeks after BCAS surgery. We hypothesized that BCAS would impair endothelium dependent dilation in PAs and induce artery remodeling compared to Sham rats. PAs from BCAS had endothelial dysfunction, assessed using a pressure myography. Inhibition of nitric oxide and prostaglandin production had no effect on dilation in PAs from Sham or BCAS rats. Surprisingly, inhibiting epoxyeicosatrienoic acid production increased dilation in PAs from BCAS rats, but not from Sham rats. Similar results were observed in the presence of inhibitors for all three dilatory pathways suggesting that epoxygenase inhibition may have restored a NO/prostaglandin independent dilatory pathway in PAs from BCAS rats. PAs from BCAS rats had remodeling with reduced wall thickness.  Impaired myogenic tone and impaired dilation was evident in posterior communicating arteries from BCAS rats. In BCAS rats these collateral arteries also exhibited hypertrophic outward remodeling. These data suggest that marked endothelial dysfunction in cerebral arteries and !113 arterioles in SHRSP with BCAS may be associated with the development of vascular cognitive impairment.   3.2 - Introduction Chronic hypertension and carotid artery stenosis are risk factors for cerebral small vessel disease, the common cause of vascular cognitive impairment (VCI) [66]. Cerebral small vessel disease is characterized by lesions in the subcortical brain regions [46,65], and dysfunction of arterioles in the parenchyma [17,64]. Parenchymal arterioles (PAs) act as bottlenecks to parenchymal perfusion [43] and interruption of blood flow in a single PA causes cognitive deficits [55]. Bilateral common carotid artery stenosis (BCAS) in stroke prone spontaneously hypertensive rats (SHRSP), a genetic model of essential hypertension, mimics cerebrovascular pathologies observed in cerebral small vessel disease [18]. Thus evaluating structural and functional changes in the PAs from SHRSP with BCAS could elucidate the pathogenesis of the disease [10,61].    Studying functional changes in PAs from SHRSP with BCAS is important because cerebrovascular endothelial failure drives many of the detrimental microvascular changes in cerebral small vessel disease [69]. In SHRSP, impaired endothelium dependent dilation is evident in pial arteries and arterioles [38,39]. While we have shown PAs from young SHRSP exhibit increased myogenic tone compared to PAs from normotensive rats [47], endothelial function in PAs from SHRSP has been largely unexplored.  !114 Endothelium dependent dilation can be mediated by nitric oxide (NO), dilatory prostaglandins, and endothelium-derived hyperpolarizing factor (EDHF). We have shown that BCAS impairs EDHF mediated dilation in PAs from normotensive rats through impaired epoxyeicosatrienoic acid (EETs) signaling [35]. In the present study, we hypothesized that BCAS in 20-week-old male SHRSP, with malignant hypertension, would impair endothelium dependent dilation and induce remodeling in the PAs. We also assessed effects of BCAS on posterior communicating arteries (PComAs) that connect the vertebrobasilar circulation to the circle of Willis and acts as the primary collateral during reduced blood flow through the middle cerebral artery in carotid occlusion models [28]. Finally, we examined maximal reserve dilatory capacity of SHRSP after BCAS. 3.3 - Methods 3.3.1 - Animals, and surgery. The experimental procedure was approved by the Michigan State University Institutional Animal Care & Use Committee and was in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals (2011). 20-week-old male SHRSP from the colony housed at Michigan State University were randomized into two groups: one group had sham surgeries and the other group underwent BCAS surgery. Rats were maintained on a 12-h light/dark cycle, with tap water and regular rat chow available ad libitum. To induce BCAS, rats were anesthetized with 3% isoflurane in oxygen and both common carotid arteries were exposed. A blunted 27-gauge needle was placed next to the artery, and two 6-0 silk sutures were used to firmly tie the carotid artery and the needle together. !115 After the ties were in place the needle was carefully removed, and the artery was reduced to the diameter of the needle (0.41mm) to induce stenosis of the artery [35]. The stenosis surgery had 100% survival rate. Post surgery, Ketoprofen (5mg/kg/day) and Combi-pen 48 (22,000 units/day) were administered subcutaneously for three and two days, respectively.  At 28-29 weeks of age, rats were anesthetized with 3% isoflurane, weighed, and euthanized by decapitation after exsanguination. Organs and tissues used in this study were harvested after decapitation.  3.3.2 - Measurement of cerebral perfusion. Pial artery perfusion before and immediately after stenosis induction was assessed using a scanning laser Doppler (PeriScan PIM 3, Perimed, Stockholm, Sweden) under 3% isoflurane anesthesia while the body temperature was maintained at 37¡C. An incision was made in the top of the head to expose the skull and the skull was cleaned for measurement of pial artery perfusion. Mean flow was analyzed using the LDPIwin 3.1 software (Perimed) [35].   3.3.3 - Novel object recognition test. Novel object recognition testing was conducted in an open box with opaque walls. Prior to testing, rats were allowed to explore the box for 15 minutes a day for 3 days. During the training phase rats were allowed to explore two identical objects for 30 seconds. 90 minutes later, during the test phase a novel object replaced one of the familiar objects in the box. The familiar and novel objects were placed in opposite corners of the box and the position was alternated between rats to prevent bias for a particular location. Exploration took place when the rat pawed at, sniffed or whisked at a distance of 1 cm from the object [24]. The time spent exploring !116 the novel object and the total exploration time was recorded.  After each trial, 70% alcohol was used to clean the objects to remove olfactory cues from the objects and box. Novel exploration quotient was expressed as a ratio of the time spent exploring the novel object to the total exploration time [41].   3.3.4 - Morris water maze. A circular tank  (29" deep and 70" across) filled with water (30¡C), was positioned in a room with external cues visible to the swimming rat. One week before surgery the rats were trained for 3 consecutive days, rats were repeatedly placed in the tank from all four possible directions (north, south, west and east), and they learned to locate a platform 1 cm above the water level in less than 60 seconds. The test phase started 4 weeks post-surgery. The test was carried out in trials of 2 from all four directions per session, and testing sessions were performed once a week until the rats were 28 weeks old. The protocol for the test was the same as training except the platform was hidden 1 inch below the surface of opaque water. Performance was measured by assessing escape latency (time required to reach the platform) [35,67].  3.3.5 - Acetazolamide challenge. Rats were anesthetized and maintained at 37¡C, acetazolamide (0.2mg/g) was injected into the tail vein and cerebral perfusion was assessed by scanning laser Doppler as described above. Flow was measured every five minutes for thirty minutes [35]. Cerebrovascular reserve capacity (CVR) over time was calculated using the formula, [(flow after acetazolamide at time x - baseline pial artery flow)/ baseline pial artery flow] *100.  !117 3.3.6 - PA isolation and cannulation. 8 weeks after BCAS rats were euthanized and decapitated. The brain was removed and kept in ice-cold Ca2+-free physiological salt solution (PSS, in mM: NaCl 140, KCl 5, MgCl2¥7H2O 1, HEPES 10, Dextrose 10) for isolation of the PAs. The section of brain tissue surrounding the middle cerebral artery was removed and placed in Ca2+-free PSS at 4¡C, with 1% bovine serum albumin. The middle cerebral artery was gently separated from the surrounding tissue, and PAs branching from the middle cerebral artery were transferred to cannulation chamber. PAs were cannulated between two glass pipettes on a small, 3 axis-micromanipulators (MT XYZ, Neport, Irvine, CA), and bathed in warm (37¡C) PSS containing 1.8mM Ca2+, and pressurized to 60mmHg to allow for the generation 20% spontaneous myogenic tone [35]. The outer diameter of the PAs was constantly tracked and recorded using MyoView 2.0 software (Danish Myo Technology, Aarhus, Denmark).    3.3.7 - Assessment of dilatory pathways in the PAs. PAs that generated at least 20% tone were used for concentration response studies. Myogenic tone was calculated using the following formula: [1 & (active external diameter/passive external diameter)] * 100. A cumulative concentration response to abluminal administration of carbachol (1nM to 100µM) was carried out. Intraluminal administration of drugs was not possible because of the small diameter and high flow resistance of the micropipettes. Each cannulated vessel was used for one concentration-response experiment. To isolate EDHF dependent dilation, PAs were incubated with the NO synthase (NOS) inhibitor N (G)-nitro-L-arginine methyl ester (L-NAME, 100 !M) and the cyclooxygenase (COX) inhibitor indomethacin (Indo, 10 !M) for 30 minutes, prior to development of myogenic !118 tone [68]. EETs mediated dilation was assessed by incubating the arteries with cytochrome P450 (CYP) epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH; 10 !M) in a similar fashion. GSK1016790A (GSK101) was used to assess function of transient receptor potential cation channel subfamily vanilloid member 4 (TRPV4), and sodium nitroprusside (SNP) was used to assess endothelium-independent dilation. % Dilation was calculated using the formula: ((external diameter at drug concentration - baseline external diameter)/(passive external diameter - baseline external diameter))*100.   3.3.8 - Cannulation and assessment of dilatory pathways in the PComAs. The PComA was carefully dissected from the brain and transferred to the pressure myograph chamber. A branchless segment of the PComA was cannulated between two glass micropipettes. The PComA was allowed to equilibrate in PSS, at an intraluminal pressure of 80mmHg and 37¡C until the development of 20% spontaneous myogenic tone. Changes in outer diameter of PComAs were tracked as described above for the PA. Endothelium dependent dilation was assessed using intraluminal carbachol (10-10 to 10-5 mol/L) at physiological flow rate (20 dynes/cm3). Myogenic tone and percent dilation were calculated using the formula used for the PAs.  3.3.9 - Assessment of the structural and mechanical properties of arteries. After completion of the concentration response curves the PAs and PComAs were placed in Ca2+-free PSS containing 2mM EGTA and 10!M SNP to assess their passive structure. Intraluminal pressure was increased from 3 to 180 mmHg in 20 mmHg increments and !119 lumen diameter and wall thickness was recorded after 5 minutes at each pressure step. Outer diameter was calculated as lumen diameter plus left and right wall thickness. The wall-to-lumen ratio, passive distensibility and circumferential wall stress were calculated as described previously [2].  