THE OXYTOCIN RECEPTOR IN VASCULAR DEMENTIA By Erin McKay A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Neuroscience-Doctor of Philosophy 2020 ABSTRACT THE OXYTOCIN RECEPTOR IN VASCULAR DEMENTIA By Erin McKay Cognitive impairment following a stroke, known as vascular dementia (VaD), is a common outcome often overlooked in lieu of the motor and verbal impairments. Nonetheless, the executive and memory deficits in VaD can be drastically impairing. A successful treatment or intervention could improve the quality of life for millions of patients. Two aims were pursued in service of this goal. The first aim was to compare the gene expression profiles of frontal cortex tissue from diagnosed cases of VaD, intermediate Alzheimer’s disease (AD), and no cognitive impairment. Gene expression products that were differentially expressed in VaD cases versus AD and controls were to be investigated for biological significance and therapeutic potential. Further validation studies were also used to identify targets worth pursuing further. The oxytocin receptor (OXTR) was identified as differentially upregulated in the frontal cortex in cases of VaD, and localization to astrocytes surrounding areas of infarction was noted. The second aim was designed to test the therapeutic potential of the OXTR in an animal model of VaD. Administration of the potent vasoconstrictor endothelin-1 (ET-1) into the prefrontal cortex of spontaneous hypertensive stroke prone rats (SHRSP) was used to create a model of focal stroke with a pre-morbidity leading to cognitive deficits without obstructing motor deficits. A pre-injury intervention of an adeno-associated virus expressing either the OXTR or green fluorescent protein (GFP) linked with the astrocytic promoter glial fibrillary acidic protein (GFAP) was applied. The animals were then subjected to a battery of cognitive tests to confirm deficits induced by the ischemic lesion and assess potential rescue by the pre-upregulation of the OXTR in astrocytes. OXTR upregulation improved performance in the novel object task compared to control, suggesting a rescue of working memory function that is mediated by the prefrontal cortex. Tissue processing assessed infarct size, white matter density, and viral expression, which showed no difference in white matter density but a reduction in the size of the infarct by the pre-upregulation of the receptor in the perimeter of the targeted prefrontal cortex site. An additional sub-aim of aim two was devised to compare the effects on known molecular participants in the pathway of ischemic injury within animals expressing ectopic OXTR or GFP in separate hemispheres to ET-1 or saline treated animals. At the 24 hour time point chosen, no difference was observed in either reactive oxygen species generation or the profiles of cytokines implicated in the course of ischemic injury. The results from the human tissue studies in aim one suggest that a specific peri-infarct specific upregulation of the OXTR on astrocytes is a hallmark of ischemic VaD. Rescue studies in a rat model in aim two suggest that this upregulation may be a compensatory response acting to salvage penumbra tissue and reduce the ensuing behavioral deficits. However, the specific mechanism remains undefined at this time. Further preclinical mechanistic studies are needed, particularly at different time points, to more rigorously evaluate the relative efficacy of targeting the OXTR as an intervention for VaD. This dissertation is dedicated to my family especially Mom, Dad, and Devin The best aspects of me I owe to you iv ACKNOWLEDGMENTS The existence of this document and the results presented could not be a reality without the support of many people. First, my scientific mentor Dr. Scott Counts for his guidance through the ups and downs of this project and letting me forge my own way in the lab and career aspirations. John Beck and Sarah Kelly for teaching me the techniques of the lab and troubleshooting advice. Gratitude to Tessa Grabinski for sharing knowledge and early morning chats. The Manfredsson lab gave our lab the virus tools and experience that made this possible and will continue to benefit the lab. Dr. Winn at the VAI for wrangling the advanced statistics. Every lab in the TSMM department and particularly the lab managers deserve my thanks for building me as a scientist and expanding my ‘tool kit’. I appreciate the Neuroscience Program for taking a chance on me and I can only hope I have lived up to their faith in me. My committee members who guided this work and made it stronger than I ever could by myself were: Dr. Anne Dorrance, Dr. Irving Vega, Dr. Caryl Sortwell, and Dr. Tim Collier, thank you all. On a personal level foremost are my parents and sister for giving me strength when I doubted myself to keep going. I love my family for forgiving the times school kept me away from them and making the times it did not count. Student friends from both Van Andel and MSU were vital for commiserating and being good weekend companions. Thank you to Maya for being my academic comrade since freshman year in college. Finally, thank you to all the individuals whose donation to scientific research formed the foundation for this research. This is your work as well. v TABLE OF CONTENTS LIST OF TABLES .................................................................................................................... vii LIST OF FIGURES .................................................................................................................. viii KEY TO ABBREVIATIONS ......................................................................................................... x CHAPTER 1: Introduction and Project Background ................................................................... 1 Vascular Dementia ......................................................................................................................... 1 Ischemic Injury ............................................................................................................................... 6 The Oxytocin Receptor ................................................................................................................. 12 Dissertation Objectives ................................................................................................................ 24 CHAPTER 2: The detection of an oxytocin receptor upregulation in vascular dementia and localization to peri-infarct astrocytes ...................................................................................................... 25 Introduction ................................................................................................................................. 25 Methods and Materials ................................................................................................................ 28 Results .......................................................................................................................................... 35 Discussion ..................................................................................................................................... 57 CHAPTER 3: The effects of viral vector-mediated upregulation of the oxytocin receptor in a rat model of vascular cognitive impairment .......................................................................................... 64 Introduction ................................................................................................................................. 64 Methods ....................................................................................................................................... 68 Results .......................................................................................................................................... 86 Discussion ................................................................................................................................... 110 CHAPTER 4: Final discussion ............................................................................................... 119 Conclusions ................................................................................................................................ 119 Future Directions ........................................................................................................................ 122 Limitations .................................................................................................................................. 125 REFERENCES ...................................................................................................................... 129 vi LIST OF TABLES Table 1.1 Effects of oxytocin receptor activation by behavioral or physiological domain ............ 15 Table 2.1 Clinical, neurological, and demographic characteristic by diagnostic category ............ 36 Table 2.2 Expanded names of key abbreviations .......................................................................... 39 vii LIST OF FIGURES Figure 1.1 The ischemic penumbra ................................................................................................ 11 Figure 1.2 Model of the oxytocin receptor .................................................................................... 13 Figure 2.1 Venn diagram representing common and disease-specific genes in CTL, VaD, and AD subjects..................................................................................................................................... 37 Figure 2.2 Pathway analysis of dysregulated genes in VaD compared to AD frontal cortex ........ 40 Figure 2.3 Network analysis of VaD-specific gene dysregulation .................................................. 42 Figure 2.4 qPCR validation of selected genes dysregulated via microarray analysis .................... 44 Figure 2.5 Western blot confirmation of OXTR protein upregulation in VaD frontal cortex ........ 46 Figure 2.6 OXTR expression in cerebrovascular endothelial cells ................................................. 48 Figure 2.7 De novo peri-infarct expression of OXTR...................................................................... 50 Figure 2.8 Peri-infarct astroglial and vascular expression of OXTR ............................................... 51 Figure 2.9 Gradient of OXTR expression in astrocytes and blood vessels relative to microinfarction site ........................................................................................................................ 52 Figure 2.10 RNAscope detection of GFAP mRNA in profiles expressing OXTR .............................. 53 Figure 2.11 Rat astrocytes in culture upregulate the OXTR in response to hypoxia ..................... 55 Figure 3.1 Schematic of Experiment 1 workflow ........................................................................... 69 Figure 3.2 Schematic of Experiment 2 workflow ........................................................................... 70 Figure 3.3 Pre-absorption and primary delete western blots demonstrate specificity of antibody ......................................................................................................................................... 79 Figure 3.4 Tests of OXTR antibody reveal specificity for the oxytocin receptor ........................... 81 Figure 3.5 No difference was detected in time mobile or distance travelled in open field .......... 87 Figure 3.6 Novel Object Task shows deficits were induced by endothelin-1 injection and was partially rescued by OXTR upregulation in male animals ....................................................... 89 viii Figure 3.7 Elevated Plus Maze shows a tendency to enter more often and spend more time in the open arms was induced by endothelin-1 injection ............................................................. 92 Figure 3.8 No differences in latency to target hole or incorrect revisits were detected with the Barnes Maze ............................................................................................................................ 95 Figure 3.9 Pre-injury upregulation of the oxytocin receptor reduces the size of final infarction in males ......................................................................................................................... 98 Figure 3.10 Luxol Fast Blue stain revealed no difference in white matter density between groups .......................................................................................................................................... 101 Figure 3.11 GFP and OXTR are expressed in the proximity of the penumbra but are not absorbed in the final lesion .......................................................................................................... 103 Figure 3.12 OXTR expression in the AAV6 OXTR hemispheres versus AAV6 GFP hemispheres is highly variable ..................................................................................................... 104 Figure 3.13 mRNA Cytokine profiles in AAV6 OXTR hemispheres versus AAV6 GFP hemispheres at 24 hours post ischemic injury ............................................................................ 106 Figure 3.14 Reactive oxygen species at 24 hours post ischemic injury in AAV6 OXTR hemispheres versus AAV6 GFP hemispheres as determined with DHE ...................................... 108 ix KEY TO ABBRIEVATIONS Adeno-Associated Virus Alzheimer’s Disease Atrial Natriuretic Peptide Autism Spectrum Disorders Adenosine Triphosphate Blood Brain Barrier Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy Cerebral Blood Flow Central Nervous System Control Cerebral Vascular Disease Diacylglycerol Dihydroethidium Eukaryotic Elongation Factor 2 Endothelial Nitric Oxide Synthase Endothelial Progenitor Cells Extracellular Signal-Regulated Kinase Endothelin-1 Glial Fibrillary Acidic Protein Green Fluorescent Protein G-Protein Coupled Receptor x AAV AD ANP ASD ATP BBB CADASIL CBF CNS CTL CVD DAG DHE eEF2 eNOS EPC ERK ET-1 GFAP GFP GPCR H&E HBEC IF IHC IL-1 IL-6 IL-10 iNOS IP3 LCM LFB MAPK MCI MID MMP NADPH NF-κβ NGS NO OXT OXTR PBS PFC PGF Hematoxylin & Eosin Human Brain-Derived Endothelial Cells Immunofluorescent Immunohistochemistry Interleukin-1 Interleukin-6 Interleukin-10 Inducible Nitric Oxide Synthase Inositol Trisphosphate Laser Capture Microdissection Luxol Fast Blue Mitogen-Activated Protein Kinase Mild Cognitive Impairment Multi-Infarct Dementia Matrix Metalloproteinase Nicotinamide Adenine Dinucleotide Phosphate Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells Normal Goat Serum Nitric Oxide Oxytocin Oxytocin Receptor Phosphate Buffered Saline Prefrontal Cortex Placental Growth Factor xi PIP PI3K PKC PLC PMI RAGE ROS rtPA SD SID SOD TBS TGFβ TNF VaD VCI VEGF Phosphatidylinositol Phosphoinositide 3-Kinases Protein Kinase C Phospholipase C Post-Mortem Interval Receptor for Advanced Glycation End Products Reactive Oxygen Species Recombinant Tissue Plasminogen Activator Standard Deviation Strategic Infarct Dementia Superoxide Dismutase Tris-Buffered Saline Transforming growth factor beta Tumor Necrosis Factor Vascular Dementia Vascular Cognitive Impairment Vascular Endothelial Growth Factor xii CHAPTER 1: Introduction and Project Background Vascular Dementia Vascular dementia (VaD) is defined as cognitive impairment caused by a multitude of cerebrovascular accidents and chronic conditions leading to low perfusion and subsequent tissue loss (Gorelick et al., 2011; Kalaria, 2016; Smith, 2017). Vascular pathology has been found to be the root cause of dementia in about 8-10% of all clinical cases of cognitive impairment and somewhere between 25-80% of Alzheimer’s disease (AD) patients also have vascular contributions to their dementia (Jellinger, 2013). VaD can be divided into several subtypes based on either the precipitating vascular injury or the pattern of tissue loss The human cost of all VaD subtypes is similar to other primary dementias, with symptoms like disorganized thoughts and emotions, gait abnormalities, and mild memory impairment progressively interfering with activities of daily living for the patient and increasing the burden for caregivers (Román, 2003). VaD has a commonality with stroke survivors without dementia in that both groups have a significantly increased risk of clinical depression, further complicating caregiving and compounding patient distress (Ballard et al., 2000; Park et al., 2007). However, this is complicated by results suggesting that depression due to vascular insufficiency might precede the observation and diagnosis of VaD (Lin, Hu, Tsai, Yang, & Shen, 2017; Loganathan, Phutane, Prakash, & Varghese, 2010; Steffens, Taylor, & Krishnan, 2003). VaD was also estimated to have the highest annual cost per patient among the most common dementias, likely due to a higher rate of recurrent hospital admissions (Hill, Fillit, & Shah, 2005). While the presentation of VaD is often debilitating, it is usually a short course as the average life expectancy of a patient does not extend past 4 years (Fitzpatrick, Kuller, Lopez, Kawas, & Jagust, 2005; Strand et al., 2018). 1 However, despite the societal toll of VaD, no approved treatment currently exists to reverse or modify the trajectory of the cognitive decline once it has become apparent (Baskys & Cheng, 2012). As the field currently stands, prevention of strokes and management of risk factors are the only universally and medically accepted means of controlling the onset of VaD (Baskys & Cheng, 2012). In fact, VaD is sometimes recognized as post-stroke dementia due to the highly connected nature of VaD to stroke (Chen et al., 2016; Mijajlović et al., 2017), particularly ischemic stroke which accounts for 80-90% of all strokes (Vijayan & Reddy, 2016). Of course, this is not to minimize the contribution of other cerebrovascular incidents, silent strokes, or transient ischemic attacks to VaD (Smith, 2017). The link between cerebrovascular disease (CVD) and cognitive impairment has a long history. Before the recognition of Alzheimer’s type pathology in the 1970s, substantial mental decline in the elderly was attributed to vascular causes, particularly atherosclerosis and hypoperfusion (Hachinski, 1990; Román, 2003). While assigning appropriate credit to an individual for this theorem is difficult, one of the most prominent proponents was Otto Binswanger, for whom a subtype of subcortical VaD, discussed shortly, is still named (Binswanger, 1894). When the contribution of the amyloid-beta protein in the 1970s and tau in the following decade (Kosik, Joachim, & Selkoe, 1986) to AD experienced a renaissance, the focus on VaD lessened, but persisted. The study of VaD, and furthermore stroke, owes much to the work of C. Miller Fisher and his rigorous study of the stenosis and embolism of carotid arteries, small vessel thrombosis, and lacunar infarcts (Fisher, 1954). He was perhaps the first, since the atherosclerotic dementia theorem fell out of favor, to link dementia to CVD in the absence of AD pathology (Fisher, 1968; Fisher, 1954). This attribution of discrete brain lesions to carotid artery disease was further supported by a large scale autopsy finding suggesting a high rate of embolism and infarcts with carotid occlusions (Torvik & Jörgensen, 1964, 1964). 