3.3.10 - Statistical analysis. Novel object recognition test, myogenic tone and resting lumen diameter data were analyzed by StudentÕs t-test or a non-parametric alternative if data were not normally distributed. Cerebrovascular reactivity, dilation, passive and mechanical properties were analyzed by two-way ANOVA followed by Sidak correction for multiple comparisons, or a non-parametric alternative. In cases of unequal variance data was transformed to homogenize variance with the following formula: y= (y. A two-way ANOVA was then performed on the transformed data and a P value less than 0.05 was considered significant. Analyses were performed using GraphPad Prizm 6.0 software (La Jolla, CA, USA).   3.3.11 - Chemical reagents. MS-PPOH was purchased from Cayman Chemical (Ann Arbor, MI, USA) and acetazolamide was purchased from X-Gen (Big Flats, NY, USA). All other chemicals and reagents were purchased from Sigma-Aldrich (Saint Louis, MO, USA).  3.4 - Results 3.4.1 - 8 weeks after BCAS cerebral perfusion was restored, but CVR was absent in both Sham and BCAS rats. BCAS caused an immediate )38% reduction in cerebral !120 perfusion whereas in Sham rats surgery had no effect (Fig 3.1A). Perfusion levels in SHRSP with BCAS were restored 8 weeks after stenosis such that there was no difference between Sham and BCAS rats at that time point (Fig 3.1B). Baseline perfusion in SHRSP before induction of BCAS was reduced compared to WKY rats from our previous study (Table 1) [35].  Acetazolamide, a carbonic anhydrase inhibitor, induces vasodilation in cerebral arteries and was used to assess CVR. BCAS impaired CVR in WKY rats, thus we expected to see BCAS impair CVR in SHRSP [35]. We have demonstrated that acetazolamide produces 8.7% increase in cerebral perfusion in Sham WKY rats (Table 1). However, surprisingly, there was a complete absence of reserve capacity in Sham SHRSP (Figure 3.1C). To test if this absence of CVR could be attributed to chronic hypertension we assessed CVR in younger (18-week-old) SHRSP. CVR in 28-week-old SHRSP and SHRSP with BCAS was significantly reduced compared to 18-week-old SHRSP.  3.4.2 - BCAS impaired memory function in SHRSP. To verify that BCAS induced cognitive impairment in SHRSP, novel object recognition testing was carried out 7 weeks post-surgery. Novel exploration quotient was reduced after BCAS indicating impaired memory function (Figure 3.2A). However, spatial learning abilities measured using Morris water maze were not impaired by BCAS (Figure 3.2B).  3.4.3 - Altered dilatory signaling and remodeling in PAs after BCAS. Myogenic tone generation was unchanged in PAs from BCAS rats (Figure 3.3A). Dilation to muscarinic receptor agonist carbachol was impaired in PAs form BCAS rats (Figure 3.3B). To !121 evaluate if impaired dilation was due to reduced sensitivity of the smooth muscle cells to NO, a concentration response curve to NO donor SNP was constructed. BCAS reduced sensitivity of NO in the PAs, as evidenced by increased EC50 value (Figure 3.3C). We also measured function of the transient receptor potential channel vanilloid 4 (TRPV4), which is a downstream target of muscarinic receptor activation[57]. There was no difference in dilation to TRPV4 agonist GSK101 between the groups (Figure 3.3D).   We assessed EDHF mediated dilation, predominant in PAs [36,56], by incubating PAs with L-NAME and indomethacin to inhibit NO and prostaglandins production respectively. In the presence of L-NAME and Indo there was no change in dilation in PAs from Sham  (Figure 3.4A) or BCAS rats (Figure 3.4B). EETs act as an EDHF in several vascular beds and are produced by CYP epoxygenase [6]. Inhibition of CYP epoxygenase with MS-PPOH enhanced dilation in PAs from BCAS rats (Figure 3.4D) while there was no change in dilation in PAs from Sham rats (Figure 3.4C). MS-PPOH in addition to L-NAME, and Indo had no effect on dilation in PAs from Sham rats (Figure 3.4E), however dilation in PAs from BCAS rats was increased (Figure 3.4F).  Passive artery structure was assessed under zero flow and Ca2+ free conditions. There was no change in the lumen diameter of the PAs between the groups (Figure 3.5A), but PAs from BCAS rats had reduced wall thickness (Figure 3.5B) accompanied by reduced wall-to-lumen ratio (Figure 3.5C). The thinner walls of PAs from BCAS rats had increased wall stress (Figure 3.5D), without a change in distensibility (Figure 3.5E).  !122 3.4.4 - PComAs exhibit impaired dilation and outward remodeling after BCAS. BCAS impaired myogenic tone generation (Figure 3.6A) and dilation to carbachol in the PComAs (Figure 3.6B). PComAs from BCAS rats exhibited hypertrophic outward remodeling, evidenced by a small but significant increase in the lumen diameter (Figure 3.6C) and increased wall cross-sectional area (Figure 3.6D). While was no difference in wall stress (Figure 3.6E) in the PComAs between the groups, distensibility was increased in PComAs from BCAS rats (Figure 3.6F).    !123  Table 1: Significant key differences in dilatory pathways and structural properties between PAs from Sham WKY rats and Sham SHRSP. * different from Sham WKY rats, # different from Sham SHRSP, ^ different from WKY rats with BCAS, p<0.05. For scanning laser Doppler studies n= 6 to 12, for cognitive studies n=11 to 18, for vessel studies n=3 to 8. Data from WKY rats have been previously published[36].    !124 Figure 3.1: Cerebral perfusion was restored in BCAS rats and cerebrovascular reserve capacity was absent in both Sham and BCAS rats. (A) BCAS surgery reduced cerebral perfusion, whereas perfusion levels were unchanged with sham surgery. (B) At the end of 8 weeks, there was no difference in cerebral perfusion between the groups. (C) Cerebrovascular reserve capacity was absent in both Sham SHRSP and SHRSP with BCAS. However, 18-week-old SHRSP had significantly higher reserve capacity compared to 28-week-old SHRSP; *p<0.05, different from Sham rats.  !125 Figure 3.2: BCAS impaired memory function with no change in spatial learning abilities. (A) Novel object recognition was reduced in SHRSP with BCAS, signifying that they spent a smaller portion of their exploration with the novel object (B) There was no difference in Morris water maze between the groups; *p<0.05, different from Sham rats. !126  Figure 3.3: Dilation was abolished in PAs from SHRSP with BCAS. (A) There was no difference in myogenic tone in PAs from the two groups, however (B) dilation to carbachol was abolished in PAs from BCAS rats. (C) LogEC50 of NO donor SNP was increased in PAs from BCAS. (D) Dilation to TRPV4 agonist, GSK1016790A was not different between Sham and BCAS; *p<0.05, different from Sham rats.   !127  Figure 3.4: Inhibition of CYP epoxygenase restored dilation in PAs from BCAS rats. (A, B) Inhibition of NOS and COX pathways did not change dilation in PAs from Sham or BCAS rats. (C) Enhanced dilation was observed in PAs from Sham rats in the presence of MS-PPOH. (D) Dilation is restored in PAs from SHRSP with BCAS in the presence of MS-PPOH. (E) There was no difference in dilation in the presence of MS-PPOH, L-NAME and Indo in PAs from Sham rats. (F) However, in PAs from BCAS rats addition of the three inhibitors enhanced dilation; * p< 0.05, different from Sham rats.   !128 Figure 3.4 (contÕd)   !129 Figure 3.5: Hypotrophic remodeling in the PAs from BCAS rats. (A) No difference in lumen diameter in the PAs after BCAS. (B) Wall thickness was reduced in the PAs after BCAS.  !130 Figure 3.5 (contÕd) (C) Wall to lumen ratio was reduced in the PAs after  BCAS. (D) There was no difference in distensibility in PAs between the groups. (E) Increased wall stress in the PAs after BCAS; *p<0.05, different from Sham rats.   !131 Figure 3.6: Impaired myogenic tone generation, dilation, and remodeling in the PComAs after BCAS. (A) Myogenic tone in PComAs from BCAS rats was impaired (B) Dilation to carbachol was abolished in PComAs after BCAS, with dilation only at the !132 Figure 3.6 (contÕd) highest concentration of the drug (10-5M). (C) Increased lumen diameter or outward remodeling was accompanied by (D) increased wall cross sectional area in PComAs from BCAS rats. (E) There was no difference in wall stress in PComA between the two groups. (F) Increased distensibility in the PComAs after BCAS; *p<0.05, different from Sham rats.    !133 3.5 Ð Discussion The current study focused primarily on the PAs since they modulate the functional hyperemia that is essential for cognitive function [16,32]. We observed several salient findings regarding dilatory pathways and structure in PAs in this hypertensive model of cognitive impairment. Our studies demonstrated that BCAS induced remodeling and impaired dilation in PAs with significant alterations in EDHF mediated dilator pathways. PComAs from BCAS rats also exhibited impaired dilation and hypertrophic outward remodeling. These changes in PAs and PComAs were associated with memory deficits in BCAS rats. By comparing these findings to our previous studies in normotensive Wistar Kyoto (WKY) rats [35] we were also able to elucidate critical differences in the responses of hypertensive and normotensive rats to chronic cerebral hypoperfusion. To facilitate our discussion we have listed these differences in Table 1.   3.5.1 - BCAS impaired memory function in SHRSP. Our initial hypothesis was that the BCAS surgery would produce more marked cognitive decline in hypertensive rats than in normotensive rats. Our rationale for this was two-fold, first, hypertension causes detrimental artery remodeling and endothelial dysfunction in large cerebral arteries that could exacerbate BCAS induced vascular injury. Second, antihypertensive drugs have the potential to prevent, or delay the onset of cognitive impairment in hypertensive patients [19]. However, we observed that memory impairment in SHRSP with BCAS was similar to WKY rats with BCAS (Table 1) [35] and BCAS had no effect on spatial learning abilities in SHRSP. This lack of effect of BCAS on spatial learning could be due to the fact that this capacity appears to be already impaired in Sham SHRSP compared !134 to Sham WKY rats (Table 1)[35]. Impaired learning-memory in SHRSP was also reported in a passive avoidance test [29]. This mildly impaired cognition may be the result of mild hypoperfusion in the SHRSP, in the current study we observed reduced blood flow in the SHRSP compared to WKY rats (Table 1)[35] at 20 weeks of age. Other labs have also reported similar reduction in perfusion in brain regions critical for cognition in SHRSP with established hypertension [23,26,40].  