2 While Fisher ascribed the focal softenings of multiple lacunes to the onset of dementia, Hackinski, Lassen and Marshall shepherded the field away from its atherosclerotic path, when they recognized small and large infarcts as a more common cause of CVD-related dementia, and introduced the term multi-infarct dementia (MID), that persists today as a subtype of VaD (Hachinski, Lassen, & Marshall, 1974). The question then arose as to why some with infarcts had dementia and some did not. Tomlison, Blessed and Roth attributed much of this to a volume based hypothesis, finding dementia to be almost certain with 100mL of tissue loss with an incremental decrease in probability down to about 50mL (Tomlinson, Blessed, & Roth, 1970). It was later recognized that an area of small tissue loss in an area with less compensatory options, such as the thalamus, caudate, hippocampus, or inferior genu can lead to dementia, called strategic infarct dementia (SID) (Katz, Alexander, & Mandell, 1987; Scheinberg, 1988; Tatemichi et al., 1992). There was also a renewed understanding that wide-spread white matter lesions can also precipitate dementia, known as subcortical vascular encephalopathy (Burger, Burch, & Kunze, 1976; Tomonaga, Yamanouchi, Tohgi, & Kameyama, 1982). We now transition to a further exploration of these subtypes and their hallmark pathology and etiology. The most common type of VaD is multi-infarct dementia (MID) (Thal, Grinberg, & Attems, 2012). Previously, MID was used nearly interchangeable with VaD before the recognition of dementias resulting from single infarcts or genetic arteriopathy led to the recognition of subtypes (Gorelick et al., 2011). It follows that despite VaD subtypes often resembling each other in clinical presentation, the subtypes are defined by the divergent underlying pathology. MID is defined as cognitive impairment caused by multiple lesions and infarcts in both white and grey matter that follow occlusions in cerebral arteries and arterioles (Iadecola, 2013). Thrombus and embolism are particularly prominent in this subtype, and as such, atherosclerosis of the cardiac branches or Circle of Willis are important risk factors (Moroney & Desmond, 1997; Suemoto et al., 2011). Strategic infarct dementia (SID) is the diagnosis when a single infarct is enough to cause dementia due to its presence in a particularly vulnerable region, 3 especially those that disrupt vital cortical-subcortical relays (Katz et al., 1987; Scheinberg, 1988; Tatemichi et al., 1992). Small vessel disease or embolism in posterior cerebral arteries and anterior cerebral arteries are particularly disruptive due to their branches into subcortical structures (Tatemichi et al., 1993). A third type is subcortical vascular encephalopathy, of which Binswanger’s is the hallmark type, arises not from grey matter injury, but from widespread damage to white matter, particularly those linking frontal and subcortical grey matter (Tomonaga et al., 1982; Yamanouchi, Sugiura, & Tomonaga, 1989). Rather than an acute occlusion, dangerous hypoperfusion due to stenosis of blood vessels is a primary factor (Kawamura, Meyer, Terayama, & Weathers, 1991). It has been suggested that the lower degree of collateral branches supplying the white matter is what makes it susceptible to this condition (Pantoni & Garcia, 1997; Tomonaga et al., 1982), although a vulnerability to inflammatory responses is also a popular hypothesis (Fowler et al., 2018). Primarily hemorrhagic or microbleed-related VaD is predominantly related to cerebral amyloid angiopathy, which presents as microbleeds occurring throughout the cerebral hemispheres due to the accumulation of Aβ peptides in the walls of microvessels (Greenberg & Charidimou, 2018). Interestingly, impaired interstitial clearance due to underlying vascular disease is an intriguing recent suggestion for this accumulation (Weller, Subash, Preston, Mazanti, & Carare, 2008), but this does not fully explain the different ratios of Aβ1-40 and Aβ1-42 between AD-related CAA and sporadic CAA (Haglund, Kalaria, Slade, & Englund, 2006). Genetic causes of VaD also exist, of which the most commonly encountered is the mutant Notch-3 linked cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, commonly referred to as CADASIL (Chabriat et al., 1995; Joutel et al., 1996). In this condition the mutation leads to an aberrantly increased number of cysteines in the extracellular domain of this receptor in the smooth muscle cells of vessel walls leading to occlusions as well as bleeds (Chabriat, Joutel, Dichgans, Tournier-Lasserve, & Bousser, 2009). Why, if the observable symptoms are so similar, are the differentiation of these subtypes important? As the pathology is what is targeted by 4 intervention, any potential therapy should consider these VaD subtypes in their sample to avoid discarding a treatment that may benefit one group of patients, but not the others (Moretti, Torre, Antonello, Cazzato, & Pizzolato, 2008). An important limitation to note with the diagnosis of VaD is found in the very name of the condition. By definition, dementia has a memory component. However, the commonest cognitive impairment following a stroke is deficits in executive function (Erkinjuntti, 1997; Pohjasvaara et al., 2002). The term executive function encompasses such domains as: attention, planning, cognitive flexibility, abstract thinking, volition, and working or procedural memory (Moorhouse et al., 2010; Sachdev et al., 2004). This may then progress into including episodic memory disruption (Wentzel et al., 2001), but many have pointed out that executive deficits are impairing on their own (Pohjasvaara et al., 2002), and that treatment only after the memory deficits occur means that only late stage disease is being considered (Dong et al., 2010; Sachdev et al., 2004). Therefore, as the clinical presentation of VaD is discussed it would be remiss not to mention that many are beginning to transfer to using the term vascular cognitive impairment (VCI) rather than VaD (O’Brien, 2006; O’Brien et al., 2003). VaD is used in this document to align with the diagnosis of the human tissue used and the deficits of the animal model. Other cognitive domains are often affected as well, particularly in stages when subcortical damage or widespread white matter lesions are present (Graham, Emery, & Hodges, 2004; Price et al., 2012). These include deficits in regards to verbal comprehension, visuoconstruction, and semantic memory (Graham et al., 2004; Lukatela, Malloy, Jenkins, & Cohen, 1998; Price et al., 2012; Vuorinen, Laine, & Rinne, 2000). By contrast, there are some cognitive domains with relative preservation in VaD, primarily recognition memory and verbal fluency and memory, although this is often reported in comparison to AD, rather than controls without cognitive impairment (Looi & Sachdev, 1999; Tierney et 5 al., 2001). Overall, VaD is a heterogenous condition that regardless of the neuropsychological profile in a single patient has disastrous implications for the quality of a patient’s life beyond the mortality. While the incidence of VaD is variable across studies, several meta-analyses have concluded that incidences of non-hereditary vascular dementia begin to occur at age 65 and increase exponentially every five years after initial risk begins (Jorm & Jolley, 1998). In addition, the rates of all dementias are expected to increase at a rate of nearly double every twenty years with an aging population and better healthcare available (Ferri, Prince, Brayne, & Brodaty, 2005). The risk factors for stroke are also gaining prevalence in the western world with no sign of relenting since more people are classified as hypertensive or prehypertensive than normotensive, and more than half of these cases are not adequately controlled (Greenlund, Croft, & Mensah, 2004). Interestingly, the chance of fatal complications upon occurrence of a stroke appears to be in decline, especially for middle-aged patients, as the access to and efficacy of intervention treatment improves (Kalaria, Akinyemi, & Ihara, 2016; Yang et al., 2017). While this is promising news, it does follow that while more individuals are surviving strokes, not all will be left without long-term disability. In fact, estimates of cognitive impairment following a first-time stroke range from a third to a half of all patients (Mijajlović et al., 2017; Pendlebury & Rothwell, 2009). Thus, the lack of valid treatment, high likelihood of a continuous increase in diagnoses, and prevalence of risk factors justifies an increased interest in understanding and treating VaD in the future. In order to better frame what a treatment for VaD might entail we must turn to the most common instigating event, ischemic injury. Ischemic Injury Beyond the immediate tragedy of lives lost in the immediate aftermath of a stroke, there is the added effect of disability adjusted life years lost, of which stroke accounts for the 3rd most common detractor (Feigin et al., 2014). As mentioned above, a significant proportion of this disability is related to 6 cognitive functioning (Pohjasvaara et al., 2002; Rasquin et al., 2004). It follows that by examining the molecular and ionic changes that follow an ischemic injury and induce death of CNS tissue, insights into how to prevent the subsequent cognitive impairment would emerge. Therefore, a discussion on what characterizes an ischemic injury is warranted. In a basic sense an ischemic injury occurs when an occlusive event starves cells of supportive oxygen and glucose, ultimately causing cell death (Astrup, Siesjö, & Symon, 1981). This is an oversimplification as the downstream reactions to this initial insult encompass the full range of consequences leading to tissue loss, and insights for the eventual development of multifactorial intervention strategies (Dirnagl, Iadecola, & Moskowitz, 1999). Lack of oxygen leads to a failure of ATP generation by mitochondria (Siesjo, 1988). The loss of aerobic respiration means that not enough energy is generated to properly maintain ionic cell gradients. Ionic fluxes and responsive osmoregularity lead to the movement of water and cell swelling or cytotoxic edema (Rosenberg, 1999). The resulting ionic flux leads to the activation of many intracellular kinases in response to release of calcium stores and the depolarization of the cell contributing to excitotoxicity (Siesjo, 1988). Some of the kinases the efflux of calcium stores trigger include the pro-apoptotic calpains, the reactive oxygen species intersecting inducible nitric oxide synthase (iNOS), and destructive proteases and lipases (Szydlowska & Tymianski, 2010). Calcium uptake by responding mitochondria can also trigger the release of the pro-apoptotic cytochrome-c after enhancing mitochondria permeability, which then triggers caspase-mediated cell death (Andreyev, Tamrakar, Rosenthal, & Fiskum, 2018; Kobayashi et al., 2003). Importantly, apoptosis requires ATP to proceed so in areas of complete metabolic collapse only liquefactive necrosis in the central nervous system will be observed (Ünal-Çevik, Kilinç, Can, Gürsoy-Özdemir, & Dalkara, 2004). The accumulation of potassium, glutamate, or more likely both in the interstitial space diffuses to other cells in the vicinity of the primarily impacted cells and depolarize these secondary cells in what is called peri-infarct depolarizations (Hartings, Rolli, Lu, & Tortella, 2003; Somjen, 2001). The metabolic failure and switch to anaerobic respiration leads to a rise in 7 lactic acid which causes tissue acidosis contributing to active necrosis, or necroptosis (Wang et al., 2015). Reactive oxygen species (ROS) are biologically active compounds containing oxygen often by either nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or xanthine oxidase (Baudry, Laemmel, & Vicaut, 2008; Shen, Rastogi, Geng, & Ding, 2019; Vreeburg & Fry, 2007). ROS cause cell damage directly through oxidizing and damaging DNA, proteins, lipids (Rodrigo et al., 2013). ROS can also contribute to BBB disruption by disrupting endothelial junctions (Kahles et al., 2007), and to inflammation, covered in the next section, by activating cytokine-linked transcription factors such as NFκβ (Taniyama & Griendling, 2003). ROS can even attenuate potentially beneficial aspects of the ischemic cascade. Timely reperfusion is vital for tissue survival, but when ROS are present the influx of oxygen can least to a burst of oxidative stress called ischemia/reperfusion injury (Sanderson, Reynolds, Kumar, Przyklenk, & Hüttemann, 2013). The vasodilatory and proangiogenic nitric oxide, that will be expanded on below can react with ROS, to create the toxic oxidant peroxynitrite (Gürsoy-Özdemir, Bolay, Saribaş, & Dalkara, 2000). Of the species and isoforms of ROS generating enzymes the NOX1 and NOX2 isoforms of NADPH oxidase are thought to be particularly important in generating ROS in the penumbra (Choi et al., 2015). Specifically, NOX1 is upregulated earliest post ischemia (6 hrs) through a Rac1 dependent mechanism with NOX2 following later (24hrs) (Choi et al., 2015). While this applies to all organs undergoing oxidative stress, the brain is especially vulnerable due to a low amount of endogenous antioxidant enzymes (Saeed, Shad, Saleem, Javed, & Khan, 2007). Destruction of neural tissue from ischemia leads to the release of damage associated molecular patterns which call to immune cells through pattern recognition receptors (Famakin, 2014). A discussion of inflammation in stroke must consider both local and peripheral immune cells. The brain has sometimes been called immune privileged, but the incidence of BBB breakdown and the upregulation of cell adhesion molecules on vascular endothelial cells mean that peripheral macrophages, neutrophils, 8 and lymphocytes contribute to the immune profile of ischemic tissue (Gelderblom et al., 2009; Yilmaz & Granger, 2008). In a reflection of the connection drawn in the previous paragraph, inflammation can also induce the production of ROS. For example, the interaction of the downstream cytokine receptor effector STAT3 with mitochondrial complexes induces ROS production (He et al., 2017), and the induction of NADPH oxidase by ischemia triggers microglia (Hur, Lee, Kim, Kim, & Cho, 2010). This is in addition to the additional production of ROS along with cytokines and chemokines by inflammatory cells (Allen & Bayraktutan, 2009). The full profile of cytokines and chemokines released by immune cells is beyond the scope of this document, but a few examples do deserve discussion. Some cytokines like IL-10 and TGFβ are considered protective and are regularly mentioned as a potential therapeutic targets in experimental stroke models (Cekanaviciute et al., 2014; Diao, Wang, Qi, Jia, & Yan, 2016; Gross, Bednar, Howard, & Sporn, 1993; Ooboshi et al., 2005). IL-1β and TNF are also commonly discussed in both human and animals studied of ischemic stroke outcomes and are typically considered deleterious (Denes, Pinteaux, Rothwell, & Allan, 2011; Hallenbeck, 2002; Kaushal & Schlichter, 2008; Liu et al., 1993). IL-6 is more controversial appearing protective in some conditions but not in others (Hotter et al., 2019; Jung, Kim, & Chan, 2011). In general, the initial response to stroke is proinflammatory, while anti- inflammatory responses mostly driven by regulatory T-cells may appear, but not until up to a week post- stroke (Chamorro et al., 2012). Without endogenous or exogenous recanalization the brain has alternative methods to attempt to return cerebral blood flow to underperfused areas (Ma, Morancho, Montaner, & Rosell, 2015). These include remodeling existing blood vessels to increase collateral blood flow through existing vessels, called arteriogenesis (Cui et al., 2009; Wei, Erinjeri, Rovainen, & Woolsey, 2001), and the sprouting of new blood vessels, called angiogenesis (Liu et al., 2013). Arteriogenesis is triggered by the detection of sheer stress upstream of an occlusion (Troidl et al., 2009). When comparing infarcted tissue to non- infarcted tissue, a greater density of small vessels have been reported in the ipsilateral hemisphere, 9 particularly in the penumbra for humans and animals (Krupinski, Kaluza, Kumar, Kumar, & Wang, 1994; Wei et al., 2001). Angiogenesis is stimulated by neurovascular factors that mobilize and guide endothelial progenitor cells (EPCs) to a peri-infarct site, such as VEGF, PGF, and TGFβ (Ma et al., 2015). The primary guidance for endothelial progenitor cells are nitric oxide produced by endothelial NOS (eNOS) from L-arginine and H2S by pyridoxal 5′- phosphate–dependent enzymes from L-cysteine (Bir, Xiong, Kevil, & Luo, 2012; Murohara et al., 1998; Papapetropoulos et al., 2009). In ischemia, the primary pathway for eNOS activation by phosphorylation is through Akt/PI3K (Bir et al., 2012). A requirement for this out-sprouting is the degradation of the extracellular matrix of blood vessels by matrix metalloproteinases, particularly MMP9 in stroke (Chopp, Zhang, & Jiang, 2007; Lee, Xue, Hao, Yang, & Young, 2009). Angiogenesis typically begins within days after stroke and can continue for weeks (Martín et al., 2012; Seevinck, Deddens, & Dijkhuizen, 2010). However, not all consequences of angiogenesis are neuroprotective, as the required disruption of the BBB can lead to hemorrhagic transformation and edema (Adamczak & Hoehn, 2015). The sum of the protective and deleterious effects of these ischemic mechanisms ultimately determines the fate of potentially salvageable tissue (Figure 1.1). This potentially salvageable tissue is called the ischemic penumbra (Astrup et al., 1981). While the core of the lesion is completely deprived of oxygen and glucose from a severe drop in CBF leading to necrotic death, the penumbra is able to subsist for up to several days before ultimately surviving or undergoing an apoptotic death (Fisher & Garcia, 1996; Uzdensky, 2019). This delineation has been observed in real time by the comparison of perfusion weighted imaging and diffusion weighted imaging to find tissue in which perfusion was impaired but the structural integrity of the tissue was still present (Lopez-Mejia & Roldan-Valadez, 2016; Røhl et al., 2001). The penumbra serves as the basis for acute intervention in ischemic stroke and prevention of the deficits, such as cognitive, that can occur as a result (Fisher & Garcia, 1996; Manning, Campbell, Oxley, & Chapot, 2014). As it stands today, the strategy for rescue of the penumbra when it is 10 present is the recanalization of occlusions through administration of recombinant tissue plasminogen activator (rtPA) within 4.5-6 hours or mechanical thrombectomy when a large artery is involved (Davis & Donnan, 2009; Goyal et al., 2016; Manning et al., 2012; Manning et al., 2014; Smith et al., 2005; Zeumer, Freitag, Zanella, Thie, & Arning, 1993). However, the previous paragraphs have indicated that the cellular response to ischemic injury and the loss of the penumbra can be ascribed to more than metabolic failure, and targets that could shift the balance of the ischemic cascade to favor a pro-survival environment are of growing interest in studies of ischemic injury (Felberg, Burgin, & Grotta, 2000). Figure 1.1. The ischemic penumbra. The basis and differentiation of the ischemic penumbra versus the ischemic core, including the factors determining how much of the penumbra is absorbed into the utlimate infraction. 11 Before we turn to the body of this document, some background on an important finding in Chapter 2 is summarized to aid in understanding its importance. In brief, in Chapter 2 of this document, primary research and evidence for an induction of the oxytocin receptor (OXTR) in VaD will be reported. Before the presentation of this data, a final section of this introduction is dedicated to the receptor itself focusing on its general biology and intersection with stroke-related pathology. The Oxytocin Receptor The OXTR is a widely expressed Gαq protein-coupled receptor (GPCR) (Figure 1.2), as well as a structurally similar nonapeptide, vasopressin, with an affinity of about 100nM-1µM (Postina, Kojro, & Fahrenholz, 1996). The OXT peptide and its full nine amino acid sequence was first detailed in 1953 by Du Vigneaud and colleagues through varied partial hydrolysis experiments combined with paper chromatography (du Vigneaud, Ressler, & Trippett, 1953). However, its existence was recognized as early as 1928 when researchers began testing the effects of OXT from pituitary extracts on peripheral reactions such as uterine contractions and blood pressure (Bourne & Burn, 1928; Griling & Eddy, 1928; Gruber, 1928; Kamm, Aldrich, Grotb, Rowe, & Bugbee, 1928). OXT has since been found to exert both central and peripheral effects via OXTR-mediated phospholipase C (PLC) activation and downstream Ca2+ signal transduction (Zingg & Laporte, 2003). OXT is synthesized in the hypothalamic magnocellular and parvocellular neurons, reaching the peripheral circulation through the posterior pituitary (Argiolas & Gessa, 1991), while central actions appear to occur through both axonal and possibly volume transmission through dendrites (Meyer-Lindenberg, Domes, Kirsch, & Heinrichs, 2011). This volume transmission and its relative contribution to OXTR activation is an ongoing subject of debate, as more OXT neuronal projections to forebrain OXTR expressing regions have become apparent in recent years (Grinevich, Knobloch-Bollmann, Eliava, Busnelli, & Chini, 2016; Knobloch et al., 2012). The very first hint of a bioactive OXTR was indirectly demonstrated by Sir Henry Dale and focused on the induction of uterine contractions by posterior pituitary gland components (Viero et al., 2010). Since that time OXTRs 12 have not only been identified in the uterus (Fuchs, Fuchs, Husslein, & Soloff, 1984), but also the mammary glands (Soloff, Schroeder, Chakraborty, & Pearlmutter, 1977), heart (Gutkowska et al., 1997), blood vessels (Thibonnier, Conarty, et al., 1999), and brain (Muhlethaler, Sawyert, Manning, & Dreifuss, 1983). The widespread expression of this receptor and its ligands underscores the continued relevance and necessity of research into its functional repertoire, even after the hundred years that have passed since Dale engaged the receptor without knowing what it was (Grinevich et al., 2016; Knobloch et al., 2012). Figure 1.2. Model of the oxytocin receptor (A) linear view of the receptor. (B) The top of the receptor as it forms a binding pocket. (C) The bottom of the receptor as it forms a binding pocket. (D) Color coded key. The general location of specialized sites are highlighted by color with key provided. Based on chimera, point mutation, and structural analysis studies as cited in the text. Created in Blender 2.8. 13 Most research with a clinical application, beyond the long established birth-related uses, has focused on modulating conditions with social deficits such as ASD and psychiatric disorders like schizophrenia (Alvares, Quintana, & Whitehouse, 2017; Cai, Feng, & Yap, 2018; Feifel et al., 2010; Zheng et al., 2019). Although areas of OXTR signaling research in the behavior and function of whole organisms including reproduction & maternal care, social affiliation, and satiety and osmoregulation are critical on and ongoing (Feldman, Monakhov, Pratt, & Ebstein, 2016; Leng & Russell, 2019; Skinner, Campbell, Dayas, Garg, & Burrows, 2019; Szymanska, Schneider, Chateau-Smith, Nezelof, & Vulliez-Coady, 2017), relevant to this dissertation are the functions of OXTR signaling related the ischemic response introduced above that might play a role in the onset or treatment of VaD (Table 1.1 for summary). 14 Table 1.1. Effects of oxytocin receptor activation by behavioral or physiological domain. Effect Induces cellular proliferation through phosphorylation of MAPK (ERK5). Citation Devost et al., 2008; Hansenne et al., 2005; Stary et al., 2019 Aids in cellular proliferation through PKC activation of eEF2 by dephosphorylation, and Src dependent mechanisms. Domain Cellular Differentiation Cellular Proliferation Cellular Migration The PLC pathway activates Akt/PI3K to influence endothelial cell migration. Reduces the transendothelial cell migration of immune cells. Synaptic Plasticity Leads to upregulation of plasticity related Vasoactivity Glucose Uptake Emotion Reading Autonomic Nervous System HPA axis Fear Facial Recognition Memory proteins and neurotrophins. Promotes vasodilation in small vessels, but might promote vasoconstriction in large vessels; dependent on endothelial versus smooth muscle contributions. Encourages the uptake of glucose in most cell types. Better judgement of the feelings of others. Increases parasympathetic tone over sympathetic tone leading to decreased reactivity. Blunts the HPA axis response. Suggested that GABAerigic OXTR neurons in central amygdala reduce the fear response. Overall heightened recognition of previously encountered faces with some specificity for facial expressions. Generally appears to be amnestic for non- social related memories with some contradictions for prefrontal cortex involvement. Learning Blood Pressure Less obvious as to whether learning is as impaired as memory retrieval, acquisition tests suggest it is not impaired. Lowers blood pressure. 15 Devost, Wrzal, & Zingg, 2008; Cassoni, 2006; Deing, Roggenkamp, Kühnl, Gruschka, Stäb, Wenck, … Neufang, 2013 Cattaneo, Lucci, & Vicentini, 2009; Viero et al., 2010; Senturk et al., 2013; Liu, Pan, Tan, Zhao, & Liu, 2017 Bakos, Srancikova, Havranek, & Bacova, 2018; Havranek et al., 2015 Thibonnier, Berti-Mattera, et al., 1999; Uvnäs-Moberg, 1998; Altura & Altura, 1984 Florian, Jankowski, & Gutkowska, 2010; Lee et al., 2008 Hollander et al., 2003, 2007 Huber, Veinante, & Stoop, 2005; Ebner, Bosch, Krömer, Singewald, & Neumann, 2005) Neumann, Wigger, Torner, Holsboer, & Landgraf, 2001; Ditzen et al., 2009; Parker et al., 2005 Huber, Veinante, & Stoop, 2005 Savaskan, Ehrhardt, Schulz, Walter, & Schächinger, 2008 Kovács & de Wied, 1994; Bohus, Urban, Van Wimersma Greidanus, & De Wied, 1978; Ibragimov, 1990; Boccia, Kopf, & Baratti, 1998; Heinrichs et al., 2004 Ferrier et al., 1980; Engelmann, Wotjak, Neumann, Ludwig, & Landgraf, 1996 Petersson, Alster, Lundeberg, & Uvnäs-Moberg, 1996; Light, Grewen, & Amico, 2005 Table 1.1 (cont’d) Heart Rate Antioxidant Protection Inflammation Decreases heart rate and cardiac output through ANP, however, some pregnant women experience increases. Suggested to support an anti- inflammatory phenotype, but it remains unclear whether the receptor or peptide is more responsible. Pushes most inflammatory cells towards an anti-inflammatory phenotype, though a specific mechanism is unknown. Candidates include activity at NF-κβ and RAGE. Gutkowska et al., 1997; Rabow, Hjorth, Schönbeck, & Olofsson, 2018; Yashpal, Gauthier, & Henry, 1987 Rashed et al., 2011; Szeto et al., 2008; Honceriu, Ciobica, Stoica, Chirazi, & Padurariu, 2016; Deing et al., 2013; Polshekan et al., 2016; Wang et al., 2018 Jankowski et al., 2010; Wang et al., 2018; Garrido-Urbani et al., 2018; An et al., 2019; Yuan et al., 2016; Metz, Kojro, Rat, & Postina, 2012 16 Activation of the OXTR typically stimulates intracellular calcium (Ca2+) mobilization through a PLC-dependent mechanism (Gutkowska & Jankowski, 2012; Park et al., 1998). While the OXTR is reported as coupling predominantly to Gαq/11 type G protein subunits, it is now established that the OXTR also couples to Gi/Go type G protein complexes (Busnelli & Chini, 2018; Hoare et al., 1999). Recently, an ambitious synthesis of over 1800 articles reporting OXTR intracellular signaling pathways was completed by Chatterjee and colleagues. To date, this open source resource on NetPath is the most comprehensive overview of the OXTR signaling cascade (Chatterjee et al., 2016). Several of the more well-established pathways and their functional role at the cellular level will be described below. Gq/11- transduced signaling is mediated by PLC -stimulated hydrolysis of the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to diacyl glycerol (DAG), which in turns activates protein kinase C (PKC), and inositol 1,4,5-trisphosphate (IP3), which stimulates the release of intracellular Ca2+ stores via IP3 receptors and also activates PKC among other Ca2+-activated kinases (Thibonnier, Berti-Mattera, Dulin, Conarty, & Mattera, 1999; Viero et al., 2010). OXTR-stimulated PKC signaling has been shown to lead to the dephosphorylation of eukaryotic translocation factor (eEF2), which aids in cellular proliferation through peptide chain elongation (Devost, Wrzal, & Zingg, 2008). Several other kinases are reported to be activated through the Gαq/11 cascade of OXTR activation including the mitogen-activated protein kinases (MAPKs) ERK1/2, which induce c-fos and c-jun expression as an early mediator of proliferation, and ERK5, which is more specific to cellular differentiation (Devost et al., 2008; Zingg & Laporte, 2003). Additionally, PLC stimulates the phosphorylation of PI3K and AKT leading to the activation of endothelial nitric oxide synthase influencing cellular migration and vasodilation (Cattaneo, Lucci, & Vicentini, 2009; Viero et al., 2010). The involvement of Rho kinases in smooth muscle uterine contractions suggests that OXTR activation of these kinases can lead to the production of phospholipase A2 and, in turn, cyclooxygenase 2 (Viero et al., 2010). The modulation of levels of Rho GTPases by OXTR leads to shifts in cell adhesion molecules, particularly in neurons (Zatkova, Reichova, Bacova, & Bakos, 17 2019). Whereas the suspected Gi/Go pathway is less well-defined, it has been demonstrated that Giβγ signaling leads to p38 MAPK activation and aids cells in adaptive processes to physiological stressors through transcriptional activators and direct effects on cell stabilizing proteins (Hoare et al., 1999). Potential hyperpolarizing effects through Gi could occur through interactions with calcium-dependent potassium channels, as proposed based on recent studies using alternative peptides and chelators (Pierce, Mehrotra, Mustoe, French, & Murray, 2019). Hence, in addition to the well-established role for OXT as a contraction influencing hormone, it appears to be involved in cellular differentiation, migration, proliferation, responses to stressors, dilation, and inflammation. In vitro studies have shown that exogenously applied OXT decreases the production of reactive oxygen species (ROS) initiated by H2O2 application to lymphocytes (Stanić et al., 2016). This observation is supported by in vivo studies finding reduced ROS production with chronic OXT treatment in a mouse model of autism (Wang et al., 2018) as well as reduced oxidative stress status in OXT-treated naïve Wistar rats (Honceriu, Ciobica, Stoica, Chirazi, & Padurariu, 2016), ischemia-reperfused Sprague-Dawley rats (Faghihi, Alizadeh, Khori, Latifpour, & Khodayari, 2012), and naïve zebrafish (Balmus et al., 2017). Candidate downstream enzymes mediating OXT’s effects on ROS production include MAPK/ERK1/2, superoxide dismutase (SOD), and glutathione as antioxidant promoting pathways (Deing et al., 2013; Polshekan et al., 2016; Wang et al., 2018). Alternatively, NADPH oxidase production of ROS is observed to be dampened with the addition of OXT, indicating an effect of attenuating prooxidative pathways (Rashed, Hashem, & Soliman, 2011; Szeto et al., 2008). Additionally, OXTR knockdown in fibroblasts led to a decrease in oxidative stress and an increase in antioxidative enzymes (Deing et al., 2013). While the bulk of the evidence available points to an decrease in ROS following OXT administration or OXTR engagement, it is unclear whether encouraging antioxidative signals is the primary cause or a reduce in prooxidative ones, and whether enzymatic subtypes, cell type, or injury state matter. 18 With respect to the role of OXTR signaling in inflammatory modulation, complementary in vitro and in vivo studies have shown that OXT administration reduces the production of pro-inflammatory cytokines such as IL-6, TNF, and IL-1β (Garrido-Urbani et al., 2018; Jankowski et al., 2010; Szeto et al., 2008), and increases anti-inflammatory cytokines such as IL-10 and TGF-β (An et al., 2019; Jankowski et al., 2010). This dampening of proinflammatory cytokines is at least partially attributed to actions at NF-kβ (Yuan et al., 2016). Alternatively, stimulating the OXTR leads to a several fold reduction in receptor for advanced glycation end-products (RAGE) which stimulates macrophage cells to produce proinflammatory cytokines (Metz, Kojro, Rat, & Postina, 2012). Additionally, cytokines like IL-6 and IL-1β appear to feed forward and increase the expression of OXTR (Schmid, Wong, & Mitchell, 2001; Young, Muns, Wang, & Insel, 1997), suggesting a protective feed-back loop. As far as specific inflammatory cell types, OXT seems to generally promote the activation of peripheral immune responses while tempering central immune activation (Li, Wang, Wang, & Wang, 2017; Stary et al., 2019). For instance, OXT increases the production of spleen leukocytes, enhances the differentiation of thymus immune cells (Hansenne et al., 2005; Stary et al., 2019), and reduces inflammation-related transendothelial migration (Senturk et al., 2013; Liu, Pan, Tan, Zhao, & Liu, 2017), whereas OXT appears to mitigate microglial activation in the brain (Inoue et al., 2019; Mairesse et al., 2019; Yuan et al., 2016). The elucidation of the full extent of OXTR signaling involved immune activation and the consequences of this activation on OXTR activity is an ongoing subject of research. While the bulk of OXTR research has focused on interpersonal and reproductive behaviors, OXTR’s vasogenic activity has been well established from the earliest use of its peptide ligand in research (Gruber, 1928), when OXT was observed to lower blood pressure. This long-term decrease in blood pressure has subsequently been confirmed in rats (Petersson, Alster, Lundeberg, & Uvnäs- Moberg, 1996) and humans (Light, Grewen, & Amico, 2005). Subsequent studies in pregnant women and rats have shown that while blood pressure drops, heart rate increases with peripheral 19 administration of OXT (Rabow, Hjorth, Schönbeck, & Olofsson, 2018; Yashpal, Gauthier, & Henry, 1987). The interest in OXTR as a candidate in the etiology of and treatment for cardiovascular conditions has recently been revived due in large part to the work of Gutkowska and Jankowski (Gutkowska & Jankowski, 2012). Subsequently, research has suggested there might be a protective effect for OXTR signaling in tissue response to infarctions OXTRs are synthesized and found in the heart contributing to the release of atrial natriuretic peptide (ANP) and a decrease in cardiac output (Gutkowska et al., 1997). OXT can induce vasodilation when acting on endothelial cells through endothelial nitric oxide synthase (eNOS) activation (Thibonnier, Berti-Mattera, et al., 1999; Uvnäs-Moberg, 1998), but can also promote vasoconstriction when acting on smooth muscle cells (Altura & Altura, 1984). Further, there is reason to believe this might not be due to OXTR signaling, but through OXT acting on vasopressin receptors (Oyama et al., 1993; Suzuki et al., 1992). These divergent findings might also be vessel dependent, as small artery vasodilation has been reported (Rabow et al., 2018), while larger peripheral arteries may respond instead with vasoconstriction (Petersson, 2002). As Petersson points out, this could be explained by the alternate effects on endothelial versus smooth muscle cells, their relative distribution in large and small vessels, and the administration method (Petersson, 2002). OXTRs in the vasculature seem to maintain many of the same attributes as central and uterine receptors such as upregulation following circulating estrogen (Gutkowska, Jankowski, Mukaddam-Daher, & McCann, 2000), and interactions with arrestins and the same G proteins (Gutkowska & Jankowski, 2012). The blood pressure lowering effects of OXT seems to be based on receptors in the periphery and not those in the CNS, as peripherally but not centrally administered OXT lowers blood pressure and heart rate (Petersson, Lundeberg, Sohlström, Wiberg, & Uvnäs-Moberg, 1998). However, a systemic rise in OXT concentration, even with intranasal delivery leads to a decrease in regional cerebral blood flow, primarily in the amygdala (Martins et al., 2020). Alternatively, this same route of delivery under an fMRI using cerebral blood volume reported an 20 increase in the hippocampus and frontal cortex (Galbusera et al., 2017). Like the memory and stress related pathways, it is probable based on the divergent results that regional, conditional, or cell-specific differences exist with respect to vascular responses. Nonetheless, OXTR signaling clearly influences vascular activity in a context-dependent manner. The potential role for OXTR signaling in cerebrovascular protection is grounded in evidence for its protective role in the periphery. With respect to cardiovascular disease, OXTR stimulation in the heart causes the release of ANP and decreased heart rate (Gutkowska et al., 1997). In addition, a decrease in pressure in the chambers of OXT-treated hearts has also been reported (Sousa et al., 2005). Stress- induced increases in blood pressure that can prove deleterious over time are also mitigated with higher plasma OXT in mothers (Grewen & Light, 2011). Since ischemic heart disease is a leading cause of death worldwide (Naghavi et al., 2015), the idea that OXTR-mediated effects might prove effective as a management strategy or as an acute rescue agent has gained traction (Nilsson, 2009). OXT treatment in rodents has been shown to induce stem cells to adopt a cardiomyocyte phenotype (Matsuura et al., 2004), which could lead to exciting prospects in cardiac regeneration. Some experiments have even tested non-invasive ways to increase OXT after heart surgery, including massage and music interventions (Nilsson, 2009). In addition to these acute treatments, long term OXT increases are thought to be a primary agent through which social ties reduce the risk for cardiovascular disease (Knox & Uvnäs-Moberg, 1998). In rats, treatment with OXT lowered blood pressure in a hypertensive strain (Petersson & Uvnäs-Moberg, 2008). However, well-controlled longitudinal studies are needed to assess whether OXTR manipulation might lead to improved cardiovascular or even cerebrovascular outcomes. OXTRs might be uniquely positioned to respond to vascular insults due to their localization on microvascular endothelial cells (Nakamura et al., 2000; Thibonnier, Berti-Mattera, et al., 1999). OXT has been shown to induce proliferation of endothelial cells, most likely through a PI3K and Src kinase dependent production of nitric oxide by eNOS (Cassoni, 2006; Cattaneo, Chini, & Vicentini, 2008). 21 Beyond these pro-angiogenic effects, the receptor appears to have potent anti-inflammatory and antioxidant properties. It both reduces the activity of NADPH oxidase isoforms on endothelial cells and innate immune cells (Rashed et al., 2011; Szeto et al., 2008) and reduces the production of pro- inflammatory cytokines in favor of anti-inflammatory cytokines (Jankowski et al., 2010; Wang et al., 2018). OXTRs also potentiate the uptake of glucose during hypoxia (Florian, Jankowski, & Gutkowska, 2010; Lee et al., 2008). Notably, these are some of the same pathways that are thought to be beneficial in the recovery of surviving tissue after an ischemic injury (Bir et al., 2012; Choi et al., 2015; Liu et al., 2013; Ooboshi et al., 2005; Rodrigo et al., 2013). OXTR activation has been mechanistically linked to the amelioration of tissue damage following cardiac infarction (Jankowski et al., 2010), renal infarction (Tuǧtepe et al., 2007), hepatic infarction (Düşünceli et al., 2008), and cerebral stroke (Moghadam et al., 2018; Karelina et al., 2012; Seo, Lee, & Oh, 2018). Cardiomyocytes can also be protected from ischemia and reperfusion injury through a reduction in mitochondrial-sourced ROS and a shift in cell signaling away from pro- apoptotic Bax towards anti-apoptotic Bcl-2 (Gonzalez-Reyes et al., 2015). In examining cerebral ischemic stroke more directly, Karelina and colleagues used social housing, OXT treatment, and OXTR antagonists to demonstrate a protective role for OXT in reducing tissue loss and deleterious inflammation while enhancing antioxidative enzyme expression following middle cerebral artery occlusion (Karelina et al., 2012). This observation has been extended to show that the neuroprotective effect of nursing in cerebral ischemia that can be mimicked with exogenous OXT administration in mice, reducing ROS production and apoptotic neuron death (Moghadam et al., 2018; Stary et al., 2019). Effects on cognitive changes post-stroke and animal models and human studies are limited. Only one human case study post-stroke has been published, wherein the authors speculated that a patient’s rapid recovery from post-partum stroke may have been due to OXT administered to reduce postpartum bleeding and increased endogenous OXT release upon contact with her newborn (Seo et al., 2018). Cognitive effects 22 have been limited to post-stroke depression and anxiety-like behavior in animals, and supposition in humans (Long & Hillis, 2016; Zhong et al., 2020). Post-stroke memory impairments are a relatively unexplored target. In this regard, a de novo up-regulation of OXTRs in astroglia within the peri-infarct space was demonstrated in patients who died with a clinical pathologic diagnosis of vascular dementia, suggesting a druggable target for quick intervention (McKay et al., 2019) (See Chapter 2). This is supportive of the detection of functional OXTR on astroglial cells in culture that can bind appropriate radioligands and trigger a release of TGF-β (Di Scala-Guenot & Strosser, 1992; Mittaud, Labourdette, Zingg, & Guenot-Di Scala, 2002) as well as reports of post-ischemic increases in OXTR for CNS tissue (Moghadam et al., 2018), though the opposite has been found in post-ischemic heart tissue (Jankowski et al., 2010). In cases of birth-related ischemic injury, OXT administration improved viability of immature hippocampal cells and reduced markers of oxidative stress (Ceanga, Spataru, & Zagrean, 2010; Kaneko, Pappas, Tajiri, & Borlongan, 2016; Tyzio et al., 2006), which may be linked to associated changes in GABAergic chlorine channels in addition to possible hemodynamic alterations (Kaneko et al., 2016; Tyzio et al., 2006). By contrast, other studies have found that OXT administered to dams of pups undergoing birth-related ischemic injury might actually exacerbate injury due to a vasodilatory reaction leading to exacerbated birth anoxia (Boksa, Zhang, & Nouel, 2015). Critically, an ischemic environment might switch the vasodilatory effect of OXT to a vasoconstrictive one based on studies of isolated cerebral arterioles (Bari, Enico, Louis, & Busija, 1997). In the case of long term management of vascular health, OXT has been found to reduce atherosclerosis in mice, rabbits and rats prone to the development of such plaques (Ahmed & Elosaily, 2011; Nation et al., 2010; Szeto et al., 2013). While this work suggests the receptor might also make a valid target for recovery of cerebrovascular insults, including those related to cognitive impairment and dementia (McKay et al., 2019), further mechanistic and validation studies must be performed, especially in reference to ischemic conditions. 