3.5.2 - CVR was absent in SHRSP. To understand the vascular component of the observed cognitive deficits we first evaluated CVR with acetazolamide. Impaired CVR in patients is associated with cognitive dysfunction and is predicative of increased risk of ischemic events [7,31,34]. Acetazolamide, a carbonic anhydrase inhibitor, causes carbonic acidosis by reducing extracellular pH to induce dilation [63]. Reserve capacity was detected in WKY rats and blunted in WKY rats with BCAS (Table 1) [35], similar to what is observed in patients with carotid artery lesions [7,31]. In SHRSP, acetazolamide did not increase blood flow in either the BCAS or the Sham rats suggesting that there was no cerebrovascular reserve at the time points studied. This may be a function of the reduced blood flow observed even in the sham SHRSP, autoregulatory vasodilation occurs in response to reduced cerebral perfusion and this may prevent further dilation of cerebral arteries to other vasoactive stimuli such as acetazolamide [48]. Inward remodeling of large cerebral arteries could reduce cerebral perfusion pressure in pial arteries from SHRSP. Similar impairment in CVR in response to acetazolamide was reported in patients with severe hypertension [13]. In healthy volunteers CVR declines with age [54]. Age dependent reductions in CVR were also observed in mice after !135 unilateral internal carotid occlusion [22]. To assess if aging could have contributed to the severely disrupted cerebral hemodynamics in 28-week-old Sham SHRSP, we carried out acetazolamide challenge in 18-week old SHRSP. These younger SHRSP had a robust cerebrovascular reserve capacity suggesting that impaired reserve capacity cannot be due to a strain or a blood pressure effect. Taken together, these data suggest that impaired reserve capacity in the 28-week-old SHRSP is related to increased age and the duration of hypertension. These differences in CVR between 28-week-old Sham SHRSP and WKY rats, from our previous study[35], provide additional support to the argument that hypertension accelerates normal aging of the brain [51]. It should also be noted that isoflurane causes cerebral artery dilation [25], thus it is possible that the cerebral arteries from the SHRSP were maximally dilated before the administration of the ACZ. 3.5.3 - Dysfunctional dilatory pathways in PAs from BCAS rats. Impaired PA function plays a critical role in the progression of cognitive impairment [3,59]. In the current study dilation to carbachol was abolished with no change in myogenic tone in PAs from SHRSP with BCAS, similar to the changes we reported in PAs from normotensive WKY rats with BCAS [35]. Reduced sensitivity to NO donor in PAs from SHRSP with BCAS also suggested that impaired dilation was not wholly endothelium-dependent and could be due to diminished ability of smooth muscle cells to relax. In resistance arterioles, Gq coupled muscarinic receptor activation stimulates TRPV4 via a protein kinase C dependent mechanism [9]. We speculated that the signaling pathway connecting muscarinic receptor activation to TRPV4 activity could be disrupted and may !136 have contributed to impaired dilation to carbachol. This does not appear to be the case as the dilator response to direct TRPV4 activation was similar in Sham and BCAS rats. It is however possible that hypertension itself affects this signaling pathway as TRPV4 mediated dilation was significantly impaired in SHRSP compared to Sham WKY rats.   In small arterioles, instead of an endothelium-derived ÔfactorÕ inducing smooth muscle cell hyperpolarization, direct electrotonic transfer of hyperpolarization occurs from the endothelial cell to the smooth muscle cells [15].  However, we observed no change in dilation in PAs from Sham SHRSP in the presence of the MS-PPOH alone or in combination with L-NAME and indomethacin suggesting that this endothelium dependent hyperpolarization (EDH) likely mediates dilation in PAs from Sham SHRSP.  TRPV4 activation produces Ca2+ sparklets that open Ca2+-activated potassium channels and initiates EDH [57]. In hypertensive mice, attenuated Ca2+ sparklet generation leads to reduced dilation to carbachol and GSK101 in mesenteric arteries [58]. It is possible that reduced dilation to carbachol and GSK101 in PAs from Sham SHRSP could be due to a similar impairment in TRPV4 function (Table 1) [35]. These findings in Sham SHRSP were in distinct contrast to our study in Sham WKY rats where inhibiting EETs production with MS-PPOH abolished dilation [35].    While impaired dilation was observed in the presence of NOS/COX inhibitors, it was restored in the presence of CYP epoxygenase in PAs from BCAS rats. Dilation remained enhanced when MS-PPOH was added in combination with NOS/COX inhibitor, suggesting that improved dilation was dependent on CYP epoxygenase and !137 independent of NOS/COX pathways in PAs from BCAS rats. The data also suggest that chronic cerebral hypoperfusion induces CYP epoxygenase dysfunction, such that enzyme activity generates vasoconstrictors. Epoxidation reactions generate large amounts of reactive oxygen species such as superoxide or hydroxyl radical [14]. Under normal conditions, antioxidants such as superoxide dismutase convert superoxide to hydrogen peroxide that dilates PAs [60]. However, under pathophysiological conditions with exhausted antioxidant levels, reactive oxygen species impairs dilation in cerebral arteries [27,37,42,70]. It is possible that after BCAS, reactive oxygen species generated during epoxidation reactions in the PAs impairs dilation via ion channel dysfunction [1,4,12,14,33,49]. Increased oxidative stress disrupted TRPV4-Ca2+ activated potassium channel signaling and impaired dilation in cerebral arteries [73].   3.5.4 - Impaired myogenic tone, and dilation in PComAs from BCAS rats. Although our primary focus was on the PAs, we also assessed dilation in PComAs since these arteries control for the severity of hypoperfusion in carotid occlusion models [28]. This is the first study examining dilatory functions in the PComAs after chronic cerebral hypoperfusion. PComAs from SHRSP with BCAS exhibited impaired myogenic tone and dilation. PComAs connect the vertebrobasilar circulation to the circle of Willis, and are exposed to increased flow after obstruction or occlusion of a large artery [11] such as in our BCAS model. Reduced carbachol dilation in PComAs from SHRSP with BCAS suggests that dilation in response to increased flow [50] could be impaired in these collateral arteries.   !138 Impaired dilation in arteries is characteristic of hypertensive models [30,38,72], thus it is likely that PComAs from Sham SHRSP also have attenuated dilation. After occlusion of a large artery and before the initiation of artery remodeling, flow mediated dilation is necessary to prevent perfusion from falling below critical levels. We speculate that impaired flow mediated dilation and inward remodeling (Table 1) in PComAs could be important for the pathogenesis of cerebral hypoperfusion in SHSRP. In pilot studies conducted in our lab we induced cerebral hypoperfusion by staggered and complete occlusion of both the common carotid arteries.  We observed a fifty percent mortality rate in SHRSP, compared to the zero mortality in WKY rats after the same procedure. The inability of the PComAs to responds the insult may have contributed to increased mortality observed in the hypertensive rats.    3.5.5 - Differential remodeling in PComAs and PAs from BCAS rats. PComAs from BCAS rats underwent outward hypertrophic remodeling evidenced by an increase in lumen diameter and wall cross sectional area. Outward remodeling is an adaptive mechanism by which blood flow is restored after induction of the hypoperfusion [11]. Outward remodeling has been reported in primary collaterals in models of common carotid artery occlusion, as well as in patients with bilateral internal carotid artery obstruction [8,20,44]. At 80 mmHg, the remodeling index was 107.6% and the growth index was 30% implying that the structural difference between the groups involves remodeling and some growth [21]. Impaired myogenic tone in PComAs may have initially increased wall stress, which acted as a stimulus for growth [21] and lead to increased wall cross sectional area and normalized wall stress in PComAs after 8 !139 weeks of BCAS.  Hypertension attenuated outward remodeling of small leptomeningeal anastomoses in response of unilateral common carotid artery occlusion [45] and our study shows that this maladaptation extends to PComAs remodeling in response to BCAS. Compared to the 14% increase in lumen diameter we reported in PComAs from WKY rats with BCAS, stenosis in SHRSP lead only to a 7% increase in lumen diameter (Table 1)[35].  The effects of BCAS on flow though the PAs and the PComAs is likely different. The PAs branch off the middle cerebral artery that receives the majority of the flow from the carotid arteries. During stenosis flow through the middle cerebral arteries to the PAs is likely reduced. While PAs from WKY rats with BCAS did not exhibit remodeling (Table 1) [35], PAs from SHRSP with BCAS had reduced wall thickness or hypotrophy. It is conceivable that after BCAS in PAs from SHRSP were exposed to reduced blood flow compared to PAs from WKYs as a result of inward hypertrophic remodeling in cerebral arteries upstream from them [52]. Models of low blood flow in the peripheral circulation have reported hypotrophic remodeling due to loss of smooth muscle cells [5]. Loss of smooth muscle cells could explain the elevated wall stress observed in PAs from BCAS rats, and could initiate degenerative changes in the vessel walls that increase the incidence of lesions and hemorrhagic transformation post-stroke [53,71].   In conclusion, our study shows that the combination of malignant hypertension and carotid stenosis impaired dilation and induced remodeling in PAs from SHRSP. These changes could initiate the development of pathological features observed during !140 cerebral small vessel disease. Additionally, impaired PA dilation could be indicative of a diminished ability of the PAs to conduct dilation and enable functional hyperemia. These findings suggest that preventing these deleterious changes in cerebral arteries and arterioles from hypertensive patients with carotid stenosis could prevent the onset of cognitive deficits.  3.6 - Limitations. Our current study has a few limitations that need to be mentioned. First, we used scanning laser Doppler on animals with an intact skull. The laser has a penetration depth of approximately 1 mm into the tissue and skull of a 28-wk-old SHRSP is around 0.9 mm thick, thus the setup could only measure flow in surface pial circulation. 