23 Dissertation Objectives The goal of the work was to identify and evaluate a potential new target for intervention in VaD based on discoveries made in human tissue from VaD patients. In service of this goal, human frontal cortex tissue from VaD, AD, and non cognitively impaired cases were compared using microarray technology. Genes of interest were subsequently analyzed via validation procedures. One of the most promising targets, the OXTR, was tested in a rat model of VaD for cognitive preservation and tissue preservation. Additionally, potential therapeutic mechanisms were also tested in-vivo. The divised aims to guide this work were as follows: Specific Aim 1 We will test the hypothesis that OXTR upregulation is a specific response to dementia resulting from vascular causes. Specific Aim 2.1 We will test the hypothesis that overexpression of the OXTR improves behavioral and pathological outcomes in a rat model of vascular cognitive impairment. Specific Aim 2.2 We will test the hypothesis that OXTR signaling modulates several deleterious pathways of the ischemic cascade in a rat model of forebrain ischmeic injury. 24 CHAPTER 2: The detection of an oxytocin receptor upregulation in vascular dementia and localization Introduction to peri-infarct astrocytes Alzheimer’s disease (AD) and vascular dementia (VaD) represent the first and second leading causes of dementia, a syndrome of pathologically diminished cognitive functioning affecting over 50 million people worldwide (World Health Organization, 2017). Recent estimates suggest a financial burden of $604 billion dollars (Wimo, Jönsson, Bond, Prince, & Winblad, 2013), and around 40% of familial primary caregivers suffer from depression or anxiety (Livingston et al., 2013). Advanced age is the greatest predictor of dementia with rates of AD doubling every 4.5 years and VaD every 5.5 years with an initial sporadic incidence at about 65 years of age (Jorm & Jolley, 1998). As more individuals live to an advanced age, the incidence and prevalence of dementia is expected to rise (Corrada, Brookmeyer, Paganini-Hill, Berlau, & Kawas, 2010). Therefore, the discovery of specific disease-modifying treatments or preventative strategies for these two most common dementias is a top priority for translational research. AD and VaD can be difficult to differentiate clinically. This is owing in part to overlapping risk factors such as advanced age, hypertension, type 2 diabetes mellitus, and hypercholesterolemia (Attems & Jellinger, 2014). A general rule states that episodic memory deficits herald the onset of AD while executive deficits predominate initially in VaD (Brookes, Hannesdottir, Lawrence, Morris, & Markus, 2012). However, results from longitudinal studies like the Gothenburg mild cognitive impairment (MCI) study suggest this may only be reliable in the early stages of MCI, as all cognitive domains showed impairment once the patients progressed to AD or VaD (Wallin et al., 2015). Behavioral differences are also unreliable as AD and VaD do not differ significantly in affective volatility or the presence of psychiatric symptoms (Bathgate, Snowden, Varma, Blackshaw, & Neary, 2001). Currently the only 25 definitive diagnosis of AD or VaD can be made postmortem based on pathological hallmarks (Román et al., 1993). The pathological hallmarks of VaD differ greatly from the amyloid plaques and neurofibrillary tangles that characterize AD (Zheng, Vinters, Mack, Weiner, & Chui, 2015). The cognitive impairment in VaD is typically preceded by clinical stroke or small vessel disease that reduces cerebral blood flow (CBF) without obvious stroke (Gorelick, Counts, & Nyenhuis, 2016; Thal et al., 2012). The most common injury leading to VaD is an infarction or restriction of CBF caused by either a local atherogenic thrombus or an embolism (Thal et al., 2012). This ischemic event initiates a cascade of deleterious effects including: inflammation, neutrophil invasion and obstruction of vessels through the endothelium lining, superoxide production, nitric oxide synthase uncoupling, and deficits in oxidative phosphorylation from oxygen and glucose depletion (Dirnagl et al., 1999). Without reperfusion or compensation by collateral branches, the surrounding tissue becomes necrotic resulting in infarcts, white matter lesions, and demyelination (Iadecola, 2013). While a single ischemic event is sufficient to produce dementia symptoms if it occurs in a particularly sensitive location like the thalamus or hippocampus, VaD may also arise from multiple, cumulative vascular accidents with increasing volume of impacted tissue predicting dementia severity (Jellinger, 2013; Tomlinson et al., 1970). An ischemic core of unsalvageable neural tissue forms in the immediate wake of a significant reduction in CBF. However, the surrounding penumbra of at-risk neurons represents a site where early therapeutic intervention may limit the ultimate cognitive impact of the ischemic event (Hossmann, 1994; Liu, 2012). As noted above, several potential pathogenic factors contribute to the loss of brain parenchyma in the event of ischemic injury, and a successful therapy to limit neuronal death would need to exert protective effects against several of these contributors (Shichinohe et al., 2015). 26 While it has been half a century since C.M. Fisher concluded that stroke alone could lead to dementia (Fisher, 1968), the high rate of mixed pathology dementias from both AD and vascular damage has confounded efforts to combat VaD resulting from stroke or cerebrovascular disease alone (Attems & Jellinger, 2014; O’Brien & Thomas, 2015). To date, several pharmacological interventions, primarily cholinesterase inhibitors, have FDA approval for the treatment of AD, yet no drug has regulatory approval for the treatment of VaD (Gorelick et al., 2011; Kavirajan & Schneider, 2007). Of the many clinical trials that have repurposed commonly used AD medications for the symptomatic management of VaD, there have been only small trends in cognitive improvement that skew to aiding suspected mixed pathology participants far more than VaD patients (Baskys & Cheng, 2012). The present study was undertaken to address these difficulties in finding potential therapeutic options for VaD (Gorelick et al., 2011). Using microarray technology, we generated gene expression profiles of frontal cortex obtained from individuals who died with AD, VaD, or age-matched, cognitively intact controls. This analysis revealed 3,495 genes that were dysregulated in AD, whereas 413 genes were selectively dysregulated in VaD. Pathway analysis and validation studies identified a VaD-specific upregulation of transcripts encoding the oxytocin receptor (Oxtr). Moreover, subsequent human tissue studies revealed focal endothelial and astroglial OXTR protein upregulation surrounding grey and white matter microinfarctions. Given the evidence for a protective role of oxytocin (OXT) signaling in peripheral ischemia (Düşünceli et al., 2008; Gonzalez-Reyes et al., 2015; Jankowski et al., 2010; Tuǧtepe et al., 2007), we posit that OXTR upregulation in VaD may represent a novel, druggable pathway for the therapeutic treatment of dementia resulting from cerebrovascular injury. 27 Methods and Materials Postmortem Human Tissue Fixed and frozen frontal cortex (Brodmann Area 10) samples collected postmortem from individuals who died with AD (n = 12), VaD (primarily of the Multi-Infarct Dementia subtype, n = 9), or without dementia (controls [CTL]; n = 10) were obtained from the University of Michigan Alzheimer’s Disease Center Brain Bank. The cases were matched for age, postmortem interval, and gender (Table 2.1). Cases were selected such that the AD samples had no infarcts present in the autopsied hemisphere, and the VaD cases had no evidence or very minimal evidence of any AD typical pathology. This allowed for exclusion of likely mixed dementia cases which could obscure VaD specific changes. mRNA Preparation and Microarray Processing 40-50 mg of frozen tissue was cut on a platform in dry ice and lysed using a TissueLyser set to 20 Hz for 2 minutes in 10x volume of lysis buffer. RNA was extracted using the mirVana miRNA isolation kit (Ambion) according to the manufacturer’s instructions. Organic extraction was performed using a 1/10 volume of the miR Homogenate Additive and the acid-phenol/chloroform method. Total RNA was quantified by NanoDrop spectrophotometry (Thermo Scientific) and RNA quality was assessed on an Agilent 2100 Bioanalyzer. RNA Integrity Number values (mean ± SD) were as follows: CTL (6.9 ± 0.6); VaD (5.9 ± 1.2); AD (6.3 ± 1.1). Agilent’s Low Input QuickAmp Labeling Kit was used with 100 ng total RNA/sample to generate cRNA labelled with Cy3 and Cy5 dyes following the supplied protocol. The cRNA was purified using Qiagen’s RNeasy MiniElute Cleanup Kit. The yield and specific activity of each product was determined by NanoDrop analysis. The cutoff used for viable yields was above 0.825 µg and the specific activity cutoff was 6 pmol/µg. For each sample, 300 ng of labelled, linearly amplified cRNA was digested and hybridized for 17 hours at 65 ○C on Agilent Human 8x60k v2 microarray slides pressed to a gasket slide using the Agilent SureHyb chamber base. The slides were then sequentially washed with 28 Gene Expression Wash Buffer 1 at room temperature and Gene Expression Wash Buffer 2, which was warmed to 37 ○C. Ajer washing, the slides were scanned on an Agilent C Microarray Scanner using the G3 microarray format with the resolution set to 3 µm. Microarray Data Processing and Analysis Raw data files were imported into R (www.r-project.org)/Bioconductor for downstream processing and analysis (Huber et al., 2015). Data were assessed for quality via intensity distributions, principal component analysis, and mean correlation with one VaD sample being removed due to poor sample and data quality. All remaining data were background corrected and quantile normalized prior to differential expression. Differential expression was performed via limma using a block design to leverage technical replicates (Ritchie et al., 2015). Genes with a False Discovery Rate (FDR)-adjusted p < 0.05 were considered differentially expressed (Gaujoux & Seoighe, 2010). Individual lists of differentially expressed genes from each comparison were imported into MetaCore version 6.23.67496 (Thomson Reuters) for Pathway Map enrichment and Shortest Paths network analysis. MetaCore Pathway Maps are rigorously curated biochemical pathways and signaling cascades. An algorithm based on the hypergeometric distribution is used to calculate enrichment p-values. Shortest Paths network analysis was performed with a maximum number of steps in the path set to 1 without the use of canonical pathways. Nodes and edges were exported from MetaCore and imported into Cytoscape v3.3.0 for final visualization (Merico, Isserlin, Stueker, Emili, & Bader, 2010). Raw and processed microarray data is available on GEO (GSE122063). Quantitative PCR validation cDNA was generated from 1 µg RNA using ThermoFisher’s RevertAid First Strand cDNA synthesis kit. TaqMan probes for Rac Family Small GTPase 1 (Rac1; probe set Hs01902432), cAMP-response element binding protein (CREB) binding protein (Crebbp; Hs00231733), 5-Oxoprolinase, ATP-Hydrolyzing 29 (Oplah; Hs00417215), Spondin 2 (Spon2; Hs00202813), and Oxtr (Hs00168573) were multiplexed with Glyceraldehyde-3-Phosphate Dehydrogenase (Gapdh; Hs02758991; see Table 2.2 for gene nomenclatures). Three technical replicates were performed. Target genes used FAM as the reporter dye while the reference gene used VIC as the reporter dye. 4 µL of cDNA was used per well and 6 µL of a mixture of the two Taqman probes and Taqman Universal Master Mix II, DNA polymerase, and dNTPs were added to each well for a total volume of 10 µL per well. The plates were briefly centrifuged and analyzed on an Applied Biosystems 7500 real time PCR system over 40 cycles. Fold change between controls and dementia samples was established using the 2-ΔΔCt method with Gapdh as the reference. Western Blotting 30-50 mg of frozen frontal cortex tissue was dissected on dry ice and collected in a round bottom 2 mL tube. Subcellular fractionation of the samples was achieved using a protocol modified from Guillemin and colleagues (Guillemin, Becker, Ociepka, Friauf, & Nothwang, 2005). 5x volume of cell lysis buffer (10 mM HEPES, 10 mM NaCl, 1 mM KH2PO4, 5 mM NaHCO3, 1 mM CaCl2, 0.5 mM MgCl2) was added to each sample that included a volume of 1% 100 nM PMSF, 1% Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific), and 1% 0.5 M EDTA. Samples were homogenized by sonication and restored to isotonic condition with a 0.4x volume of 2.5 M sucrose. After centrifuging for 10 minutes at 6300 rpm at 4 ○C, the supernatant was collected and centrifuged again for 150 minutes at 14,000 rpm at 4 ○C. The supernatant representing the cytosolic fraction was reserved, and the pellet representing the membrane fraction was resuspended with 1x phosphate buffered saline pH 7.4 at a volume of 50 µl per pellet and the EDTA, PMSF, and inhibitor cocktail were spiked in at a 1% concentration. Protein quantification was determined with the Pierce bicinchoninic acid assay kit (BCA, Thermo Scientific). Proteins of the membrane fraction (30 µg/sample) were separated on 4-20% PAGE gels (Criterion, Bio-Rad) for 25 minutes at 250 mV. They were transferred to a PVDF membrane at 400 mA 30 for 50 minutes. After blocking with 5% dry milk in 1x Tris-buffered saline, pH 7.4, with 0.05% Tween-20 (TBST), membranes were incubated overnight at 4 ○C with a rabbit primary OXTR anmserum (ProteinTech; 1:500) in TBST. The following day, membranes were blocked for ten minutes and washed twice with TBST, incubated with goat anti-rabbit 680 secondary antibody (LiCor; 1:10,000) in TBST for two hours, followed by washes in TBST (1 x 10 minutes) and TBS (2 x 10 minutes). Blots were visualized on an Odyssey scanner (LiCor) at a resolution of 169 µm and an intensity of 5.0 in the 700 nm channel. The blots were then stripped in Restore western blot stripping buffer (Thermo Scientific) for 15 minutes, washed briefly with TBS and blocked for one hour with TBST/5% milk. Membranes were then incubated with rabbit primary calnexin antiserum (ProteinTech; 1:1000) in TBST overnight at 4°C. The next day, the membranes were washed and incubated with goat anti-rabbit 680 secondary antibody (Licor; 1:10,000) and imaged on the Odyssey scanner. OXTR protein levels were normalized to calnexin levels for quantitative analysis (Odyssey Image Studio, LiCor). Immunohistochemistry and Immunocytochemistry For immunohistochemistry (IHC), 20 µm sections of paraffin-embedded frontal cortex tissue from the same cases used in the microarray analysis were deparaffinized, cleared in xylenes, and rehydrated using a 100/95/70% ethanol series. Antigen retrieval was achieved by submerging the slides in a 65°C citric acid buffer (0.1 M, pH 6.0) for 20 minutes. Slides were cooled to room temperature, washed in TBS, and incubated in a solution of 90% methanol/10% H2O2 for 10 minutes to quench endogenous peroxidase activity. The tissue was then permeabilized with TBS/0.25% Triton X-100 (Tx) for 3 x 10 minutes, blocked in TBS/0.25% Tx/1% normal goat serum (NGS) for 30 minutes, and incubated overnight at 4°C with the OXTR antiserum (1:100) in blocking buffer. Adjacent tissue sections were incubated in the absence of primary antibody. The following day, the slides were rinsed in TBS and incubated with biotinylated goat anti-rabbit secondary antibody (Vector Labs, 1:500) in TBS/1%NGS for 31 one hour at room temperature. Afterwards, the slides were rinsed in TBS and incubated for one hour in Vectastain ABC solution (Vector). Once this was completed the slides were rinsed in imidazole acetate buffer (pH 7.4) and developed for two minutes with the ImmPact DAB peroxidase substrate kit (Vector). Once sufficient staining was observed, the slides were washed in TBS, dehydrated in an 70/95/100% ethanol series, cleared in xylenes, and coverslipped with Cytoseal 60 (Thermo Scientific). For dual-labelling of both GFAP and OXTR in the same sections, GFAP antiserum (Cell Signaling; 1:200) was used following the protocol described for OXTR IHC up to the development step, when Immpact VIP peroxidase substrate kit (Vector) was used instead. After washing with TBS, the slides were blocked with avidin and biotin using Vector’s Avidin/Biotin Blocking kit. Slides were washed in TBS 3 x 10 minutes, placed in 90% methanol/10% H2O2 for 10 minutes, exchanged to TBS/0.25% Triton X-100 (Tx) for 3 x 10 minutes, blocked in TBS/0.25% Tx/1% normal goat serum (NGS) for 30 minutes, and incubated overnight at 4 ○C with the OXTR antiserum (1:100) in blocking buffer. Detection and development of the OXTR antiserum was performed the following day as described above. For immunocytochemistry, HBEC-5is human brain-derived endothelial cells (ATCC) were seeded in 8-well chamber slides at a density of 10,000 cells/well in 200 µL of DMEM/F12 media (Gibco) with L- Glutamine, 15mM HEPES, 10% calf bovine serum, and 40 µg/mL endothelial growth supplement. The CTX TNA2 rat immortalized cortex-derived astrocytes (ATCC) were seeded on 6-well cell culture dishes at a density of 300000 cells/well in 3 mL of DMEM/F12 media (Hyclone) with low glucose, 10% calf bovine serum, and 1% penicillin/streptomycin. The media was exchanged three times with 1x phosphate-buffered saline (PBS; 0.1M, pH 7.4). Cultures were fixed with PBS containing 4% paraformaldehyde for fifteen minutes, permeablized with PBS/0.2% Tx for 10 minutes and blocked with PBS/0.2% Tx/10% NGS for one hour (all steps at room temperature with gentle agitation). OXTR antiserum incubations were performed at 1:100 as well as no- 32 primary antibody control overnight at 4 ○C with gentle agitation. The GFAP antiserum used was a mouse monoclonal at 1:100 (Proteintech, 60190-1-Ig) also performed overnight at 4°C with agitation. The next day, wells were rinsed 5 x 5 minutes with PBS at room temperature followed by incubation with goat anti-rabbit 488 fluorescent secondary antibody for OXTR and anti-mouse 568 fluorescent secondary antibody for GFAP (Life Technologies, 1:200) in blocking buffer for one hour at room temperature. Afterwards, the wells were washed 5 x 5 minutes with PBS and chamber slides were processed for coverslipping using Vectashield antifade mounting media with DAPI (Vector). Hypoxia for Rat Astrocytes An incubator with the potential to backfill with nitrogen to reduce oxygen was used (ThermoFisher). The incubator was sterilized and set to 2% O2 before the cells were placed within. The time points chosen were 15 minutes, 1 hour, 2 hours, 6 hours, and 12 hours. Matching plates in the standard culture chamber were used as controls for each time point. At the end of each time point the plates were quickly retrieved and placed on ice to preserve the cells’ condition. The media was removed and the cells were retrieved using 1mL 0.5% Trypsin with EDTA (Gibco) in 9mL 1xPBS and agitation into a 15mL falcon tube. The cells were pelletized through centrifugation at 500rpm for 5 minutes at 4°C. The fluid was aspirated and the cells were suspended in 500µL Trizol. After a vigorous vortex, 100µL of chloroform was added to each tube. Incubation for three minutes at room temperature was followed by centrifugation at 4°C for 10 minutes at 12,000rpm. The supernatant was transferred to a clean tube and placed on ice. 250µL isopropanol plus 2µL GlycoBlu (Thermos Fisher) were added and mixed. A second centrifugation step at 4°C for 20 minutes at 12,000rpm followed. The supernatant was discarded, after which the pellet was washed with 80% ethanol. The ethanol was removed and the remaining pellet was dried for 5 minutes and then resuspended with 15µL nuclease-free water. RNA samples were stored at - 80°C. The RNA was quantified with a nanodrop and the qPCR methods were then employed using the 33 same methods as described above and with a rat OXTR (Rn00563503_m1) and GAPDH (Rn01462662_g1) Taqman probe in FAM and VIC respectively. RNAscope 20 µm sections of fixed paraffin-embedded frontal cortex tissue were deparaffinized, cleared in xylenes, and submerged in 100% ethanol. All steps were performed with Advanced Cell Diagnostics (ACD) reagents (RNAscope 2.5 HD Reagent Kit-BROWN Catalog No. 322300) according to the manufacturer’s instructions. Slides were incubated in H2O2 for 15 minutes at room temperature, washed twice in water, and then submerged in boiling antigen retrieval buffer for 15 minutes followed by washes with water. The slides were then incubated with ACD Protease Plus reagent for 30 minutes at 40 °C, followed by four washes in 40 °C Wash Buffer and incubation with the human GFAP probe (ACD No. 311801) for two hours at 40 °C. The slides were removed and washed four times. The amplification steps proceeded 1-4 at 40 °C alternating between 30 and 15 minutes with washes between each step. The final two amplification steps, step 5 for 30 minutes and step 6 for 15 minutes, were performed at room temperature. After the final amplification, a premixed 150 μL of premixed DAB reagents A and B were applied to the slides for 10 minutes. The DAB reaction was halted by submersion in distilled water. The slides were then dried and processed for OXTR IHC as described above. Hematoxylin and Eosin Histology After deparaffination and rehydration in ethanol series as described above, slides were submerged in Harris Modified Hematoxylin (Thermo Scientific) for six minutes, dipped in 1% acid alcohol (hydrochloric acid in 70% ethanol), differentiated in ammonia water (1/100 dilution of 28% ammonium hydroxide in water) for 30 seconds, and placed in Eosin working solution (Fisher Scientific) for one minute before dehydration and cover slipping. 34 Power and Statistical Analysis Based on our previous gene expression studies with human tissue samples from three diagnostic groups (Counts, Alldred, Che, Ginsberg, & Mufson, 2014; Counts et al., 2007; Kelly et al., 2017; Tiernan et al., 2016, 2018) and α = 0.05, we calculated that a sample size of 8-10 cases/group would have ~85% power to detect an effect size of 1.25 standard deviations. Subject demographics and postmortem variables were compared by Kruskal-Wallis test or Fisher’s Exact test. Microarray-based differential expression was performed using limma for R/Bioconductor with technical replicates being leveraged via a block design, as described above. qPCR results were compared by one-way ANOVA with Tukey’s correction. Western results were compared via Kruskal-Wallis test to account for irregularities in variance, with Dunn’s post hoc test used for multiple comparisons. For all analyses the significance was set at p < 0.05. Results Subject characteristics Demographic and neuropathological characteristics of the 31 subjects (10 CTL, 9 VaD, and 12 mild/moderate AD) are summarized in Table 2.1. There were no significant differences in age, gender balance, or postmortem interval. When scored for Braak stage, subjects with AD displayed significantly greater tau pathology compared to the CTL and VaD groups (p < 0.001). By contrast, there were significantly more subjects in the VaD group displaying infarctions (p < 0.00001), with 89% of the VaD cases exhibiting lacunes, large infarcts, or microinfarctions compared to 20% of CTL cases and none of the AD cases. Furthermore, 100% of the VaD cases exhibited some form of age-related cerebrovascular pathology (e.g., atherosclerosis, white matter rarefaction) compared to 67% of AD cases and 50% of CTL cases (p = 0.02). 35 Table 2.1 Clinical, neurological, and demographic characteristic by diagnostic category 36 Differential gene expression in VaD and AD Frontal cortex tissue was used for microarray analysis as frontal executive function and working memory deficits are predominant in the initial stages of VaD compared to AD (Stuss & Levine, 2002). Differential expressions analysis of AD versus controls, VaD versus controls, and AD versus VaD identified 4564, 1482, and 319 significantly differentially expressed genes, respectively (Figure 2.1). Data are accessible at the NCBI GEO database (accession GSE122063). Figure 2.1. Venn diagram representing common and disease-specific genes in CTL, VaD, and AD subjects. Comparisons are based on significantly differentially expressed genes (FDR-adjusted p < 0.05) in frontal cortex from the three clinical diagnostic groups. 37 Pathway analysis across the diagnostic groups Pathway analysis was performed using differentially expressed genes from the following comparisons: AD versus controls, VaD versus controls, and AD versus VaD (Figure. 2.2). Twenty pathways were significantly enriched among differentially expressed genes in AD versus control. Among these AD-related functional pathways were synaptic processes such as vesicle fusion and recycling (e.g., Syn1, Syn2, Snap25) (Table 2.2). Oxidative phosphorylation (Uqcr10, Uqcrfs1, Cox5a), cytoskeletal remodeling (Efnb1, Efnb3, Cxcr4), and NMDA receptor trafficking (Grin2b, Grin2d, Cdk5) were also robustly implicated in AD. By comparison, only three pathways were significantly enriched among differentially expressed genes in VaD compared to controls, including oxidative phosphorylation (Ndufa7, Ndufb7), cytoskeletal remodeling (Rac1, Trio), and clathrin-coated vesicle cycling (Aak1, Rab5a), whereas there were no significantly enriched pathways among genes specifically differentially expressed between VaD and AD. While these results did not provide a pathway unique to VaD, this was not unexpected since neurodegenerative processes likely involved many common functional gene families in these two dementia subtypes. Moreover, cerebrovascular comorbidities in the control and AD groups might also lead to the expression of overlapping pathways in the absence of frank vascular pathology. 38 Table 2.2 Expanded names of key abbreviations 39 Figure 2.2. Pathway analysis of dysregulated genes in VaD compared to AD frontal cortex. Horizontal bar graph shows that gene expression alterations in AD were heavily enriched for synaptic function, cytoskeletal remodeling, and glutamatergic signaling compared to controls. By contrast, VaD displayed an enrichment of genes dysregulated in oxidative phosphorylation and clathrin-coated transport compared to controls. Pathways most strongly differentiating AD and VaD were associated with cell adhesion and RhoA-mediated G protein-coupled signaling. Red hashed line: p < 0.05 (FDR-adjusted). 40 VaD-specific network analysis reveals genes of interest A network analysis was performed using all genes that were found to be up- or downregulated in VaD cases but not in AD (n = 413) (Figure 2.3). Two hub genes and a highly upregulated receptor were noted. Rac1, a Rho-type small GTPase implicated in both reducing apoptosis-mediated cell death and, by contrast, contributing to higher incidence of myocardial infarction (Marei & Malliri, 2016), was downregulated in VaD (logFC=-0.40, p=0.01) (see Table 2.2). Interestingly, several RAC1 protein activators such as Plekhg4 (logFC=-0.78, p=0.02) and Sh3rf1 (logFC=-0.33, p=0.04) were also downregulated, whilst RAC1 inhibitors such as Rcc2 (logFC=0.41, p=0.04) and Ralbp1 (logFC=0.40, p=0.02rfrf) were upregulated. On the other hand, Crebbp mRNA was upregulated in VaD (logFC=0.37, p=0.02), as were several core histones targeted by CREBBP acetyltransferase activity (Ogryzko, Schiltz, Russanova, Howard, & Nakatani, 1996) such as Hist1h4b (logFC=0.59, p=0.01), Hist1h4d (logFC=0.53, p=0.02), and Hist1h2bm (logFC=0.60, p=0.04). Finally, Oxtr, which encodes the OXTR G-protein coupled receptor linked to phospholipase C activation, was significantly upregulated in VaD (logFC=0.86, p=0.03) (Figure. 2.3). 41 Figure 2.3. Network analysis of VaD-specific gene dysregulation. Two major interaction hubs were identified centering on Rac1 and Crebbp (see also Table 2.2). Rac1 was significantly down-regulated in VaD compared to AD and controls (log fold change = -0.4, FDR adjusted p<0.01). Likewise, several genes encoding RAC1 inhibitors (e.g., Ralbp1) were upregulated whereas genes encoding RAC1 activators (e.g., Plekhg4) were downregulated in VaD. By contrast, Crebbp gene expression was significantly upregulated in VaD samples (log fold change = 0.4, FDR-adjusted p < 0.02). Crebbp is a modulatory hub for several upregulated genes encoding core histones 0e.g., Hist1h2bm, Hist1h4b) and regulators of chromatin remodeling 002e.g., Satb1). Notably, Oxtr was among the most strongly upregulated genes within the VaD network (log fold change = 1.86, FDR-adjusted p < 0.04). 42 Validation of VaD-specific gene changes qPCR analysis of frontal cortex samples was used to validate microarray-based observations in VaD-related gene expression. In addition to Rac1, Crebbp, and Oxtr, we selected two additional genes for analysis based upon their differential expression in VaD compared to AD and controls, Oplah and Spon2. The OPLAH gene product is an enzyme that aids in the conversion of 5-oxoproline to glutamate and in cases of genetic insufficiency has been associated with oxidative stress and delayed motor skills (Almaghlouth et al., 2012). SPON2 is a cell adhesion protein important for neurogenesis (Feinstein et al., 1999; He et al., 2004). As shown in Figure 2.4, levels of Rac1 (Figure 2.4A), Crebbp (Figure 2.4B), and Oplah (Figure 2.4D) did not differ across the diagnostic groups, whereas Spon2 was significantly downregulated in AD compared to controls (Figure 2.4C). By contrast, Oxtr was significantly upregulated in VaD compared to AD and controls (Figure 2.4E), suggesting a VaD-specific alteration in this receptor. Western blot analysis of frontal cortex membrane fractions revealed that OXTR protein levels were significantly upregulated in VaD compared to controls, with intermediate levels detected in AD cases (Figure 2.5) 43 Figure 2.4. qPCR validation of selected genes dysregulated via microarray analysis. A) qPCR analysis of Crebbp mRNA in the same tissue blocks of frontal cortex examined by microarrays revealed a nonsignificant trend of upregulation in VaD and AD samples compared to control levels (one-way ANOVA [F = 2.99, p = 0.06]). B) Rac1 levels did not differ between the three diagnostic groups (F = 1.22, p = 0.31). C) Spon2 was significantly downregulated in AD compared to control samples (F = 11.18, p = 0.0003). D) Oplah mRNA levels were unchanged among the three diagnostic groups (F = 0.4850, p = 44 Figure 2.4 (cont’d) 0.6209). E) Oxtr mRNA levels were significantly upregulated in the VaD samples (F = 3.886, p = 0.03). *, p < 0.05. 45 Figure 2.5. Western blot confirmation of OXTR protein upregulation in VaD frontal cortex. Membrane fractions of the same tissue blocks of frontal cortex were used to detect protein levels of OXTR. A) Representative western blot showing OXTR and calnexin immunoreactivity in CTL, VaD, and AD cases.. B) Quantitative analysis revealed that protein levels were significantly different among the three diagnostic groups (Kruskal-Wallis test [H =7.426., p = 0.03]). OXTR levels in VaD were significantly different than CTL (Dunn's multiple comparison's test [mean rank = -10.63, multiplicity-adjusted p = 0.033), with intermediate but nonsignificant levels detected in AD. *, p < 0.05. Samples were run as technical replicates with means used for statistical analysis. 46 OXTR expression in neocortex and human cerebrovascular endothelial cell culture OXTR IHC revealed immunopositive profiles in frontal cortex consistent with a predominant vascular localization (Figure 2.6A and B), supporting previous work suggesting that OXTR is abundantly expressed by cerebrovascular endothelial cells (Thibonnier et al., 1999). Accordingly, fluorescence immunocytochemistry using an immortalized line of human brain derived endothelial cells revealed robust OXTR positive staining that was not observed in control primary-delete experiments (Figure 2.6C). Hence, OXTR expression may be enriched in the brain cerebrovasculature. 47 Figure 2.6. OXTR expression in cerebrovascular endothelial cells. A, B) OXTR immunoreactivity is enriched in vascular profiles in human frontal cortex tissue (left). Deletion of primary OXTR antibody supports antigen specificity (right). Images shown at 20X (A) and at 40X (B). C) Fluorescence immunocytochemical detection of OXTR with DAPI counterstain in cultures of human brain derived endothelial cells, shown with (left) and without (right) primary antibody. 48 De novo OXTR expression surrounding grey and white matter microinfarctions Although we identified specific and significant regional increases in Oxtr gene and OXTR protein expression in frozen frontal cortex tissue samples from individuals who died with VaD, these experiments did not test the extent to which OXTR upregulation was associated with microvascular lesions. We hypothesized that a specific OXTR response to ischemic injury would be expected to peak in the penumbra region of an infarction and dissipate with distance. To test this possibility, we processed adjacent paraffin-embedded frontal cortex tissue sections from the same cases using hematoxylin and eosin staining to identify microinfarcts (Figure 2.7A and B) and IHC to label OXTR-expressing profiles (Figure 2.7C and D). A strong, de novo expression of the receptor was noted around sites of both white matter (Figure 2.7A and C) and grey matter (Figure 2.7B and D) infarcts. Notably, OXTR antibody labeling was observed in vessels, but also in cells resembling polarized astrocytes (Figure 2.7C and D). Subsequently, dual labelling for OXTR and the reactive astrocyte marker glial fibrillary acidic protein (GFAP) was performed on additional series of tissue. Around established microinfarcts (Figure 2.8A and B), we found prominent overlap of OXTR and GFAP labeling on both astrocytic and vascular profiles (Figure 2.8C and D), the latter likely reflecting astrocytic end-feet. Upon further examination, colocalization of OXTR and GFAP was at its highest in fields close to the infarction and minimal to nonexistent in fields remote from the infarction (Figure 2.9). 49 Figure 2.7. De novo peri-infarct expression of OXTR. A, B) H&E staining identifies both white (A) and grey (B) matter infarcts in paraffin embedded frontal cortex tissue from VaD cases. Shown at 4X magnification. C, D) OXTR IHC in adjacent sections revealed prominent expressions surrounding the white (C) and grey (D) matter microinfarcts. Shown at 4X (top) and 10X (bottom) magnification. The OXTR-immunoreactive profiles showed an astroglial typical morphology. 50 Figure 2.8. Peri-infarct astroglial and vascular expression of OXTR. A, B) Photomicrographs show white matter microinfarction via H&E staining at 4X (A) and 10X (B, panel A inset). C, D) Photomicrographs show dual-label IHC of OXTR (brown reaction product) and GFAP (black reaction product) at 4X (C) and 10X (D, panel C inset) in an adjacent section. 51 Figure 2.9. Gradient of OXTR expression in astrocytes and blood vessels relative to microinfarction site. A) Photomicrograph shows dual-label IHC of OXTR and GFAP from Figure 2.8 with numbered insets as shown in (B). B) OXTR expression is enriched in GFAP-immunopositive profiles in a field close to the microinfarction (3) and in the left part of fields (1) and (4) proximal to the lesion. By contrast, OXTR is minimal in GFAP-immunopositive profiles in a field farther from the microinfarction (2) and in the right part of fields (1) and (4) distal to the lesion. All fields shown at 20X. C) OXTR-expressing astrocytes are indicated with black arrows, whereas vessels labeled with OXTR are indicated with black arrowheads and OXTR-negative astrocytes are labeled with asterisks. All fields shown at 40X. D) Another field from the same section further demonstrates dual-labeled GFAP positive astrocytes and vessels expressing OXTR (40X). 52 OXTR Expressing Cells with Astrocytic Profiles Also Contain GFAP mRNA In order to further validate the presence of OXTR positive astrocytes within the penumbra of microinfarctions, we performed RNAscope combining GFAP mRNA probe amplification with OXTR IHC. OXTR was detected in both vascular profiles and strongly arborous cells, which also exhibited the presence of GFAP mRNA (Figure 2.10A). While the GFAP mRNA signal remained with omission of the OXTR primary, the vessels and arborous limbs of the cells did not, supporting the concept that de novo expression of OXTR occurs within parenchymal microvessels and astroglia at the sight of microinfarction (Figure 2.10B). Figure 2.10. RNAscope detection of GFAP mRNA in profiles expressing OXTR. A) Photomicrograph shows OXTR IHC (blue-black reaction product) combined with GFAP mRNA amplification via RNAscope (brown reaction product) in a per-infarct field (20X). Insets 1 and 2 are shown at 40X magnification. B) GFAP mRNA labeling in the absence of OXTR primary antibody validates specificity of co-expression at 20X (left) and 40X (right, insert). 53 Astrocytes in Culture Exposed to Hypoxia and Reperfusion Upregulation OXTR mRNA Astrocytic cells isolated and immortalized from neonatal rat cortex were used for additional validation of OXTR upregulation in response to hypoxic insults and to examine the time course. The cells chosen maintained the expression of GFAP with a fibroblast-like appearance and express the OXTR endogenously (Figure 2.11A). At early time points, 15 minutes (Mann-Whitney [n1=1,38, n2=6.967, U=5, p=0.0823]) and 1 hour (Mann-Whitney [n1=0.6363, n2=1.58, U=13, p=0.7922]), no significant difference was observed for the OXTR mRNA between hypoxic and normoxic astrocytes (Figure 2.11B and C). A significant upregulation of the OXTR in the hypoxic astrocytes was observed starting at 2 hours (Mann- Whitney [n1=0.9211, n2=10.59, U=1, p=0.0043]) and remained elevated at 6 hours (Mann-Whitney [n1=0.9862, n2=5.867, U=0, p=0.0022]) and 12 hours (Mann-Whitney [n1=1.125, n2=6.329, U=0, p=0.0022]) (Figure 2.11D-F) 54 Figure 2.11. Rat astrocytes in culture upregulate the OXTR in response to hypoxia. A) Rat astrocytes in culture express OXTR and maintain their expression of astrocytic marker GFAP. B) At 15 minutes of hypoxia and reperfusion there was no difference in normoxic versus hypoxic astrocytes in expression of OXTR (Mann-Whitney [n1=1,38, n2=6.967, U=5, p=0.0823]). C) At 1 hour of hypoxia and reperfusion there was no difference in normoxic versus hypoxic astrocytes in expression of OXTR (Mann-Whitney [n1=0.6363, n2=1.58, U=13, p=0.7922]). D) At 2 hours of hypoxia and reperfusion there was significantly more OXTR mRNA in hypoxic versus normoxic astrocytes (Mann-Whitney [n1=0.9211, n2=10.59, U=1, p=0.0043]). E) At 6 hours of hypoxia and reperfusion there was significantly more OXTR mRNA in hypoxic 55 Figure 2.11 (cont’d) versus normoxic astrocytes (Mann-Whitney [n1=0.9862, n2=5.867, U=0, p=0.0022]). F) At 12 hours of hypoxia and reperfusion there was significantly more OXTR mRNA in hypoxic versus normoxic astrocytes (Mann-Whitney [n1=1.125, n2=6.329, U=0, p=0.0022]). 