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Soluble epoxide hydrolase metabolizes epoxyeicosatrienoic acids (EETs), which are part of the arachidonic acid signaling cascade. Inhibition of the enzyme elevates EETs levels and potentiates their dilatory effects in cerebral arteries. We hypothesized that treatment with the soluble epoxide hydrolase inhibitor trifluoromethoxyphenyl-3 (1propionylpiperidin-4-yl) urea (TPPU) for 8 weeks would prevent cognitive dysfunction and ameliorate impaired dilation in PA dilation in 28-week-old male SHRSP with BCAS. TPPU prevented CCH induced memory deficits, evaluated by novel object recognition test. TPPU also partially prevented impaired dilation in PAs from BCAS rats, as evidenced by increased dilation to carbachol compared to vehicle treated BCAS rats. Inhibition of nitric oxide and prostaglandin production enhanced endothelium dependent hyperpolarizing factor (EDHF) dilation in PAs from TPPU treated BCAS rats. Similarly inhibition of EETs production increased dilation in both PAs from TPPU treated and vehicle treated BCAS rats. Inhibiting the production of nitric oxide, prostaglandin and EETs completely blocked dilation in PAs from TPPU treated BCAS rats. Taken together, these data suggest that in PAs from TPPU treated BCAS rats inhibition of one dilatory !151 pathway leads to a compensatory increase in alternative dilatory pathways. TPPU treatment also increased brain mRNA expression of superoxide dismutase-3, uncoupling protein-2 and doublecortin, markers of neuronal survival and neurogenesis. These data suggest that both vasoprotective and neuroprotective effects of chronic TPPU administration prevent CCH induced memory deficits.   4.2 - Introduction Vascular cognitive impairment (VCI), the second most common cause of dementia after AlzheimerÕs disease, is a spectrum of cognitive deficits that have a cerebrovascular origin [37]. According to the World Health Organization 2016, an estimated 47.5 million people live with dementia and the number is projected to rise to 135.5 million by 2050. While there are drugs to alleviate the symptoms of VCI, there are no reliable therapeutic options to halt the progression of this condition [15]. VCI patients usually have co-morbidities such as hypertension and carotid stenosis that makes it difficult to find successful therapeutic options. Regardless of the exact cause, chronic cerebral hypoperfusion (CCH) is an underlying theme in the pathogenesis of VCI [15,32,48]. Thus, therapies aimed at enhancing cerebral blood flow and improving the vascular health of the brain may have potential therapeutic effects on VCI patients.   There are strong arguments that soluble epoxide hydrolase (sEH) inhibitors may enhance blood flow to the cerebral microcirculation in VCI models and reduce cognitive deficits. sEH metabolizes epoxyeicosatrienoic acids (EETs) [12,44] that are produced in erythrocytes, endothelial cells, astrocytes and perivascular nerves[19]. EETs act as an !152 endothelium-derived hyperpolarizing factor (EDHF) to induce vasodilation [13,17,24]. Reducing EETs level reduces blood flow in the cerebral microcirculation[1]. Overexpression of sEH in cerebral endothelial cells impaired dilation to acetylcholine assessed using a cranial window preparation [58]. This suggested that enhanced sEH activity could decrease EETs levels to reduce blood flow in the microcirculation, and this could lead to the development of VCI. Indeed, enhanced activity of sEH in brains from VCI patients indicated a role for sEH in the progression of the disease[35]. The study also reported higher intensity of sEH immunoreactivity in arterioles near microinfarcts [35] raising the possibility that detrimental effects of sEH targeting the cerebral microvasculature of patients could have aided the progression of VCI.   Cerebral parenchymal arterioles (PAs) regulate functional hyperemia, defined as increases in blood flow in response to an increase in neuronal activity and is essential for cognitive functions[14,31,61]. In fact, Kleinfeld et al showed that occlusion in a single PA induced cognitive impairment[46]. Bilateral common carotid artery stenosis (BCAS), a CCH model of VCI, abolished dilation in PAs from stroke prone spontaneously hypertensive rats (SHRSP). We have shown that EDHF dilation is pronounced in PAs[28]. Since sEH is present predominately in the cerebral arterioles compared to arteries [35] it is possible that sEH inhibition could elevate EETs levels and enhance EDHF dilation in the PAs.    We know that BCAS impairs dilation in PAs from SHRSP. In the present study, we hypothesized that sEH inhibition in SHRSP with BCAS would prevent CCH induced !153 dilatory impairment in PAs and alleviate cognitive deficits. To test the hypothesis we treated SHRSP with the sEH inhibitor trifluoromethoxyphenyl-3 (1propionylpiperidin-4-yl) urea (TPPU) for 8 weeks. We assessed cognitive functions, and PA dilatory pathways and structure. We also measured cerebrovascular reserve capacity in this model. Furthermore, we evaluated the chronic effects of TPPU on cognitive function and PAs dilatory functions and structure from sham SHRSP.   4.3 - Methods 4.3.1 - Animals and surgery. 20-week-old male SHRSP from the colony housed at Michigan State University were used for this study. BCAS surgeries were carried out as previously described by our laboratory [28]. Briefly, rats were anesthetized using isoflurane, and placed in a supine position, the skin over the common carotid arteries was shaved and cleaned. An incision was made in the skin and the common carotid arteries were exposed. A 27-gauge blunt needle was place alongside the left carotid artery and two 6-0 silk sutures were used to tie the carotid artery and the needle firmly together. The needle was then carefully removed to induce partial occlusion or stenosis of the artery. The same procedure was carried out on the right common carotid artery. Sham surgeries were carried out in an identical manner as the BCAS surgeries but without tying the common carotid arteries. The experimental protocol was approved by the Michigan State University Institutional Animal Care & Use Committee and was in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals (2011).  !154 4.3.2 - TPPU treatment. After surgeries the rats were randomized into two groups: one group received TPPU (3mg/kg/per day) while the other received vehicle (20% PEG400) in their drinking water until euthanasia 8 weeks later.   4.3.3 - Blood pressure measurement. In the BCAS group, blood pressure was measured one week before the surgery and 7 weeks after the surgery by tail-cuff plethysmography using a RTBP1001 tail-cuff blood pressure system (Kent Scientific, Torrington CT) as described previously by our laboratory [40].    4.3.4 - Novel object recognition test. Novel object recognition test was carried out in an open box with opaque walls. Rats were acclimatized to the box for 15 minutes/day for three days. During the familiarization phase the rats were allowed to explore two identical objects for 30 seconds. Memory function was evaluated either 90 minutes or 24 hours later by replacing one of the sample objects with a novel object. The color, size and texture of the familiar objects and the novel objects were similar to prevent any bias. Total exploration time and the time spent exploring the novel object was recorded. Exploration took place when the rat pawed at, sniffed or whisked with its snout directed at the object from a distance of under !1 cm [20]. Between each rat and each trial, 10% alcohol was used to clean the objects and the box to remove any olfactory cues. Novel exploration quotient was defined as a ratio of the time spent exploring the novel object to the total exploration time[34].   4.3.5 - Morris water maze. A circular tank filled with water was positioned in a room with external cues visible to the swimming rat. 4 weeks after BCAS the rats were trained !155 for a week and the Morris water maze (MWM) test was performed once a week for three consecutive weeks. During the training phase the rats were placed in the tank from all four possible directions (north, south, west and east), and could only escape by locating a platform 1 cm above the water level. The protocol for the MWM test was the same as the training phase and had trials of 2 from all four directions per session. However, during the testing phase the platform was hidden 1 inch below the surface of water colored opaque with non-toxic white tempera paint. To evaluate the ratÕs spatial learning ability, latency time or time required to locate the escape platform was measured [28].   4.3.6 - Cerebral tissue perfusion and acetazolamide challenge. 8 weeks after BCAS surgery, cerebral perfusion was measured with a scanning laser Doppler (PeriScan PIM 3, Perimed, Stockholm, Sweden). Rats were anesthetized and placed on a heated platform. The scanning laser Doppler was positioned )18 cm above the exposed and cleaned skull, and cerebral perfusion was recorded. Acetazolamide, a carbonic anhydrase inhibitor, was used to assess cerebrovascular reserve capacity [22]. Cerebral perfusion was recorded immediately after tail vein injection of acetazolamide (0.2mg/g) [28] and then for every five minutes for twenty-five minutes. A total of 4 consecutive scans were performed. Mean perfusion was analyzed using the LDPIwin 3.1 software (Perimed) and expressed as mean perfusion units. Cerebrovascular reserve capacity was calculated as the percentage change in cerebral tissue perfusion after administration of cerebral artery dilator compared to baseline perfusion.  !156 4.3.7 - PA isolation and cannulation. After 8 weeks of vehicle/TPPU treatment, rats were anesthetized using isoflurane and thoracotomized. After exsanguination the brain was removed and placed in ice-cold Ca2+-free physiological saline solution (PSS, in mM: NaCl 140, KCl 5, MgCl2¥7H2O 1, HEPES 10, Dextrose 10) for PA dissection. To isolate PAs, a section of brain tissue containing the middle cerebral artery (MCA) was removed and PAs branching from the MCAs were carefully dissected and transferred to a cannulation chamber. PAs were cannulated between two glass micropipettes (<40 !m) mounted on small, 3-axis micromanipulators (MT-XYZ, Newport, Irvine, CA) that could be adjusted in three dimensions [9]. PAs were bathed in warm (37¡C) PSS containing 1.8mM Ca2+ and pressurized at 60 mmHg until myogenic tone developed. The perfusion chamber was positioned on the stage of an inverted microscope (Leica DMIL, Wetzlar, Germany) with a 20x objective (Leitz Wetzlar objective, numerical aperture: 0.3). PA outer diameter was constantly tracked and recorded using MyoView 2.0 software (Danish Myo Technology, Aarhus, Denmark). To assess endothelium dependent dilation, PAs were incubated with increasing concentrations of carbachol (1nM to 100µM) in the bath. To assess the role played by EDHF in the carbachol-induced dilation, PAs were incubated with inhibitor of nitric oxide (NO) synthase (NOS)  inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 100 !M) and cyclooxygenase (COX) inhibitor indomethacin (Indo, 10 !M) prior to development of myogenic tone. To evaluate the contribution of EETs in endothelium dependent hyperpolarization, PAs were incubated in a similar fashion with cytochrome P450 (CYP) epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH; 10 !M). !157 Endothelium-independent dilation was studied by incubating PAs with nitric oxide (NO) donor, sodium nitroprusside (SNP: 1nM to 100 !M). Only one concentration-response experiment was performed on each cannulated PA. Myogenic tone was calculated as: [1 & (active external diameter/passive external diameter)] * 100. % Dilation was defined as: ((external diameter at drug concentration -baseline external diameter)/(passive external diameter-baseline external diameter))*100.  