56 Discussion Currently, there is no FDA approved therapy for VaD and only rapid thrombolysis is indicated for acute ischemic stroke (Tsivgoulis et al., 2015). Failures to find interventions of significant benefit in experimental and clinical studies of VaD have been partially attributed to a lack of emphasis on probable VaD (Herrmann, Lanctôt, & Hogan, 2013; Knopman et al., 2003; Perry, Ziabreva, Perry, Aarsland, & Ballard, 2005). This study was designed to identify gene expression changes reflecting putative pathologic or protective mechanisms unique to VaD that might be amenable to therapy. Using microarray analysis to quantify differences in gene expression patterns and highly connected networks among individuals who died with VaD, AD or no dementia, we identified and prioritized several dysregulated genes of interest for further validation including Rac1, Crebbp, Oxtr, Opah, and Spon2. To our knowledge, this is the first microarray study comparing cortical gene expression profiles among VaD, AD and CTL subjects. The main finding of this study is the novel observation via independent, complementary methods that Oxtr gene expression is differentially upregulated in VaD and appears to manifest as de novo, peri-infarct OXTR expression localized to cerebrovascular endothelial cells and reactive astroglia surrounding microinfarctions. Given the multiple lines of evidence supporting a protective role for OXTR signaling in peripheral infarction (Düşünceli et al., 2008; Jankowski et al., 2010; Tuǧtepe et al., 2007), as well as studies suggesting that OXT may protect against cerebral damage in stroke in both rats (Karelina et al., 2012) and postpartum women (Seo et al., 2018), we posit that this phenomenon is a compensatory response to rescue the ischemic penumbra and that manipulations of OXTR signaling may provide a therapeutic benefit in VaD and perhaps mixed dementia, as well. The OXTR is a G-protein coupled receptor linked to Gαq/11 -mediated activation of phospholipase C (PLC), which in turn activates a variety of calcium-dependent intracellular signaling pathways through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) (Zingg & Laporte, 57 2003). Its endogenous ligand OXT is a neuropeptide synthesized primarily in the paraventricular nucleus and supraoptic nucleus of the hypothalamus (Gimpl & Fahrenholz, 2001). Fiber projections to the posterior pituitary allows for the centrally produced OXT to be stored and released into the general circulation (Gimpl & Fahrenholz, 2001). The activation of OXT signaling has been routinely studied for its role in social affiliation and reproduction (Argiolas, Melis, & Gessa, 1986; Bale, Davis, Auger, Dorsa, & McCarthy, 2001; Insel & Shapiro, 1992; Kosfeld, Heinrichs, Zak, Fischbacher, & Fehr, 2005), as well as uterine contractions (Akerlund et al., 1987; Caldeyro-Barcia & Poseiro, 1959) during labor and milk ejection during nursing (Newton & Newton, 1948; Nishimori et al., 1996). In fact, the OXTR antagonist Atosiban is effective in halting premature labor (Akerlund et al., 1987), and OXTR agonists such as Carbetocin can be used to induce labor and minimize the incidence of postpartum hemorrhage (Phaneuf, Rodríguez Liñares, TambyRaja, MacKenzie, & López Bernal, 2000). Intranasal OXT has been found to increase the likelihood of adapting to the opinions of one’s in-group when presented with diverging opinions of an in-group and out-group member and thus has been closely tied to social conformity (Stallen, De Dreu, Shalvi, Smidts, & Sanfey, 2012). In contrast, far less is known about the vascular effects of OXTR signaling, but the reported effects of OXT signaling during experimental peripheral infarction (Jankowski et al., 2010; Kobayashi et al., 2009) suggest a striking propensity to counter many of the deleterious mechanisms of the ischemic cascade in stroke. In this regard, while the current study does not address the functional consequences of increased OXTR expression surrounding infarction, an examination of the available literature supports a protective role for OXTR upregulation in VaD. The ischemic cascade comprises an array of negative consequences following oxygen and glucose deprivation that leads to apoptotic tissue death. These consequences include NADPH oxidase production of superoxide free radicals (Manzanero, Santro, & Arumugam, 2013), induction of pro-inflammatory cytokines leading to immune cell activation and adhesion (Siniscalchi et al., 2014), endothelial disruption weakening the blood brain barrier (Khatri, 58 McKinney, Swenson, & Janardhan, 2012), and disturbances in nitric oxide synthase (NOS) isoforms resulting in toxic uncoupling by inducible NOS (Rodrigo et al., 2013) and impairment of pro-angiogenic endothelial NOS (eNOS) (Poittevin et al., 2015). Significantly, several studies have suggested an ability of OXTR signaling to modify several of these aspects of the ischemic cascade that might ultimately prove beneficial in preventing cognitive impairment due to strokes. For instance, OXTR stimulation by OXT was shown to decrease NADPH oxidase-mediated superoxide production in endothelial cells by 24-48% at baseline and by ~40% upon tumor necrosis factor (TNF) stimulation (Szeto et al., 2008). Due to its ability to attenuate oxidative stress in the vasculature, OXT has been linked to the noted protective effect of social affiliation in atherosclerosis-prone animal models as a probable physiological intermediator (Szeto et al., 2013). Moreover, Jankowski and colleagues found that OXT treatments after surgical induction of myocardial infarction in rats led to a significant decrease in pro-inflammatory cytokines (e.g., IL-6 and TNFα), and an increase in anti-inflammatory cytokines (e.g., IL-10) (Jankowski et al., 2010). This ability to regulate the inflammatory environment may explain promising results that led to previous interest in OXT as a pro- survival factor in tissue grafts (Petersson et al., 1998). OXTR activation is also known to contribute to eNOS phosphorylation via the activation of the phosphatidylinositol-3-kinase(PI3-K)/AKT pathway, which stimulates cell migration and angiogenesis to promote reperfusion (Cattaneo et al., 2008). By contrast, the noted ability of OXTR activation to stimulate tube formation in endothelial cell cultures at a similar rate to that experienced with vascular endothelial growth factor has led to suggestions that its repression may be a viable method to control aberrant angiogenesis such as occurs in endometriosis (Cattaneo et al., 2009). A final putative, protective consequence of OXTR activation in the ischemic penumbra is its ability to induce proteolytic shedding of full-length receptor for advanced glycation end- products (RAGE) up to 5-fold over basal turnover through PLC and PI3-K controlled mechanisms (Metz, Kojro, Rat, & Postina, 2012). This induction of RAGE shedding has attracted interest as a means to 59 prevent diabetic complications by limiting the number of receptors for AGEs to bind to and provoke inflammation and oxidative stress particularly in an early metabolic syndrome stage (Koyama et al., 2005). We identified astrocytes and the cerebrovascular endothelium as the likely cellular sources of de novo OXTR upregulation surrounding ischemic injury. This is intriguing as both cell types have attracted attention as mediators of acute and chronic responses to ischemia. The endothelium, or the innermost monolayer of endothelial cells comprising the first line of the blood brain barrier in cerebral blood vessels, has noted vasoactive and immunity mediating responses to ischemic damage (Hiroi et al., 2018; Laufs et al., 2002). The vasodilation of blood vessels due to enhanced eNOS activity promotes tissue preservation in ischemic injury and has been suggested to be a secondary means through which statins protect against cerebral ischemia (Hiroi et al., 2018; Laufs et al., 2002). In the acute phases of hypoxia, the endothelium produces cytokines and reactive oxygen species similar to innate immune cells (Kahles et al., 2007; Mai, Virtue, Shen, Wang, & Yang, 2013; O’Carroll et al., 2015; Taniyama & Griendling, 2003). Basal levels of cytokines are often minimal in endothelium, but in response to hypoxic signals they can adopt either a pro-inflammatory ( IL-1α, IL-1β, IL-6, TNFα) or anti-inflammatory (IL-10, transforming growth factor β (TGFβ) phenotype (Mai et al., 2013). The environmental conditions, particularly transmitters and cytokines secreted by other cells, have a vital role in driving endothelial cell phenotype (O’Carroll et al., 2015). Reactive oxygen species, particularly the superoxide anion O2-, are produced by both the NAPDH oxidase enzyme and uncoupled NOS in dysfunctional endothelial cells and further damages the blood brain barrier in cerebral vascular disorders (Kahles et al., 2007; Taniyama & Griendling, 2003). In later stages of post-hypoxic injury, the endothelium can take an important reparative role through angiogenesis and the secretion of neurotrophic factors, which re-establishes blood perfusion and supports neurogenesis to restore damaged tissues (Krupinski et al., 1994; Qin et al., 60 2014; Ruan, Wang, Zhuge, & Jin, 2015). Hence, OXTR upregulation and signaling in the cerebrovasculature may provide a mechanism for rescuing these deleterious pathways following stroke. Astrocytes also play a role in the acute and chronic stages of post-ischemia. Although there is less evidence for potential OXTR signaling consequences in astrocytes versus endothelial cells, astrocytes have been shown to express the OXTR (Di Scala-Guenot & Strosser, 1992; Mittaud et al., 2002), and it is exciting to speculate on OXTR function given canonical astrocyte-mediated pathways in hypoxic conditions. The most commonly known aspect of astrocytic intervention in cerebral injury is the formation of a glial scar through the interaction of astrocytic processes produced by intermediate filaments with the extracellular matrix to isolate the lesion from salvageable tissue and sometimes provide paths for neuroblasts to restore vital circuits (Cekanaviciute & Buckwalter, 2016; Pekny et al., 2016; Roy Choudhury & Ding, 2016; Ruan et al., 2015). Astrocytes also act as inflammatory mediators in hypoxia, producing cytokines and ROS through both a p38 mitogen-activated protein kinase pathway and nuclear factor-kappa β (NF-kβ) (Roy Choudhury & Ding, 2016). Importantly, astrocytes can adopt either pro-inflammatory (NF-kβ) or anti-inflammatory (gp130) responses (Cekanaviciute & Buckwalter, 2016). Given the anti-inflammatory signaling profile for OXTR described above, we posit that de novo OXTR expression in peri-infarct reactive astrocytes supports neuroprotection. Moreover, in a more acute protective role, astrocytes can limit excitotoxicity through increased glutamate uptake and metabolism via TGFβ activation (Li et al., 2008; Choudhury & Ding, 2016), which leads to an increase in glutamate transporter 1 (Li et al., 2008) and the glutamate-aspartate transporter (Choudhury & Ding, 2016). In contrast, we cannot rule out the possibility that de novo expression of the OXTR in astrocytes and the endothelium in the wake of ischemia is deleterious. For instance, it has been suggested that OXT enhances proliferation of endothelial cells (Cattaneo et al., 2008), yet this also includes an increase in matrix metalloproteinases to degrade extracellular matrix proteins (Kobayashi et al., 2009). While 61 necessary for angiogenesis, the role of these enzymes in infarction has also raised concerns about hemorrhagic transformation due to blood brain barrier degradation (Macrez et al., 2011; Montaner et al., 2003; Rosell & Lo, 2008) and destructive immune responses (Amantea et al., 2015; Worthmann et al., 2010). Hence, OXTR signaling in endothelial cells in the proximity of the ischemic lesion could lead to expansion of the lesion rather than reduction, especially after the therapeutic administration of tissue plasminogen activator (Montaner et al., 2003). Moreover, while reactive astrocytes can have a neuroprotective role in cerebral ischemia, there are also opposing mechanisms by which reactive astrocytes can lead to expansion of the lesion (Matsui et al., 2002; Tateishi et al., 2002), or hinder plasticity during recovery (Galtrey & Fawcett, 2007; Yiu & He, 2006). Reactive astrocytes can interdigitate and express inhibitory molecules like chondroitin sulfate proteoglycans that restrict axonal regeneration (Galtrey & Fawcett, 2007). They can also contribute to oxidative stress and inflammation through inducible nitric oxide synthase (Matsui et al., 2002) and the transcription factor NF-kβ (Zhang et al., 2013). Like many ischemic responses, controversy exists as to whether signaling of the endothelium and activated astrocytes ultimately fall under neuroprotective or neurotoxic (Caleo, 2015; De Pablo, Nilsson, Pekna, & Pekny, 2013). Ultimately, timing, degree of injury, and the presence of multiple modulators in the tissue likely contribute to the sum effect (Pekny, Wilhelmsson, & Pekna, 2014; Choudhury & Ding, 2016). Further research on the pathways affected by the OXTR on endothelium and astrocytes under hypoxic and metabolic stress is necessary to support or refute our belief hypotheses that peri-infarct expression of OXTRs represents a compensatory response. In summary, we have identified OXTR upregulation as a novel, potentially protective phenomenon associated with microinfarction lesions, which may modify many of the negative outcomes associated pathways of the ischemic cascade though increased OXT signaling. Moreover, the persistence of peri-infarct OXTR expression in vessels and astrocytes at autopsy suggests that this phenomenon is a protracted rather than acute response to injury with a wide therapeutic window for target engagement. 62 This is particularly compelling given the safety profile of its centrally acting properties in clinical settings and the availability of FDA-approved OXTR ligands (Finger et al., 2015; Tachibana et al., 2013). Significantly, our results may have therapeutic implications for stroke recovery irrespective of putative cognitive outcomes, as well as for mixed AD dementia. A reduction in ROS production by NADPH oxidase could reduce the oxidation of DNA in impacted regions and enhance the percentage of newborn neurons surviving in the environmentally harsh penumbra (Choi et al., 2015). Increased eNOS production of nitric oxide by OXTR signaling could contribute to angiogenic vessel remodeling after a stroke to restore sufficient blood flow to the penumbra region (Bir et al., 2012). A reduction in pro- inflammatory cytokines in favor of anti-inflammatory cytokines, and initiation of full-length RAGE shedding could limit the activation of innate immune cells and their extravasation through the vessel wall into the brain parenchyma where they can produce cytotoxic and self-reactive damage (Chamorro et al., 2012). Future studies will dissect the precise intracellular pathways mediated by astroglial and endothelial OXTR that might represent a novel protective mechanism unique to vascular dementia with translational potential. 63 CHAPTER 3: The effects of viral vector-mediated upregulation of the oxytocin receptor in a rat model of vascular cognitive impairment Introduction Ischemic stroke is the most common type of stroke estimated to account for somewhere between three quarters to nearly 90% of all recorded strokes (Shiber, Fontane, & Adewale, 2010). Upon survival of a stroke many patients are left with residual deficits that hinder daily functioning and can lead to the loss of independence. In fact, it is estimated that at least a third of stroke survivors are left with permanent disability (Hankey, Jamrozik, Broadhurst, Forbes, & Anderson, 2002). This high prevalence has galvanized clinical, research, and patient advocacy groups around the critical need to reduce the incidence of stroke and decrease their associated disabilities. For many years, the prominent deficits noted and targeted for therapy and recovery beyond survival were motor deficits (Bernhardt et al., 2017; Cumming et al., 2011; Dobkin, 2004; Wolf et al., 2006). Despite centuries-old work linking cerebrovascular disease and stroke to subsequent dementia by Binswanger (Binswanger, 1894) and Thomas Willis (Román, 2003; Willis, 1672) it was not until landmark autopsy (Hachinski et al., 1974) and longitudinal studies (Snowdon et al., 1997) that ischemic infarcts were linked to the emergence of dementia. Since then, the recognition of vascular dementia (VaD) (Gorelick et al., 2016; Hachinski, 1990; Iadecola, 2013) and the broader term, vascular cognitive impairment (VCI), for cognitive impairment in the absence of memory impairment (O’Brien, 2006; O’Brien et al., 2003) have inspired renewed interest in stroke intervention strategies focused on cognitive rescue. Much of this work has focused on the aftermath of an ischemic injury and the salvaging of the partially perfused penumbra region (Bastianello, Del Sole, Bosone, Cavailini, & Nappi, 2006; Liu, 2012; Manning et al., 2014). Often the cell death in the penumbra versus the core differ in their primary pathways and the temporal resolution (Chavez, Hurko, Barone, & Feuerstein, 2009; Hakim, 1998). As 64 such, the penumbra area can survive for much longer than the core and ultimately succumbs based on the overall tissue environment assuming reperfusion occurs (Felberg et al., 2000; Liu, Yuan, Yuan, & Yang, 2012; Siniscalchi et al., 2014). The inflammatory profile and degree of oxidative stress are among the main determinants of this cell survival (Horváth et al., 2018; Lambertsen, Biber, & Finsen, 2012; Ramos-Cabrer, Campos, Sobrino, & Castillo, 2011). Therefore, finding a therapy that can encourage an anti-inflammatory profile and lower oxidative stress in the penumbra region might result in less tissue loss and lower the chance of cognitive decline after an ischemic injury. We previously identified a novel upregulation of the oxytocin receptor (OXTR) on astrocytes surrounding infarcts in identified VaD cases (McKay et al., 2019). The OXTR is a G protein-coupled receptor that responds to the nonapeptide oxytocin (OXT) and the similarly structured vasopressin, albeit with less affinity (Gimpl, Fahrenholz, & Gene, 2001; Jurek & Neumann, 2018; Zingg & Laporte, 2003). Notably several lines of evidence suggest that OXTR signaling has protective effects against ischemic injury, including studies showing that: 1) the receptor reduces infarct size in peripheral ischemic injury models (Düşünceli et al., 2008; Gonzalez-Reyes et al., 2015; Jankowski et al., 2010; Tuǧtepe et al., 2007), 2) OXTR activity modifies deleterious pathways like oxidative stress and inflammation following ischemia (Gutkowska & Jankowski, 2012; Jankowski et al., 2010; Szeto et al., 2008, 2013), 3) OXT administration is protective in animal stroke models (Moghadam et al., 2018; Kaneko et al., 2016; Karelina et al., 2012), and 4) a single nucleotide polymorphism in the receptor is linked to increased risk for VaD (Kim, Park, & Lee, 2009). These results and a consideration of the literature led us to consider whether the upregulation of the OXTR on peri-infarct astrocytes was a compensatory response that could be leveraged by manipulating receptor expression prior to an ischemic injury in order to reduce the lesion size through modification of the deleterious ischemic cascade, and prevent the emergence of cognitive deficits. 65 An ischemic injury results in a rapid response of central and peripheral immune cells, which are major contributors to the cytokines and reactive oxygen species that drive the ischemic cascade (Jones et al., 2018; Offner et al., 2006). Among these are astrocytes, which become reactive and change their morphology and function (Chen & Swanson, 2003; Panickar & Noremberg, 2005). Astrocytes are supportive glial cells with long branched processes that envelop both synapses and blood vessels, making them an integral part of the neurovascular unit and tripartite synapse (Sofroniew & Vinters, 2010). Additionally, one of the primary responses of the reactive astrocytes is to form a glial scar, where these cells interact with each other and other cell types to isolate and contain the core area of ischemic cell death and prevent the spread of toxins and pro-apoptotic signals (Anderson, Blomstrand, Blomstrand, Eriksson, & Nilsson, 2003; Huang et al., 2014). A hallmark of these reactive astrocytes is the upregulation of intermediate filaments like glial fibrillary acid protein (GFAP), nestin, and vimentin (Anderson et al., 2003; Duggal, Schmidt-Kastner, & Hakim, 1997; Kindy, Bhat, & Bhat, 1992). Astrocytes release multiple substances in response to ischemia and some are deleterious or supportive with respect to cell survival. Astrocytes are capable of releasing both pro- and anti- inflammatory cytokines (Kim, Min, Seol, Jou, & Joe, 2010; Ting Lau & Cheung-Hoi Yu, 2001) and reactive oxygen species (Barreto, White, Ouyang, Xu, & Giffard, 2012; Ouyang, Voloboueva, Xu, & Giffard, 2007), which contribute both individually to the toxic post-stroke environment and cross-cellularly through the induction of similar responses in neighboring cells like microglia (Cekanaviciute & Buckwalter, 2016; Ma, Wang, Wang, & Yang, 2017), as well as the attraction of invading peripheral immune cells (Che, Ye, Panga, Wu, & Yang, 2001; Panenka et al., 2018). As the previous research mentioned above suggests a convergence of OXTR signaling on these responses, it is possible that the upregulation of the receptor on astroglia could be a protective influence in driving astrocytes towards expressing and promoting the expression of more pro-survival mediators than deleterious molecules. 66 We hypothesized that upregulation of the OXTR in astrocytes in the immediate vicinity of an acute ischemic injury would reduce the subsequent tissue loss and prevent cognitive deterioration through inducing anti-inflammatory and antioxidant responses. To test this hypothesis, we used the spontaneous hypertensive stroke prone rat (Okamoto & Aoki, 1963; Saito et al., 1995), a model with a pre-existing comorbidity common in human patients, hypertension. This strain of rat was chosen due to the high rate of hypertension in human patients prior to stroke as discussed in the introduction. It is conceivable that the chronic experience of hypertension on blood vessels and the subsequent response could affect therapeutic interventions and is considered in our preclinical model using these rats. The OXTR or control green fluorescent protein (GFP) was introduced to astrocytes near the identified site of a subsequent, targeted ischemic lesion using the adeno-associated virus, AAV6, which is a good option for rodent astrocyte transduction under the control of the GFAP promoter (Schober et al., 2016). We induced a focal ischemic lesion linked to the development of executive dysfunction, the most common cognitive deficit encountered post-stroke (Moorhouse et al., 2010; Royall & Roman, 2000), by administering the potent vasoconstrictor endothelin-1 (Cordova, Jackson, Langdon, Hewlett, & Corbett, 2014; Déziel, Ryan, & Tasker, 2015; Déziel & Tasker, 2017) into the prefrontal cortex. Animals were assessed for behavioral functioning after their ischemic injury and the tissue damage was quantified. A separate experiment with a within-animal design was used in addition to examine and quantify the consequence of the astrocytic OXTR upregulation on several stroke-related cytokines and reactive oxygen species generation at 24 hours, when the most overlap of expression was expected (Abe et al., 2015; Clark et al., 1994; Perera et al., 2006; Yang et al., 2019). These studies were intended to examine the therapeutic potential for targeting astrocytic OXTR in addition to reperfusion strategies and inquire as to the underlying in-vivo response of astrocytes to this manipulation. 67 Methods Animals 3-4 month old stroke prone spontaneous hypertensive rats (n=96, 36 females, 60 males) were used for this study. 36 females and 40 males were used for Experiment 1 (Figure 3.1). Experiment 1 was performed in 5 cohorts to enable behavioral testing on such a large number of animals. 20 males were used for Experiment 2 (Figure 3.2). This age was chosen to result in the rats being 4-5 months old at the time of the endothelin-1 surgeries after their hypertension has steadied (Yamori & Horie, 1977), while remaining unlikely to have had an independent ischemic injury at the time of the behavioral testing (Fredriksson, Nordborg, Kalimo, Olsson, & Johansson, 1988). The rats were kept on a reverse light cycle (12pm-12am) and given access to food and water ad libitum. 68 Figure 3.1. Schematic of Experiment 1 workflow. 76 rats of each were randomly and evenly separated into two different viral groups. Half of each group received endothelin-1 and half received saline in equal volumes. They were put through four behavior tests to assess functioning. Pathological stains were used to confirm virus presence and assess tissue damage. 69 Figure 3.2. Schematic of Experiment 2 workflow. 20 male SHRSP rats were bilaterally injected with the AAV6-OXTR virus on the left and the AAV6-GFP virus on the right. Half of this group received endothelin- 1 and half received saline in equal volumes. After 24 four hours the brains were retrieved and frozen. 70 Figure 3.2 (cont’d) Tissue punches around the infarct were used for qPCR-based cytokine analysis and 10mm sections were taken for DHE-based ROS detection. 71 Production of AAV6-GFAP-OXTR and AAV6-GFAP-GFP The GFP and OXTR constructs were a generous gift of the Manfredsson lab. A heat shock method was used for transfection into Sure 2 competent cells and grown at 30°C on LB with AMP antibiotics. Colonies for individual clones were selected and put through mini-prep kits. Inserts were verified for sequence and orientation via restriction enzyme assay on agarose gel. HEK 293T were grown in DMEM with 5% fetal bovine serum and 1% penicillin/streptomycin. until the transfection was to begin. The cells were then plated on a 15cm culture dish and supplemented with the same media. In separate experiments, when 80% confluence was reached, in separate experiments, ptr-MS plasmids containing either a construct for GFP or the rat OXTR along with the sequence for the rat GFAP promoter obtained through IDT gBlocks were transfected with pXX6 helper plasmid and rAAV6 plasmid containing the capsid and replication site (a generous gift of the Manfredsson lab) and incubated for 72 hours. Afterwards, cell lysis was performed using the freeze- thaw alternative method, followed by the removal of the media, collection with trypsin, and centrifugation to collect the supernatants containing the two viral constructs. These were purified using iodixanol gradient and chromatography. The titers of the viruses were obtained using qPCR. The completed viruses were stored at 4 degrees Celsius and sealed with parafilm until they were ready to be injected. A more detailed overview of the virus preparation methods can be found in recently published work by Dr. Manfredsson’s lab (Sandoval, Kuhn, & Manfredsson, 2018). Experiment 1 Virus Surgeries Rats were anaesthetized with 3% isoflurane and 22µL of Rimadyl (carprofen) (recommended 4.4mg/kg from 50mg/mL stock for a 250gram rat) was added to Ringer’s solution to a total volume of 72 1mL administered subcutaneously to manage postoperative pain and continued for two days afterwards. They were immobilized in a stereotaxic device after having the fur removed from the surgical site and cleansed with 70% ethanol. Lubricant eye gel was dotted onto the eyes and lidocaine was applied to the wound as a local anesthetic. A circulating water warming pad was used during surgery and recovery to prevent hypothermia. Stereotactic coordinates for viral injections into prefrontal cortex were as follows: A-P:+2.5, M-L:±0.6, D-V:-4.0 and A-P:+1.5, M-L:±0.6, D-V:-4.0. These were based on previously established coordinates and adjusted to match our rats (Cordova et al., 2014). Needles for the viral injections were made from siliconized microcapillaries attached to a 10mL Hamilton syringe. Needles were flushed with distilled water and 3% hydrogen peroxide between each injection. 1µL at a titer of 1xE12 for each virus was infused for each of the four sites at a rate of 0.250µL per minute and rested for 5 minutes. After the last injection, the wound was cleaned with betadine and closed with wound clips before the rat was removed from the stereotaxic stage and placed in a bin with a circulating water pad covered with a bench pad. The rat was observed and not returned to its cage until it was ambulatory. No rats were lost during the virus surgeries. Endothelin-1 Surgeries Identical pre- and post-surgical procedures as those for the virus surgeries were used. Due to the supposed protective effect of the hormones estrogen and progesterone in stroke models (Gibson, Gray, Murphy, & Bath, 2006; Roof & Hall, 2000), we chose to check the female rats’ estrous cycle before this surgery to account for this difference within the females. Using a lavage technique as described previously for mice (McLean, Valenzuela, Fai, & Bennett, 2012), we did not perform surgery on any rats that were in estrous or proestrous. These surgeries were performed on the same rats three weeks after the virus surgeries to allow for sufficient expression of the gene products. Endothelin-1 (Tocris, 100µg) was reconstituted with 100 μL distilled water. A different Hamilton needle without the microcapillary 73 tube was used for the injection of endothelin-1 at a volume of 1μL per site using the same burr holes art the same sites: A-P:+2.5, M-L:±0.6, D-V:-4.0 and A-P:+1.5, M-L:±0.6, D-V:-4.0. The rate of infusion was 0.500 µL per minute and the needle was left in place for 5 minutes before it was retrieved. Eight rats were lost due to the endothelin-1 surgeries they consisted of four GFP animals and four OXTR animals, six of which were females and two males. Open Field Test During the dark active phase, the rats were retrieved from the colony room and allowed to acclimate in the behavior testing room for 1 hour before testing was to begin. An opaque black open field box (Stoelting) having the total dimensions of 90 x 90 x 40 cm with four dividers was used for this task. Before and between each rat the box was cleaned with an alcohol-based wipe. Each rat was allowed to explore the box for an hour while their movement, distance, and immobile episodes were tracked by the Anymaze software and video was recorded. Due to concerns about anxiety-related movement influencing the initial half hour of the test statistics were performed using data from the last half hour of the test which is more reflective of spontaneous activity (Gould, Dao, & Kovacsics, 2009). Novel Object Task Each day the animals were given one hour to acclimate to the room before testing began. The same opaque box was used for the novel object task as for the open field task. For two days the rats were given fifteen minutes to further explore the empty box to extinguish the novelty of the environment. On the third day the rats were given fifteen minutes to explore the box while two red plastic cubes were present in opposite corners. After an hour the rats were again allowed to explore the box with the objects changed to one of the previously encountered cubes and one novel green pyramid. The interaction time of the rat with each object was scored based on how long the animal’s nose was in contact with the object or sniffing or pawing at the object within one-inch distance. To account for 74 differences in innate curiosity the score was determined as the time spent with one object divided by the time spent with the second object. On all days the box and objects on the final day were cleaned with an alcohol-based wipe before and between tests. Elevated Plus Maze An elevated plus maze with a central platform was used for the following experiment. The maze reached 50 cm off the floor. The rats were acclimated to the room for 1 hour before the test began. For each animal five minutes was given to explore the maze. At the start of each experiment the rat was placed facing one of the open arms in the direct center of the maze. The Anymaze software was used to record the full test and entry to an open arm was recorded only when all four feet crossed the edge of the closed arms and fully entered the open arms. Time spent in the open arms and the number of separate entries to the open arms was recorded. The maze was cleaned with an alcohol-based wipe before each animal’s test. Barnes Maze As in the previous tests the rats were retrieved and brought to the behavior room during their active phase and given an hour to acclimate to the room. The Barnes maze protocol used was adapted from a published protocol for a shortened Barnes maze (Attar et al., 2013), one that was previously used successfully in our lab (Kelly et al., 2019). The procedure lasted for five day and consisted of a habituation day, two training days, a day off, and a final probe day. A bright clamp light and a white noise effect were used to encourage the rats to enter the target hole. Once the escape pod beneath the target hole was entered these aversive stimuli were shut off. For every day, the surface of the maze, the cylinder and the escape pod were cleaned with ethanol-based wipes before each run. On the habituation day each rat was guided slowly to the target hole in a clear, plastic cylinder and given two minutes to enter the hole, if necessary, after two minutes the rat was coaxed in gently. Large laminated 75 shapes were present on the walls to aid in spatial navigation. On the first trial day the rats were given 2 minutes to explore the maze freely to find the target hole and escape after being placed on the maze in the cylinder with the sides covered until the start of the task. After 2 minutes if the rat had not found the target hole, the cylinder, now uncovered, was used to guide the rat to the escape hole as on the habituation day. On this first training day each rat underwent three trials in rotating groups of four. The second training day followed the protocol of the first, but with only two trials per rat. On the probe day the box attached to the target hole was removed, but the location of the target hole was not changed. Each rat was placed on the maze in a covered cylinder with the aversive stimuli enacted. Each rat was given two minutes to find the target hole. After two minutes the rat was retrieved from the surface of the maze and returned to the home cage. On the training and probe days the latency for the rat to find the target hole was recorded and the number of incorrect revisits to previously explored non-target holes was recorded. Anymaze software tracking the rat’s nose and body was used to record and score this task. Tissue Processing for Behavioral Rat Cohorts On the day following the last behavioral test the rats were euthanized with a lethal dose of FatalPlus that was diluted using saline to minimize discomfort. They were perfused for 18 minutes intracardially with ice cold 0.9% saline with anti-clotting heparin added at 10,000 units per liter. After decapitation by guillotine the brain was removed and post-fixed in 4% paraformaldehyde (diluted from 16%, Alfa Aesar) for 48 hours in 20mL scintillation vials. A sucrose gradient was used to reconstitute the brains: 15% sucrose for 24 hours, and 30% sucrose until the brains sank into the sucrose to the bottom of the vials. The brains were cut on a sledge microtome at a section thickness of 40 microns. The sections were dispersed into a 24 well culture dish (CytoOne) in serial sections and stored at -20°C preserved in a cryoprotectant (600g sucrose, 600mL ethylene glycol in 2L Tris-buffered saline). 76 Hematoxylin and Eosin Staining One well of tissue representing each animal was washed 6 times for 10 minutes each in Tris buffered saline (8.76g NaCl, 6.05g Tris, pH to 7.4 in distilled water, Cold Spring Harbor Protocols). The sections were mounted using the same buffer onto Histobond plus slides (VWR VistaVision) and allowed to dry overnight. The sections were placed in Harris Modified Hematoxylin (Thermo Scientific) for six minutes, washed in distilled water, dipped in 1% acid alcohol (hydrochloric acid in 70% ethanol), washed again in distilled water, and differentiated in ammonia water (1/100 dilution of 28% ammonium hydroxide in water) for 30 seconds, washed again, and transferred to the Eosin working solution (Fisher Scientific) for one minute. The slides were dehydrated in one minute each 70%, 95% and 100% ethanol, cleared in 2 changes of xylene and cover slipped with Cytoseal 60 mounting media. Luxol Fast Blue The same prewashing and mounting protocol was undertaken as for the H&E protocol. After drying overnight, the slides were placed in 1% luxol fast blue solution (1g luxol fast blue, 5mL glacial acetic acid in 1L 95% ethanol) overnight on a slow setting Orbi-Blotter. The following day, the slides were rinsed in 95% ethanol for 30 seconds followed by distilled water. A solution of 0.05% lithium carbonate in distilled water was used to differentiate the stain for 30 seconds followed by 70% ethanol for 30 seconds more. The slides were dipped in distilled water. The slides were returned to the lithium carbonate and the protocol from that point was repeated. Afterwards, the slides were taken from the distilled water wash step and dehydrated in 95% and then 100% ethanol for 1 minute each. The slides were submerged in two changes of xylene for 1 minute each and cover clipped with Cytoseal 60 mounting media. 77 Immunohistochemistry for OXTR and GFP One well for each animal was washed in tris buffered saline (pH 7.4) in 6 well plates with staining nets for four changes at ten minutes each. Antigen retrieval was achieved by heating 6 well plates with 5mL citric acid buffer (0.1 M, pH 6.0) to 65°C for 20 minutes. The sections were then washed by transferring through four changes of TBS. The peroxidase activity was blocked using 3% hydrogen peroxide (1:10 30% hydrogen in TBS). Another set of four washes with TBS containing 0.5% Triton X was followed by blocking with TBS containing 0.5% Triton X, 2% bovine serum albumin, and 10% normal goat serum for 1 hour, and incubated overnight at 4°C with either the OXTR antibody (1:500, Proteintech 23045-1-AP) or the GFP antibody (1:1000, Abcam ab290) in TBS/0.5% Triton X/1% normal goat serum. The specificity of the OXTR antibody was confirmed with a pre-absorption test (Figure 3.3) described in the next section and by an examination of the OXTR-rich hypothalamus of untreated male and female rats using the same procedure described in this section (Figure 3.4). After twenty-four hours the sections were washed with TBS containing 1% normal goat serum four times for ten minutes each and then incubated with biotinylated goat anti-rabbit secondary antibody (Vector Labs, 1:500) in the same wash buffer for one hour. The sections were then washed in TBS for four changes during which the Vectastain ABC solution was made and let sit. The sections were then transferred to the premade Vectastain ABC solution (Vector) for one hour. Once this was completed the slides were rinsed in TBS for four changes after which ImmPact DAB peroxidase substrate kit (Vector) was used for two minutes to visualize any detected OXTR or GFP. The sections were finally washed in TBS, mounted on Histobond plus slides and allowed to dry overnight. The following day they were cleared in xylenes, and cover slipped with Cytoseal 60 (Thermo Scientific). 78 Figure 3.3. Pre-absorption and primary delete western blots demonstrate specificity of antibody. A) Pre=absorption of antibody with 1:10 mass of recombinant protein and unabsorbed antibody show the ability of antibody to be competitively blocked by the protein. Protein presence confirmed with calnexin. B) Withholding of the primary antibody and addition of the antibody in parallel blots shows lack of nonspecific binding. Protein presence confirmed with calnexin. 79 Antibody Pre-absorption and Western Blot The OXTR antibody (Proteintech 23045-1-AP) was pre-absorbed using a recombinant oxytocin receptor protein (US Biological 156208) at a concentration difference of 1:100 overnight at 4°C. This was mixed in a block of 1x TBS with 0.5% Tween 20 and 5% dried milk. The concentration of OXTR antibody to block was 1:500. At the same time a mix of the antibody and block without the pre-absorption was prepared. The following day 5 samples of human frontal cortex tissue were run in duplicate. The samples at 30 µg were combined with 2x Laemmli buffer and sat at room temperature for 30 minutes. These were separated along with a Precision Plus dual colored ladder (Bio-Rad) for each set on a 4-20% PAGE gels (Criterion, Bio-Rad) for 25 minutes at 250 mV in run buffer (Bio-rad). The gels were transferred with cold buffer (Bio-rad) onto PVDF membranes after the activation of the membrane with methanol. This took place for 50 minutes at 400 mA. The same blocking solution for the antibody suspension, minus the antibodies, was used to block the membrane for 1 hour. Afterwards the two sets of the same protein samples were split by bisecting the membrane. One was incubated overnight with the pre-absorbed antibody mix and one with the antibody mix, as described in the previous paragraph. The following day the blots were washed first with the block for 10 minutes, then 1x TBS+Tween20 for 2 changes of 10 minutes each. The same secondary antibody, anti-Rabbit 680 (Li-Cor) was applied to both blots at a concentration of 1:10000 in the block for 2 hours covered. The blots were washed once in TBS with Tween20 and twice with TBS for 10 minutes each. The blots were imaged on an Odyssey scanner (LiCor) using a resolution of 169 µm and an intensity of 5.0 in the 700 nm channel. Afterward, the blots were tested for calnexin as a control protein. First, the blots were stripped using Restore western blot stripping buffer (Thermo Scientific) for 15 minutes at room temperature. After 80 washing with TBS the blots were re-blocked as before and the procedure proceeded as it had for the previous antibody test, this time using a rabbit anti-calnexin at 1:2000 (Proteintech 10427-2-AP). Figure 3.4. Tests of OXTR antibody reveal specificity for the oxytocin receptor. A) Male 1 year old SHRSP hypothalmus for a positive control. B) Male 1 year old SHRSP with the primary antibody withheld. 