4.3.8 - Assessment of structural and mechanical properties in PAs. After the end of the concentration response experiments, the arteries were bathed in Ca2+-free PSS containing 2mM EGTA + 100!M SNP to assess changes in passive structure of the PAs. A CCD camera (Hitachi Kokusai Electric Inc., Japan) connected to a video dimension analyzer (Living Systems Instrumentation, Burlington, VT) was used to assess structural changes. Intraluminal pressure was increased from 3 to 180 mmHg in 20 mmHg increments and lumen diameter and wall thickness was measured after 5 minutes at each pressure step. Outer diameter was calculated as lumen diameter + left wall thickness + right wall thickness. The cross-sectional area, wall-to-lumen ratio, circumferential wall stress and passive distensibility were calculated as described previously[4].  4.3.9 - High-performance liquid chromatography coupled with tandem mass spectrometry for TPPU measurement. Plasma samples of TPPU were liquid-liquid extracted with 200 µL of ethyl acetate and the residues were resuspended in 50 µL of internal standard solution (200 nM CUDA) for LC-MS/MS analysis[51]. Samples were analyzed using an Agilent 1200 SL Series HPLC with a 2.1x150mm Eclipse plus 1.8µm !158 particle size C18 column (Agilent, Santa Clara, CA). The liquid chromatography system was coupled with an AB Sciex 4000 QTRAP hybrid, triple-quadrupole mass spectrometer (Redwood City, CA). The solvent system consisted of water/acetic acid (999/1 v/v, solvent A) and acetonitrile/acetic acid (999/1 v/v; solvent B).   4.3.10 - Real-time polymerase chain reaction. Total mRNA was isolated from the brain tissue surrounding the MCAs using Qiagen RNeasy lipid tissue kit (Qiagen Sciences). RNA was also extracted from the MCAs and PAs using TRIzol reagent. RNA concentrations were evaluated using a NanoDrop spectrophotometer. Identical amounts of RNA were reverse transcribed using a qScript cDNA Synthesis Kit (Quanta Biosciences, Gaithesburg, MD). Real-time PCR was performed using a 96-well plate containing the cDNA and TaqMan primers and probes (Applied Biosystem, Foster City, CA) for sEH, superoxide dismutase-3 (SOD-3), uncoupling protein-2 (UCP-2), doublecortin (DC), transient receptor potential cation channel subfamily vanilloid member 4 (TRPV4) and gap junction proteins, connexin 40 (Cx40), connexin 43 (Cx43). Fold changes in mRNA expression compared to the vehicle group were calculated using the 2-''CT method [26] and #-2-microglobulin was used as endogenous control[41].  4.3.11 - Statistical analyses. Novel object recognition test, myogenic tone data were analyzed by StudentÕs t-test or a non-parametric alternative when the data were not normally distributed. Cerebrovascular reactivity, endothelium-dependent dilation, passive structural properties were analyzed by two-way ANOVA followed by Sidak correction for multiple comparisons, or a non-parametric alternative. Analyses were carried out using the software GraphPad Prizm 6.0 (La Jolla, CA, USA). !159  4.3.12 - Chemicals and reagents. MS-PPOH was purchased from Cayman Chemical (Ann Arbor, MI, USA) and acetazolamide was purchased from X-Gen (Big Flats, NY, USA). TPPU was a gift from Dr. Bruce Hammock at the Department of Entomology, University of California (Davis, CA). All other chemicals and reagents were purchased from Sigma-Aldrich (Saint Louis, MO, USA).  4.4 - Results 4.4.1 - Physiological parameters, and cerebrovascular reserve capacity were unchanged in TPPU treated BCAS rats. Plasma level of TPPU after chronic administration for 8 week was approximately 1276 ± 45.77 ng/ml in BCAS rats. There was no difference in pial artery perfusion between the vehicle-treated and TPPU-treated groups 8 weeks after BCAS surgery (Figure 4.1A). There was also no change in cerebrovascular reserve capacity, assessed using acetazolamide challenge, between the groups (Fig 4.1B). TPPU had no effect on systolic arterial pressure measured a week before drug administration and throughout the 8 weeks of the study (pre-TPPU vs post-TPPU at week 8: 219.4 ± 3.7 vs 228.7 ± 6.4 mmHg). Rats were weighed before euthanasia and TPPU administration did not affect body weight (vehicle vs TPPU: 361.3 ± 5.9 vs 377.4 ± 7.4 g).   4.4.2 - TPPU prevented memory deficits in BCAS rats. BCAS impaired memory in SHRSP (novel exploration quotient for BCAS+ vehicle vs Sham+ vehicle, 90 minutes retention time: 0.48± 0.06 vs 0.65±0.02; and 24 hours retention time: 0.46 ± 0.08 vs !160 0.7±0.06, p<0.05). TPPU treated BCAS rats had increased novel exploration quotient for retention times of 90 minutes and 24 hours (Fig 4.2A) compared to vehicle treated BCAS rats. TPPU prevented CCH induced memory deficits in BCAS rats (novel exploration quotient for BCAS+ TPPU vs Sham+ vehicle, 90 minutes retention time: 0.65± 0.05 vs 0.65 ± 0.02; 24 hours retention time: 0.70 ±0.07 vs 0.70± 0.06, p>0.05). In MWM test there was no difference in time required to find the escape platform between the groups suggesting that TPPU had no effect on spatial learning abilities (Fig 4.2B).  4.4.3 - TPPU improved dilation in PAs from BCAS rats without affecting artery structure. There was no change in PA myogenic tone between the groups (Figure 4.3A). 8 weeks of BCAS impaired dilation in the PAs (% dilation at 10-5M of carbachol, Sham+ vehicle vs BCAS+ vehicle: 23.56  ± 3.69 vs 3.64 ± 2.36, p<0.05). Dilation to carbachol was increased in PAs from TPPU treated BCAS rats compared to PAs from vehicle treated BCAS rats that had negligible dilation (Figure 4.3B). While maximal dilation to SNP remained unchanged, EC50 for the NO donor was increased in PAs from TPPU treated BCAS rats (Figure 4.3C). The dilator response to nifedipine was unchanged between the groups (Figure 4.3D). TPPU treatment had no effect on passive structure. There was no change in lumen diameter (Figure 4.3E) or wall thickness (Figure 4.3F) between the groups. TPPU treatment also had no effect on biomechanical properties (data not shown) of the PAs.   To assess changes in endothelium dependent dilatory pathways PAs were incubated with inhibitors of NO, prostaglandins and EETs production before constructing carbachol !161 concentration response curves. L-NAME, Indo, or MS-PPOH had any effect on myogenic tone in PAs from either group (Figure 4.4A and 4.4B). Dilation after blockade of NOS and COX is EDHF mediated [7]. Dilation in PAs from vehicle treated BCAS rats did not change after co-inhibition of NOS and COX with L-NAME and Indo respectively (Figure 4.4C). However, in the presence of the inhibitors dilation in PAs from TPPU treated BCAS rats were enhanced (Figure 4.4D). In the presence of MS-PPOH, inhibitor of CYP epoxygenase, dilation was enhanced in PAs from vehicle and TPPU treated groups (Figure 4.4E and 4.4F). In the presence of all three inhibitors, dilation in PAs from vehicle treated BCAS rats was enhanced whereas in PAs from TPPU treated rats dilation was abolished (Figure 4.4G and 4.4H).  4.4.4 - Chronic TPPU treatment enhanced PA dilation in Sham rats and had no effect on memory. Chronic treatment with TPPU in Sham rats did not have any effect on novel exploration quotient for retention times of 90 minutes and 24 hours (Fig 4.5A). Myogenic tone in the PAs remained unchanged between vehicle treated and TPPU treated Sham rats (Figure 4.5B). Dilation to carbachol was increased in PAs from TPPU treated sham rats compared to PAs from vehicle treated Sham rats (Figure 4.5C). There was no change in lumen diameter (Figure 4.5D), wall thickness (Figure 4.5E), or any biomechanical properties (data not shown) in the PAs with TPPU treatment.  4.4.5 - Chronic administration of TPPU altered mRNA expression in the brain from BCAS rats. There was increased sEH mRNA expression in the brain region surrounding the MCAs and the PAs from TPPU treated BCAS rats (Figure 4.6A). Chronic TPPU treatment also increased superoxide scavenger SOD-3 (Figure 4.6B) !162 and neuroprotective mitochondrial UCP-2 (Figure 4.6C) mRNA levels in the brains of BCAS rats. Similarly, mRNA expression of DC, marker for neurogenesis, was increased in the brain of TPPU treated rats (Figure 4.6D).   Total mRNA was also extracted from the MCAs and the PAs of BCAS rats. Chronic TPPU treatment did not effect mRNA expression of sEH (Figure 4.7A), and TRPV4 (Figure 4.7B). mRNA levels of gap junction proteins, Cx40 (Figure 4.7C), and Cx43 (Figure 4.7D) were also unchanged between the groups.   Figure 4.1: TPPU treatment did not change mean cerebral perfusion or cerebrovascular reserve capacity 8 weeks after BCAS. (A) 8 weeks after BCAS mean cerebral perfusion, measured using a scanning laser Doppler, was not different between TPPU and vehicle treated BCAS rats. (B) There was no difference in cerebrovascular reserve capacity between the groups. !163  Figure 4.2: TPPU treatment improved memory in BCAS rats. (A) Chronic TPPU treatment increased time spent with novel object evidenced by increased novel exploration quotient with a retention time of 90 minutes (left) and 24 hours (right). (B) There was no difference in latency time from the MWM test between the groups. Each data point is mean ± SEM; * p<0.05, different from vehicle-treated rats.   !164  Figure 4.3: Increased dilation in PAs from TPPU treated BCAS rats compared to vehicle treated BCAS rats. Chronic TPPU treatment in SHRSP with BCAS (A) did not affect myogenic tone generation (B) and increased PA dilation assessed using !165 Figure 4.3 (contÕd)  carbachol. (C) Sensitivity to the NO donor, SNP was reduced: EC50 for SNP was significantly higher in PAs from TPPU treated BCAS rats. (D) There was no difference in response to Ca2+ channel blocker nifedipine in between the groups. There were no differences in structural properties in PAs from vehicle treated and TPPU treated BCAS rats, evidenced by no differences in (E) lumen diameter or (F) wall thickness at 60mmHg; *p<0.05, different from vehicle-treated rats. !166  Figure 4.4: Enhanced endothelium dependent dilation and altered dilatory pathways in PAs from TPPU treated BCAS rats. (A, B) There was no change in myogenic tone generation with addition of the different inhibitors in PAs from either group. (C) Dilation to carbachol remained unaltered in the presence of the Indo and  !167 Figure 4.4 (contÕd)  L-NAME in PAs from vehicle treated BCAS rats. (D) Inhibition of NO and prostaglandin production enhanced dilation in PAs from TPPU treated BCAS rats. In the presence of CYP epoxygenase inhibitor, MS-PPOH, dilation was enhanced in (E) PAs from vehicle treated BCAS rats and from (F) TPPU treated BCAS rats. In the presence of MS-PPOH, L-NAME, and Indo (G) dilation in PAs from vehicle treated BCAS rats was enhanced, whereas in  (H) in PAs from TPPU treated BCAS rats dilation was abolished; *p<0.05, different from vehicle-treated BCAS rats. !168  Figure 4.5: TPPU enhanced PA dilation and had no effect on memory function in Sham rats. Chronic TPPU treatment (A) did not have any effect on memory functions in    !169 Figure 4.5 (contÕd)  Sham SHRSP. (B) In the PAs there was no difference in myogenic tone generation between the groups however (C) TPPU treated Sham rats increased dilation to carbachol. TPPU treatment did not have any effect on passive structure as evidenced by no difference in (D) lumen diameter or (E) wall thickness between the groups at 60 mmHg; *p<0.05, different from vehicle-treated Sham rats.   Figure 4.6: Increased expression of mRNA levels in brains from TPPU treated BCAS rats. (A) There was increased mRNA expression of sEH in the brains of BCAS rats after 8 weeks of TPPU treatment. TPPU treated rats had increased mRNA levels of   !170 Figure 4.6 (contÕd) (B) SOD-3 and (C) UCP-2. Chronic TPPU treatment also (D) increased expression of DC mRNA, in the brain; *p <0.05, different from vehicle-treated BCAS ratsÕ brains.   Figure 4.7: No difference in mRNA expression of sEH and mediators of EETs signaling in MCAs and PAs from the BCAS groups. (A) There was no difference in mRNA levels of sEH in MCAs and PAs from vehicle and TPPU treated BCAS rats. (B) Chronic TPPU treatment did not affect mRNA level of TRPV4 in the MCAs and PAs.   !171 Figure 4.7 (contÕd) Similarly there was no difference in mRNA expression of gap junction proteins (C) Cx40, and (D) Cx43 in the MCAs and PAs between the groups.! 4.5 - Discussion We demonstrated, for the first time, that chronic inhibition of sEH with TPPU prevents memory deficits after BCAS in SHRSP. This was associated with improved endothelial functions in PAs from TPPU treated BCAS rats; at 10-5M carbachol, dilation in PAs from TPPU treated BCAS rats was ) 63% of the dilation in PAs from vehicle treated Sham SHRSP.  In PAs from TPPU treated rats dilation was enhanced when EETs or NO/prostaglandin production was inhibited and was only abolished when EETs and NO/prostaglandins production were blocked simultaneously. A possible explanation for these data is that both EETs and NO/prostaglandins contributed to carbachol dilation in PAs from TPPU treated BCAS rats; in contrast primarily EDHF dilation was observed in vehicle treated BCAS rats. Chronic TPPU administration also enhanced dilation in the PAs from Sham SHRSP but did not have any effect on memory function.  Together, these results suggest that chronic inhibition of sEH may promote dilation in PAs from different models of cerebrovascular disorders.   Plasma TPPU levels in BCAS rats suggests that sEH in endothelial cells, erythrocytes and smooth muscle cells in the cerebral vasculature were exposed to a concentration of the lipophilic drug higher than its IC50 value (5nM) [38]. Extrapolating from a study published by the Hammock lab where they measured brain levels of TPPU [38] it is !172 likely that with a dose of 3mg/kg/per day TPPU also inhibited sEH in the brain, where it is expressed in neurons, astrocytes and oligodendrocytes [49]. While sEH mRNA levels in the MCAs and PAs were unchanged with TPPU treatment, mRNA levels of the enzyme were increased in brain samples that contained blood vessels, neurons and glial cells. It is possible chronic sEH inhibition lead to a compensatory increase in sEH mRNA levels in neurons and glial cells.   4.5.1 - TPPU treatment prevented CCH-induced memory impairment in BCAS rats. Our results suggest that amelioration of impaired PA dilation could be responsible, at least in part, for the absence of memory deficits in TPPU treated BCAS rats. Studies have shown that EETs enhance blood flow during functional hyperemia by improved neurovascular coupling [25,45]. Therefore, increased duration of EETs activity in the PAs and neurons could have prevented impaired neurovascular coupling and associated memory deficits after BCAS in TPPU treated rats. While our study focused on the effects of TPPU on the PAs, it is possible that memory function was preserved due to enhanced neuronal plasticity as well as vasodilatory effects. Based on studies that suggest that endothelial dysfunction can lead to loss of endothelium derived trophic signaling essential for survival and growth of neurons and oligodendrocytes [2,23] it is possible that improved endothelial function in PAs from TPPU treated BCAS rats could enable a supportive environment for neuronal survival. In support of our hypothesis, brains from TPPU treated BCAS rats had elevated levels of UCP-2 mRNA, an inducible mitochondrial protein that activates cellular redox signaling and prevents neuronal death [29]. Furthermore, increased mRNA levels of neurogenesis marker DC in TPPU treated !173 BCAS rats suggest that TPPU could be enhancing formation of new neurons to compensate for CCH induced neuronal death. Neuroprotective effects of sEH inhibitors have been delineated in ischemic stroke and depression[59,60]. TPPU potentiated nerve growth factor induced neurite outgrowth in vitro[43] and increased the levels of brain derived neurotrophic factor, which is critical for memory acquisition and long-term potentiation [21,33,43,56]. Thus TPPU could prevent memory impairment in BCAS rats by partially preventing PA dilatory function, and enhancing neuronal growth and survival.   Interestingly, TPPU did not improve memory function in Sham SHRSP, nor did it improve spatial learning abilities in SHRSP with BCAS. We have shown that SHRSP have impaired spatial learning abilities even without BCAS, and BCAS did not exacerbate the impairment. These data suggests that while TPPU prevented the effects of BCAS on cognitive functions, it did not have any effect on cognitive deficits already present in Sham SHRSP. SHRSP are a model of malignant hypertension[11] and accelerated brain aging[50]. Study showed that 20-week-old SHRSP had impaired memory and learning [50] suggesting that Sham SHRSP had measurable cognitive deficits before TPPU was administered and that the drug could not reverse hypertension associated deficits.  4.5.2 - TPPU treatment prevented endothelial dysfunction in PAs from BCAS rats. There was no difference in myogenic tone generation in PAs from BCAS rats. Myogenic tone generation in PAs is dependent on Ca2+ influx through L-type voltage-gated Ca2+ !174 channels[42]. We assessed the function of these channels with nifedipine and did not find a difference between the groups. Chronic treatment of TPPU improved dilation in PAs from SHRSP with BCAS and enhanced dilation in PAs from Sham SHRSP underscoring the potential beneficial effects sEH inhibition in the microcirculation of different cerebrovascular disease models. Improved dilation could be due to elevated EET levels after chronic inhibition of sEH in endothelial cells and the smooth muscle cells.   Since EETs act upstream of various signaling cascades to induce smooth muscle cell hyperpolarization and relaxation [10], dilation of PAs from TPPU treated SHRSP can occur by multiple mechanisms; attenuated signaling in a dilatory pathway may activate a compensatory dilatory pathway. We assessed mRNA expression of TRPV4, and gap junction proteins Cx40, and Cx43, implicated in EETs-mediated dilation[10]. There was no change in mRNA levels of the proteins in the MCAs and PAs from TPPU treated BCAS rats. Further studies are required to tease out which pathways are active in PAs from TPPU treated SHRSP.   4.5.3 - NO/prostaglandins, and EETs mediate dilation in PAs from TPPU treated BCAS rats. In PAs from TPPU treated BCAS rats enhanced dilation after inhibition of NOS/COX could be due to two possible reasons: 1) the pathways were producing vasoconstrictors or 2) they were suppressing an alternative dilatory pathway. Since TPPU ameliorates CCH induced impairment in PA dilation, the second reason is more likely. As evidence for this argument, studies show that NO inhibits EETs-mediated !175 dilation[3,36]. In coronary arteries, NO inhibited EETs-mediated smooth muscle cell hyperpolarization via a protein kinase G dependent pathway[57]. Thus it is possible that inhibition of NO could have enhanced EETs dilation in PAs from TPPU treated BCAS rats. Further studies assessing PA dilation in the presence of inhibitors of NO downstream signaling such as protein kinase G could provide insight into this mechanism.   PAs from vehicle treated BCAS rats also demonstrated dysfunctional CYP epoxygenase activity since dilation was enhanced when in the presence of MS-PPOH, alone and in combination with L-NAME and Indo. In PAs from SHRSP with BCAS we proposed that this enhanced dilation could be attributed to CYP epoxygenase dysfunction that results in inhibition of ion channels that are involved in vasodilation. In contrast, in PAs from TPPU treated rats while dilation was enhanced in the presence of MS-PPOH alone, it was abolished when the inhibitor was added in combination with L-NAME and Indo. Reactive oxygen species, generated by dysfunctional CYP epoxygenase, can impair function of large conductance Ca2+ activated potassium channels [8]. NO dilation can be mediated by activation of large conductance Ca2+ activated potassium channels [6]. While we have not evaluated PA dilation in the presence of antioxidants and inhibitors of large conductance Ca2+ activated potassium channels, it is possible that inhibition of CYP epoxygenase enhances large conductance Ca2+ activated potassium channels mediated NO dilation in PAs from TPPU treated BCAS rats.   !176 Taken together, these vasoreactivity studies using different inhibitors suggest that carbachol dilation in PAs from TPPU treated BCAS rats exhibits both EETs and NO/ prostaglandin components. EETs enhance expression of endothelial NOS [16,53], thus it is conceivable that NO production could be a result of elevated EETs levels in PAs from TPPU treated BCAS rats. Interestingly, the PAs from TPPU treated BCAS rats were less sensitive to SNP than vehicle treated BCAS rats. Increased NO production in PAs from TPPU treated BCAS rats could have reduce responsiveness of soluble guanylyl cyclase[39], and could be a critical homeostatic mechanism to regulate PA dilation.   4.5.4 - TPPU treatment had no effect on PA passive structure. There was no change in passive structure in PAs from BCAS or sham rats with chronic TPPU treatment. 6 weeks of inhibition of sEH with AUDA reduced the wall thickness but had no effect on lumen diameter in middle cerebral arteries from 12-week-old SHRSP [47], highlighting the role of sEH inhibitors in attenuating smooth muscle cell proliferation. While reduced wall thickness in MCA provided vascular protection against cerebral ischemia, such hypotrophic remodeling in PAs from BCAS rats could have detrimental effects. BCAS induces hypotrophy in PAs evidenced by reduced wall thickness (BCAS+ vehicle vs Sham+ vehicle: 5.5 ± 0.7 vs 7.1 ± 0.8 µm at 60mmHg, p<0.05). Hypotrophy was associated with increased wall stress (BCAS+ vehicle vs Sham+ vehicle: 409.0± 62.8 vs 267.3 ± 25.1 dynes/cm2 at 60mmHg, p < 0.05) in PAs from BCAS rats.   !177 4.5.5 - TPPU treatment had no effect on cerebrovascular reserve capacity in BCAS rats. TPPU has no effect on cerebrovascular reserve capacity in pial arteries and arterioles, a clinical assessment that can be used to predict ischemic cerebrovascular events[27]. Acetazolamide inhibits carbonic anhydrase and retards the conversion of H+ and HCO3- to carbonic acid to induce a rapid decrease in the pH of cerebrospinal fluid [54]. In normotensive rats BCAS abrogates cerebrovascular reserve capacity [28]. We previously demonstrated that cerebrovascular reserve capacity was severely impaired in 28-week-old SHRSP, independent of BCAS. Hypertension associated structural and functional changes in pial arteries and arterioles induce chronic reduction in cerebral perfusion pressure [5,30]. Impaired cerebrovascular reserve capacity suggests that the arteries are maximally dilated in response to reduced cerebral perfusion pressure and cannot dilate any further to vasoactive stimuli. A caveat of this study was that we used scanning laser Doppler that has a penetration depth of 0.5 - 1 mm, on a closed skull preparation to assess perfusion. Thus, we were only able to measured reserve capacity in the pial circulation and not the intraparenchymal circulation.   In summary, these data demonstrates that memory function in SHRSP with BCAS is associated with dilatory capacity of PAs. Even a small increase in dilatory capacity can substantially enhance blood flow to downstream capillaries during functional hyperemia given that flow is proportional to the fourth power of radius changes (as per Poiseuille equation of fluid dynamics).  Moreover, inhibition of sEH may protect against memory deficits not only improving dilatory pathways in the Pas but may also promote neurogenesis and neuronal survival.  !178 4.6 - Limitations. Our study has a few limitations and caveats that need to be mentioned. First, our behavioral assessments were limited to novel object recognition test and MWM. More cognitive tests could have helped thoroughly characterize the cognitive profile of BCAS rats with chronic TPPU treatment. Second, we focused on vascular changes in the PAs and did not measure neurovascular coupling that is associated with cognitive function [14]. Third, we did not assess changes in inflammatory markers with TPPU treatment. 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I also hypothesized that sEH inhibitor TPPU would prevent CCH induced cognitive impairment in SHRSP with BCAS by preventing endothelium dysfunction, and artery remodeling in the PAs. The data presented in this dissertation show that BCAS impairs dilation in PAs, which was associated with memory impairment in SHRSP. Chronic treatment with TPPU in SHRSP with BCAS improved endothelium dependent dilation the PAs and prevented the development of memory deficits as further evidence for this association. However, contrary to my hypothesis the studies described here demonstrate that SHRSP have impaired cognitive abilities and PA dysfunction compared to WKY rats, and BCAS did not exacerbate these impairments in SHRSP. The hypotheses were tested through three specific aims: BCAS in WKY rats, BCAS in SHRSP and chronic TPPU treatment in SHRSP with BCAS.   PAs regulate parenchymal perfusion[10], and PA dysfunction is one of the primary feature of SVD that cause cognitive impairment in 45% of VCI patients [11]. BCAS is widely accepted as a model of VCI that induces hippocampal damage, white matter lesions and cognitive deficits [6].  Using this VCI model, the first aim was designed to !188 assess endothelial functions (Figure 5.1) and the structural properties of PAs from normotensive WKY rats. In WKY rats BCAS was associated with impaired spatial learning abilities, impaired memory formation and diminished PA dilation via endothelium dependent and independent mechanisms. The predominance of EETs signaling was demonstrated by drastically impaired dilation in the presence of a selective CYP epoxygenase inhibitor in PAs from Sham rats. PAs from BCAS rats had impaired dilation to a TRPV4 channel agonist, and TRPV4 mRNA expression was reduced in PAs and MCAs from BCAS rats. These changes in dilatory functions were not accompanied by structural changes in PAs from WKYs with BCAS. However outward remodeling was observed in PComAs that serve a primary collateral after BCAS. We also showed reduced CVR capacity in WKY rats after BCAS that is predicative of increased risk for ischemic events (Figure 5.2).      Figure 5.1: Dilatory pathways assessed in parenchymal arterioles. Muscarinic receptor activation by carbachol stimulates the production of NO, prostaglandins (PGs) !189 and EDHF (not shown). My studies focused on the contribution of EETs in EDHF mediated dilation. MS-PPOH, Indo, and L-NAME were used in combination or individually to inhibit the production of EETs, PGs, and NO respectively. GSK101 (not shown), a selective agonist for TRPV4 channel (pink channels) was also used to assess channel function. TRPV4 channels are downstream mediators of both EETs and carbachol dilation. Endothelium independent dilation was assessed using NO donor SNP and voltage-gated Ca2+ channel inhibitor nifedipine was also used to assess channel function.   Impaired EETs-mediated dilation and TRPV4 function in PAs from BCAS rats raises a few additional questions regarding this study. Regioisomers of EETs have differential selectivity for TRPV4 and thus presence or absence of regioisomer in PAs from WKY rats could provide mechanistic insight about impaired EETs signaling after BCAS. 11,12-EET increases Ca2+ influx by translocation of TRPC6 to the plasma membrane in vitro [2]. TRPC6 are implicated in smooth muscle cell depolarization (chapter 1, section 1.3.1), thus EETs dependent TRP Ca2+ influx may not necessarily produce dilation but could also facilitate vasoconstriction. Investigation into the spatial and temporal integrity of TRP channels mediated [Ca2+]i microdomains could provide further insight into the mechanism of the EETs signaling dysfunction in PAs from BCAS rats.    The second aim of this project attempted to combine two risk factors of VCI, CCH and malignant hypertension, and study their effects on the cerebral vasculature. In this study several notable differences between Sham SHRSP and Sham WKY rats were observed that bolstered our second hypothesis that SHRSP would have worse outcome after !190 BCAS compared to WKY rats. Sham SHRSP had impaired spatial learning abilities and cerebrovascular reserve capacity compared to Sham WKY rats. Moreover, PAs from Sham SHRSP had reduced dilation to carbachol, reduced response to TRPV4 channel agonist and wall hypertrophy.   BCAS completely abolished dilation in PAs from SHRSP. Dysfunctional CYP epoxygenase may have played a role in impaired dilation in the PAs, since dilation was restored to Sham levels in the presence of MS-PPOH. PAs from BCAS rats also underwent hypotrophic remodeling with no change in lumen diameter. PComAs from BCAS rats had impaired myogenic tone and dilation. PComAs also exhibited hypertrophic outward remodeling, however the percent increase in lumen diameter after BCAS was smaller in SHRSP compared to WKY rats. CVR to acetazolamide challenge was abolished in 28-week-old SHRSP, independent of BCAS and in stark contrast to 18-week-old SHRSP. These results highlight that prolonged hypertension disrupts cerebral autoregulation and has a negative effect on CVR.   The third aim assessed the effects of chronic inhibition of sEH with TPPU in SHRSP with BCAS. sEH inhibitors increase levels of EET that have been implicated in modulating neurovascular coupling and increasing CBF. TPPU partially prevented impaired dilation in PAs after BCAS in SHRSP. EETs, and NO/prostaglandin dilatory pathways were active in PAs from TPPU treated BCAS rats unlike PAs from vehicle treated BCAS rats where we observed NO/prostaglandin independent dilation. Passive structure of the PAs did not change with TPPU from BCAS rats. Chronic TPPU treatment prevented CCH induced memory impairment in SHRSP assessed using novel !191 object recognition test, and this effect could be associated with improved dilatory functions in the PAs and increased mRNA expression of neuroprotective markers. Taken together, these results validate other studies that show that sEH inhibitors improve dilatory signaling and exert neuroprotective effects[13,17]. Inhibitors of sEH have also shown potent anti-inflammatory, and angiogenic properties that could mediate the beneficial effect of TPPU in SHRSP with BCAS. Moreover, anti-depressant effects of TPPU[12] may give it a significant advantage over other treatment option since depression is both a risk factor[4] and a symptom of VCI[7].   The work in this dissertation highlights the importance of PA function in CCH models of VCI. Interestingly, enhanced dilation in PAs from TPPU treated Sham SHRSP compared to vehicle treated Sham SHRSP was not associated with improved memory. These findings suggest that chronic sEH prevented cognitive impairment induced by BCAS and had no effect on cognitive functions in Sham SHRSP.   5.2 - Considerations about the model used, and treatment regimens used. Occlusion of common carotid arteries is a well-established model of inducing VCI.  A 27-gauge needle was used to induce BCAS by CCH. This partial occlusion surgery in SHRSP had hundred percent survival rates, as opposed to complete bilateral common carotid artery occlusion surgery that had only a fifty percent survival rate. An interesting possibility that is worth exploring would be changes in the carotid arteries after stenosis. Study shows that permanent occlusion of the carotid artery destroys the endothelium and promotes accumulation of fat and aggregation of platelets around the arteries[1]. In the BCAS model even a diluted version of this inflammatory response and phenotypic !192 change in smooth muscle cells could lead to further reduction in lumen diameter of carotid arteries.   As described in the chapter 1, section 1.4.3, 20-week-old SHRSP had malignant hypertension thus it is likely that the cerebral vasculature was unable to compensate for the severe drop in cerebral perfusion associated with complete bilateral common carotid occlusion surgeries. The time frame for CCH was chosen based on data that showed that at the end of 8 weeks measurable cognitive deficits could be observed. It is important to note, that while SHRSP are a good model to study cerebrovascular diseases assessing cognitive functions in this strain requires subtlety. SHRSP are also used as a model of attention deficit hyperactivity disorder [15]; thus it is highly possibly that memory functions and spatial learning abilities could be affected by impaired attention in SHRSP and may mask the true extent of CCH induced cognitive deficits.   In SHRSP with BCAS, TPPU was administered chronically for 8 weeks after BCAS to test if inhibition of sEH could prevent CCH induced dilatory impairments in PAs and cognitive dysfunction. Most of the studies using sEH inhibitors, including work in this dissertation, focused on the CYP epoxygenase products of AA metabolism, however other lipids feed into the same CYP/sEH axis. CYP epoxygenases preferentially metabolize *-3 polyunsaturated fatty acids such as eicosapentanoic acids and docosahexaenoic acid to epoxides products, which can be hydrolyzed by sEH into respective diols. Epoxides of *-3 fatty acids have more potent vasodilatory and anti-inflammatory effects [3] than EETs and could have had significant contributions towards the observed effects of TPPU. !193  Figure 5.2: Summary of novel findings. There were notable differences between Sham WKY rats and Sham SHRSP (grey box). However after BCAS, SHRSP (blue) and WKY rats (red) had similar end outcomes. Chronic TPPU administration in SHRSP with BCAS (blue box) partially prevented some of the detrimental effects of CCH.   5.3 - Novel findings My studies are the first to show an association between dilatory functions in the PAs and cognitive function. These findings were bolstered by mechanistic insight into impairments in PA dilation following CCH and after chronic sEH inhibition (Figure 5.2). The findings were, as follows:   !194 5.3.1 - The effects of BCAS on WKY rats.  ¥ 8 weeks of BCAS lead to significant impairment in PA dilation evidenced by reduced dilation to carbachol, an endothelium dependent dilator, and NO donor SNP.  ¥ EDHF mediated dilation was prominent in PAs from Sham WKY rats. In the presence of MS-PPOH, an inhibitor of CYP epoxygenase, dilation to carbachol was completely abolished.  ¥ CCH impaired dilation in PAs from WKY rats with BCAS by impairing EETs dependent dilation. In WKY rats with BCAS mRNA expression of TRPV4 channels, one of the mediators of downstream EETs signaling was reduced in the MCAs and the PAs and response to TRPV4 channel agonist was diminished in PAs.  ¥ There was no change in passive structure of the PAs from WKY rats with BCAS. However, the PComAs from WKY rats with BCAS had outward remodeling.   5.3.2 - The effects of BCAS on SHRSP. ¥ 8 weeks of BCAS drastically reduced endothelium dependent dilation to carbachol in PAs from SHRSP with BCAS. There was a concomitant reduction in SNP sensitivity in PAs from SHRSP with BCAS.  ¥ Similar to our observation in PAs from Sham WKY rats, EDHF mediated dilation was prominent in PAs from Sham SHRSP. However, in the presence of MS-PPOH dilation in Sham WKY rats remained unaltered, suggesting EETs-mediated dilation may not be significant. Similarly dilation remained enhanced in the presence of L-NAME, Indo and MS-PPOH implying that dilation in PAs from Sham SHRSP was not a result of cross-talk between the three dilatory pathways.  !195 ¥ Dilation was enhanced in the presence of MS-PPOH and in the presence of L-NAME, Indo and MS-PPOH in PAs from SHRSP with BCAS. These data suggests diminished endothelium dependent dilation in PAs from BCAS rats may be mediated by dysfunctional cytochrome P450 epoxygenase.  ¥ In SHRSP with BCAS, PComAs had impaired myogenic tone and dilation.  ¥ In SHRSP with BCAS while PAs had hypotrophic remodeling, PComAs had hypertrophic outward remodeling suggesting that CCH induced remodeling varies along the cerebrovascular tree.    5.3.3 - Sham WKY rats versus Sham SHRSP. ¥ Baseline perfusion in 20-week-old SHRSP was reduced compared to WKY rats. Other notable differences between the WKY rats and SHRSP were observed when the rats were 28 weeks old. Sham SHRSP had impaired spatial learning abilities compared to Sham WKY rats. While CVR capacity was observed in WKY rats, it was completely absent in SHRSP. An age dependent impairment in CVR was observed in SHRSP underscoring the malignant effects of prolonged hypertension.  ¥ Compared to PAs from Sham WKY rats, Sham SHRSP had reduced dilation to carbachol. In PAs from Sham SHRSP dilation to carbachol was not EETs dependent, unlike in PAs from WKY rats. Similarly, PAs from Sham SHRSP had reduced response to the TRPV4 channel agonist.  These changes were accompanied by hypertrophic remodeling in PAs from Sham SHRSP compared to PAs from Sham WKY rats suggesting that chronic hypertension induces profound functional and structural changes in these arterioles.   !196 5.3.4 - The effects of TPPU on SHRSP with BCAS, and on Sham SHRSP. ¥ TPPU treatment prevented CCH induced memory function in SHRSP with BCAS. This finding was associated with attenuation of impaired PA dilation in BCAS treated with TPPU, possibly via a mechanism dependent on improved NO, prostaglandins and EETs signaling.  ¥ Chronic TPPU treatment enhanced dilation in PAs from Sham SHRSP compared to vehicle treated rats but had no effect on memory function.   5.4 - Limitations ¥ There were a few caveats in our animal model. The rats used were of a relatively young age (28-week-old) and were not the most clinically representative model since increased age is a major risk factor VCI. Moreover, inducing BCAS using the blunted needle method has a considerable acute effect on cerebral perfusion. We did not separate the acute and chronic effects of BCAS. Assessing the acute effects of BCAS on PA dilation and cognitive deficits could have provided more insight into the nature of the association between PA function and cognitive abilities.  ¥ Pressure myograph is a widely used ex vivo system to assess vascular structure and function. Pressurized arteries are flushed with and kept in PSS, thus the effects of blood viscosity are lost. Additionally, the precision of measurement of media thickness is not optimal in this system. To assess remodeling in vessels, it is possible to calculate biomechanical properties such as stress, and distensibility. However, increased margins of error of derived measurements make statistical tests difficult to interpret.  !197 ¥ Scanning laser Doppler was used to assess cerebral perfusion. The laser has a depth of penetration of approximately 1 to 2 mm depth and since the skull was not thinned it is highly likely that we only assessed blood flow in the pial circulation. Thus we were unable to measure or detect any regional differences in perfusion thorough the brain parenchyma.  ¥ We used acetazolamide challenge in rats, a routinely used technique to assess CVR. A scanning laser Doppler was used to measure changes in perfusion during the acetazolamide challenge and thus the data is limited to CVR in the pial circulation. During acetazolamide challenge we also did not measure physiological parameters that could alter cerebral perfusion such as systemic blood pressure, heart and respiratory rates, arterial pH, or arterial CO2 pressure. More importantly, during the experiment the rats were anaesthetized with isoflurane, a known cerebral dilator [14] and thus the data can only be used comparatively. ¥ Morris water maze is a well-established neurological test to assess predominantly hippocampal dependent cognitive abilities. For our studies we used latency time to tease out difference in spatial learning abilities between groups. We manually recorded the latency time because video recording was not available. We also did not assess swimming patterns, time spent in each quadrant or carry out probe trials. Thus we were unable to tease out the range of cognitive abilities that can be obtained from Morris water maze such as latent discrimination, reference memory, and working memory.      !198 5.5 - Perspectives VCI is a devastating disease that is associated with large financial cost to society[16].  It not only takes away the ability of the patient to lead an independent life but also puts a tremendous burden on patientsÕ families and primary caregivers. CCH increases the incidence of VCI development and frequently co-exists with hypertension, a major risk factor of VCI. I proposed that impaired vasodilation and remodeling in the PAs were involved in the progression of VCI. Thus pharmacological interventions to attenuate or prevent these changes in the PAs would be invaluable. We show that sEH inhibitor TPPU might be a viable treatment option since it prevents CCH induced memory impairment and diminishes associated dilatory dysfunction in PAs from SHRSP. Our claim is substantiated by studies that show that sEH inhibitors have protective effects on both the vasculature and the neurons[13,17]. However, the beneficial effects of TPPU might be applicable to patients who are at risk of developing CCH induced by carotid stenosis, cardiac arrest, arrhythmias, cardiac failure, or hypotension. Further studies are need to carried out to assess if TPPU has any desired cognitive effects in hypertensive patients who have cognitive deficits.         !199            REFERENCES            !200 REFERENCES  1. Bhawan J, Joris I, DeGirolami U, Majno G. Effect of occlusion on large vessels. I. A study of the rat carotid artery. Am J Pathol 88: 355-380, 1977.  2. Fleming I, Rueben A, Popp R, Fisslthaler B, Schrodt S, Sander A, Haendeler J, Falck JR, Morisseau C, Hammock BD, Busse R. Epoxyeicosatrienoic acids regulate Trp channel dependent Ca2+ signaling and hyperpolarization in endothelial cells. Arterioscler Thromb Vasc Biol 27: 2612-2618, 2007.  3. Fromel T, Fleming I. Whatever happened to the epoxyeicosatrienoic Acid-like endothelium-derived hyperpolarizing factor? 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