81 Figure 3.4 (cont’d) C) Female 1 year old SHRSP hypothalamus for a positive control. D) Female 1 year old SHRSP with the primary antibody withheld. 82 Experiment 2 Bilateral Virus Surgeries The pre- and post-operative care were the same for these rats as for those in Experiment 1. The rats received an injection of the AAV6 GFAP OXTR virus on the left side at a volume of 1µL at a titer of 1xE12 and the same volume and titer for the AAV6 GFAP GFP virus on the rats’ right side. The stereotactic coordinates of injection for the OXTR virus was: A-P:+2.5, M-L:+0.6, D-V:-4.0 and for the GFP virus was: A-P:+2.5, M-L:-0.6, D-V:-4.0. The precoating of the needles and washing in-between injections were performed as they were for Experiment 1. The infusion rate was 0.250µL per minute and the rest before removal of the needle was 5 minutes. No rats were lost during these virus surgeries. Endothelin-1 Surgeries for Bilateral Rat Cohort Pre- and post-operative care was performed as already described for the previous surgeries. Three weeks after the virus surgeries, half of the rats received an injection of saline and half received an injection of endothelin-1 (1µg/µL) into both of the previous coordinates: A-P:+2.5, M-L:±0.6, D-V:-4.0 at a volume of 1µL. The infusion rate was 0.5µL per minute and the needle was allowed to sit for 5 minutes before extraction. One of the rats receiving the endothelin-1 injection was lost during this surgery. Tissue Processing for Bilateral Rat Cohort Twenty-four hours after the previous surgery the rats were sacrificed via rapid decapitation and the brains were frozen using dry ice and ethanol. The brains were stored at -80°C wrapped in tin foil. The brains were retrieved and mounted on a chuck using Tissue Tek OCT Compound on dry ice. At -20°C the brains were cut in coronal sections on a cryostat until the site of injection and tissue damage was reached. Several frozen sections were cut 10µm and mounted to room temperature slides using static attraction. These sections were stored at -80°C for use in reactive oxygen species detection by 83 dihydroethidium. Four 1mm tissue punches were then taken from around the vicinity of the visible lesion or needle mark. Two were from the OXTR virus side and two from the GFP virus side. These were collected in 1.5mL Eppendorf tubes and stored at -80°C until they were to undergo RNA extraction for cytokine detection. RNA extraction, cDNA creation and qPCR for an array of cytokines The frozen tissue pellets were suspended in 500µL Trizol and sonicated for 5 short bursts on ice. Each was vortexed vigorously after 100µL of chloroform was added to each tube. After a short incubation for three minutes at room temperature the samples were centrifuged at 4°C for 10 minutes at 12,000rpm. The top layer supernatant was transferred to a clean tube for each sample and 250µL isopropanol plus 2µL GlycoBlu (Thermos Fisher) were mixed in through a short vortex. The samples were then centrifuged at 4°C for 20 minutes at 12,000rpm. The liquid was discarded, and the pellet was washed with 80% ethanol which was then also removed. The pellet was dried for 5 minutes and then resuspended and solubilized with the addition of 10µL nuclease-free water and exposure at 45°C for 5 minutes. RNA samples were stored at -80°C. A nanodrop was used to quantify and validate the purity of the RNA. The RNA samples were combined with an appropriate volume of nuclease-free water to reach 1µg for each sample. ThermoFisher’s RevertAid First Strand cDNA synthesis kit was used to generated cDNA for each sample. The cDNA was diluted in nuclease free water at 1:3 volume for the qPCR analysis. 4µL of this diluted cDNA was added in duplicate to a 96 well, opaque, skirted PCR plate. Different plates were run for a variety of molecule of interest using the Taqman platform, OXTR (Rn00563503_m1), tumor necrosis factor alpha (TNFα) (Rn01525859_g1), transforming growth factor beta (TGFβ) (Rn00572010_m1), interleukin-1β (Rn00580432_m1), and interleukin-10 (Rn00563409_m1). A probe for rat GAPDH (Rn01462662_g1) was multiplexed in each well for a control. The target gene probe in FAM 84 and the GAPDH probe in VIC were added to Taqman Universal Master Mix II no UNG at 1:10 for each. A volume of 6µL of this master mix was added to each well plus one blank. The plates were centrifuged for a short burst to ensure mixing and uniformity. An Applied Biosystems 7500 real time PCR system was used to detect the amplification over 40 cycles. The results were analyzed using 2-ΔΔCt method with Gapdh as the reference to establish a fold change for each product relative to the cycling times for the GFP Saline hemisphere samples. Dihydroethidium for Reactive Oxygen Species The protocol for detection of reactive oxygen species by the oxidation target dihydroethidium was adapted from previous work supporting its use in frozen aortic tissue (Wang & Zou, 2018). Due to the volatility of the staining, one slide at a time had to be taken through the entire process rather than processed all at once. The frozen sections were quickly dried to the slides after retrieval from the -80° freezer with a blow drier on a low speed, cold setting. The slides were then taken and submerged in the DHE solution (50mL of 5µM DHE in distilled water, Sigma Aldrich) for five minutes at room temperature while covered. The slides were washed twice in ddH2O and cover slipped with Vectashield containing DAPI and placed in a dark place. After fifteen minutes to let the media dry the slides were taken to a light microscope and imaged in the red and blue excitation filters at 4x and 10x magnifications. The images were opened in ImageJ, the channels were split, converted to 8 bit and the mean intensity of the red channel was taken as a measurement of the reactive oxygen species (O2 -) present in the tissue. Statistics In cases of four group comparisons either a one-way ANOVA or a Kruskal-Wallis test, if normality assumptions were failed, were used. Subsequent comparisons of the means of each column to the other were performed with multiple comparison test to obtain multiplicity adjusted p values. For the comparisons of the differences in infarction size, outcomes from the two endothelin-1 lesioned groups 85 (OXTR or GFP pretreatment) were normalized to the non-lesioned groups and an unpaired t-test was used. For Experiment 2, one-way ANOVAS were used. Results Experiment 1 Endothelin-1 Injured Rats in the Open Field Test There were no differences in the locomotor activity between the four treatment groups either in the total distance travelled (one-way ANOVA [F (3,64) =1.378, p=0.2574]) (Figure 3.5A) or the time the animals spent moving (one-way ANOVA [F (3,63) = 2.778, p=0.0484], Holm-Sidak's multiple comparisons test=ns) (Figure 3.5D). There were also no differences noted in these two measurements when the animals were separated by sex. The male (one-way ANOVA [F (3,33) =1.071, p=0.3746]) and female (one-way ANOVA [F (3,63) = 2.778, p=0.0484], Holm-Sidak's multiple comparisons test=ns) subgroups had no differences in distance travelled (Figure 3.5B and C). The separate male (Kruskal-Wallis test [H = 7.953, p = 0.0474], Dunn's multiple comparisons test=ns) and female (Kruskal-Wallis test [H=3.044, p=0.3849]) divisions also revealed no differences in time mobile (Figure 3.5E and F). These results suggest that the endothelin-1 surgeries did not induce significant mobility impairments in either male or female animals. Therefore, the results of subsequent tests can be considered without the confounding variable of a mobility impairment. 86 Figure 3.5. No difference was detected in time mobile or distance travelled in open field. A) Distance traveled in meters by all animals in the last half hour of an hour-long exploration (one-way ANOVA [F (3,64) =1.378, p=0.2574]). B) Distance traveled in meters by male animals in the last half hour of an hour-long exploration (one-way ANOVA [F (3,33) =1.071, p=0.3746]). C) Distance traveled in meters by female animals in the last half hour of an hour-long exploration (one-way ANOVA [F (3,27) =0.805, p=0.5021]). D) Time in seconds that animals were mobile in the last half hour of an hour-long exploration (one-way ANOVA [F (3,63) = 2.778, p=0.0484], Holm-Sidak's multiple comparisons test=ns). E) Time in seconds that male animals were mobile in the last half hour of an hour-long exploration (Kruskal-Wallis test [H = 7.953, p = 0.0474], Dunn's multiple comparisons test=ns). F) Time in seconds that female animals were mobile in the last half hour of an hour-long exploration (Kruskal-Wallis test [H=3.044, p=0.3849]). 87 Endothelin-1 Injured Rats with OXTR Upregulation Retain Novel Object Recognition Deficits in the recognition of a never-before encountered (i.e. novel) object versus a familiar object after a brief previous encounter and delay were induced by endothelin-1 injection (Dunn's multiple comparison's test [mean rank = 20.75, multiplicity-adjusted p = 0.0206]). This deficit was attenuated in endothelin-1 animals that were pretreated with the AAV6 GFAP OXTR virus (Dunn's multiple comparisons test [mean rank = -18.73, multiplicity-adjusted p = 0.0395]) (Figure 3.6D). Upon further investigation it was found that this preservation of object recognition memory was driven by and limited to the male animals (Tukey's multiple comparison's test [mean diff =-1.134, multiplicity-adjusted p = 0.0319) (Figure 3.6E) with no differences in the females (Kruskal-Wallis test [H=0.8067, p=0.8479]) (Figure 3.6F). In the previous trial when both objects presented were the same, there were no baseline differences in the tendency to explore one of the familiar objects versus another (Kruskal-Wallis test [H=3.134, p=0.3715]) (Figure 3.6A). This also held for the separation of the male animals (one-way ANOVA [F (3,34) =1.157, p=0.3405]) (Figure 3.6B) and the female animals (Kruskal-Wallis test [H=3.323, p=0.3445]) (Figure 3.6C). Thus, the preemptive upregulation of the OXTR in astrocytes in the vicinity of the ischemic injury appeared to rescue recognition and working memory for a briefly encountered familiar object, which is referable to prefrontal cortex function, in the male but not the female animals. 88 Figure 3.6. Novel Object Task shows deficits were induced by endothelin-1 injection and was partially rescued by OXTR upregulation in male animals. A) Time spent exploring one similar object versus the other for all animals (Kruskal-Wallis test [H=3.134, p=0.3715]) . B) Time spent exploring one similar object versus the other for male animals (one-way ANOVA [F(3,34)=1.157, p=0.3405]). C) Time spent exploring one similar object versus the other for female animals (Kruskal-Wallis test [H=3.323, p=0.3445]) . D) Time spent exploring the novel object versus the familiar one for all animals (Kruskal- Wallis test [H=10.63, p=0.0139]). GFP ET-1 animals were significantly impaired compared to GFP Saline animals (Dunn's multiple comparison's test [mean rank = 20.75 , multiplicity-adjusted p = 0.0206]). GFP ET-1 animals were significantly impaired compared to OXTR ET-1 animals (Dunn's multiple comparisons test [mean rank = -18.73, multiplicity-adjusted p = 0.0395]). E) Time spent exploring the novel object versus the familiar one for male animals (one-way ANOVA [F (3,34) = 4.285, p=0.0114]). GFP ET-1 animals were significantly impaired compared to GFP Saline animals (Tukey’s multiple comparison's test [mean diff = 1.328, multiplicity-adjusted p =0.0116). GFP ET-1 animals were significantly impaired compared to OXTR ET-1 animals (Tukey's multiple comparison's test [mean diff =-1.134, multiplicity- 89 Figure 3.6 (cont’d) adjusted p = 0.0319). F) Time spent exploring the novel object versus the familiar one for female animals (Kruskal-Wallis test [H=0.8067, p=0.8479]). 90 Endothelin-1 Injured Rats in the Elevated Plus Maze The GFP ET-1 animals spent more time in the open arms of the maze than the saline animals (Kruskal-Wallis test [H=12.14, p= 0.0069]), both in comparison to the GFP Saline animals (Dunn's multiple comparisons test [mean rank =-19.31, multiplicity-adjusted p = 0.0388]) and OXTR Saline animals (Dunn's multiple comparisons test [mean rank = 22.28, multiplicity-adjusted p = 0.0064]) (Figure 3.7A). In contrast to the previous test results this difference was not driven by the male animals (Kruskal-Wallis test [H=6.446, p=0.0918]) (Figure 3.7B), but by the female animals (one-way ANOVA [F (3,27) = 4.543, p=0.0106]) (Figure 3.7C). For the female animals, the GFP ET-1 group spent more time in the open arms of the maze than the GFP Saline (Holm-Sidak's multiple comparisons test [mean diff=- 129.9, multiplicity-adjusted p=0.0272), OXTR Saline (Holm-Sidak's multiple comparisons test [mean diff=133.6, multiplicity-adjusted p=0.0166), and OXTR ET-1 female groups (Holm-Sidak's multiple comparisons test [mean diff=116.3, multiplicity-adjusted p=0.0376) Alternatively, the difference in the entries into the open arms for all animals (Kruskal-Wallis test [H= 10.11, p=0.0177 ]) (Figure 3.7D), which upon multiple comparisons was attributable to GFP ET-1 animals entering the open arms significantly more often than the OXTR Saline animals (Dunn's multiple comparisons test [mean rank = 19.63, multiplicity-adjusted p=0.0223]), was maintained in the male animals (Kruskal-Wallis test [H=6.446, p=0.0918], Dunn's multiple comparisons test [mean rank = 14.67, multiplicity-adjusted p= 0.0217]) (Figure 3.7E), but not in the female animals (one-way ANOVA [F (3,27) = 2.454, p=0.0848]) (Figure 3.7F). It is possible that the ET-1 injection caused some sex-specific dysfunction in corticolimbic connectivity resulting in a reduction of anxiety-like behavior. Interpretation of other behavioral results should take this into account. 91 Figure 3.7. Elevated Plus Maze shows a tendency to enter more often and spend more time in the open arms was induced by endothelin-1 injection. A) Time spent in the open arms for all animals in seconds (Kruskal-Wallis test [H=12.14, p= 0.0069]). GFP-ET-1 animals spent significantly more time in the open arms relative to GFP Saline animals (Dunn's multiple comparisons test [mean rank =-19.31, multiplicity-adjusted p = 0.0388]) and OXTR Saline animals (Dunn's multiple comparisons test [mean rank = 22.28, multiplicity-adjusted p = 0.0064]). B) Time spent in the open arms for male animals in seconds (Kruskal-Wallis test [H=6.446, p=0.0918]). C) Time spent in the open arms for female animals in seconds (one-way ANOVA [F (3,27) = 4.543, p=0.0106]). GFP ET-1 females spent significantly more time in the open arms than GFP saline females (Holm-Sidak's multiple comparisons test [mean diff=-129.9, multiplicity-adjusted p=0.0272), OXTR Saline females (Holm-Sidak's multiple comparisons test [mean diff=133.6, multiplicity-adjusted p=0.0166), and OXTR ET-1 animals (Holm-Sidak's multiple comparisons test [mean diff=116.3, multiplicity-adjusted p=0.0376) D) Entries into the open arms for all animals (Kruskal-Wallis test [H= 10.11, p=0.0177 ]). GFP ET-1 animals entered the open arms significantly more often than the OXTR Saline animals (Dunn's multiple comparisons test [mean rank = 19.63, multiplicity- adjusted p=0.0223]). E) Entries into the open arms for male animals (Kruskal-Wallis test [H=6.446, 92 Figure 3.7 (cont’d) p=0.0918]). GFP ET-1 males entered the open arms significantly more often than the OXTR saline males (Dunn's multiple comparisons test [mean rank = 14.67, multiplicity-adjusted p= 0.0217]). F) Entries into the open arms for female animals (one-way ANOVA [F (3,27) = 2.454, p=0.0848]). 93 Endothelin-1 Injured Rats in the Barnes Maze No significant differences were observed on the Barnes maze. No difference was observed in the time it took for animals to find the target hole on the probe day (one-way ANOVA [F (3,64) = 0.974, p=0.4106]) (Figure 3.8D). However, all animals were able to improve their time to find the target hole over the course of the five training days indicating they were able to learn (Figure 3.9A and B and C). This lack of significant difference held when examining the male (Kruskal-Wallis test [H = 0.8131, p= 0.8463]) (Figure 3.8E) and female animals (one-way ANOVA [F (3,27) = 0.3229, p=0.8087]) separately (Figure 3.8F), ruling out any sex difference obscuring a difference. Moreover, there were no significant difference in the number of incorrect revisits to nontarget holes for any animal groups (one-way ANOVA [F (3,64) = 0.9907, p= 0.4029]) (Figure 3.8G). Again, like the latency trial, no sex difference was found among this data as neither the male (one-way ANOVA [F (3,34) = 1.089, p= 0.3670]) (Figure 3.8H) nor female animals (one-way ANOVA [F (3,26) = 0.3031, p= 0.8229]) (Figure 3.8I). 94 Figure 3.8. No differences in latency to target hole or incorrect revisits were detected with the Barnes Maze. A) All four experimental groups’ average latency to target hole on each training day. B) All four experimental groups’ average latency to target hole on each training day for male animals. C) All four experimental groups’ average latency to target hole on each training day for female animals. D) The latency to target hole on the probe day for all animals. No differences detected (one-way ANOVA [F (3,64) = 0.974, p=0.4106]). E) The latency to target hole on the probe day for male animals. No differences detected (Kruskal-Wallis test [H = 0.8131, p= 0.8463]). F) The latency to target hole on the probe day for female animals. No differences detected (one-way ANOVA [F (3,27) = 0.3229, p=0.8087]). G) The number of incorrect revisits to nontarget hole on all training and probe days for all animals. No differences detected (one-way ANOVA [F (3,64) = 0.9907, p= 0.4029]). H) The number of incorrect revisits to nontarget hole on all training and probe days for male animals. No differences detected (one- way ANOVA [F (3,34) = 1.089, p= 0.3670]). I) The number of incorrect revisits to nontarget hole on all 95 Figure 3.8 (cont’d) training and probe days for female animals. No differences detected (one-way ANOVA [F (3,26) = 0.3031, p= 0.8229]). 96 OXTR Upregulation Coincides with Reduced Infarct Size The injection of ET-1 into the prefrontal cortex resulted in a significant infarction in both OXTR and GFP groups in comparison to the Saline animals (Kruskal-Wallis test [H=48.9, p<0.0001]); GFP (Dunn's multiple comparisons test [mean rank = -36.75, multiplicity-adjusted p<0.0001]); and OXTR (Dunn's multiple comparisons test [mean rank = -29.63, multiplicity-adjusted p<0.0001]) (Figure 3.9A). Separation of male and female animals revealed that this was true for both male (Kruskal-Wallis test [H=28.9, p<0.0001]) (Figure 3.9B) and female animals (Kruskal-Wallis test [H= 20.69, p=0.0001]) (Figure 3.9C) with the exception of male OXTR ET-1 animals not differing from GFP Saline (Dunn's multiple comparisons test [mean rank = -13.39, multiplicity-adjusted p=0.0524]). The volume of the infarct relative to the whole section was normalized to the coinciding Saline group for GFP and OXTR animals in order to directly compare the reduction in infarct size. The examination of all the subjects together revealed a significant reduction in infarct size due to OXTR upregulation (Unpaired t-test [t (33) = 2.407, p=0.0218]) (Figure 3.9D). When the male and female animals were analyzed separately, a the significant reduction was observed in the infarct size in the OXTR virus treated ET-1 group relative to the GFP virus treated ET-1 group was preserved for the male animals (Mann-Whitney test [n1Med=0.1206, n2Med=0.08569, U=78, p=0.0136]) (Figure 3.9E). It was not preserved in the female animals (Unpaired t-test [t (14) = 1.04, p=0.5271]) (Figure 3.9F). This suggests that a potential protective effect of the OXTR upregulation on peri-infarct astrocytes does exists but is driven primarily by male animals, resulting in male specific improvement on the novel object recognition task. 97 Figure 3.9. Pre-injury upregulation of the oxytocin receptor reduces the size of final infarction in males. A) Animals receiving the ET-1 Injection had greater infarct sizes relative to the saline animals (Kruskal-Wallis test [H=48.9, p<0.0001]) including GFP ET-1 relative to GFP Saline (Dunn's multiple comparisons test [mean rank = -36.75, multiplicity-adjusted p<0.0001]) and OXTR Saline (Dunn's multiple comparisons test [mean rank = 38.41, multiplicity-adjusted p<0.0001]) as well as OXTR ET-1 relative to GFP Saline (Dunn's multiple comparisons test [mean rank = -27.97, multiplicity-adjusted p=0.0004]) and OXTR Saline (Dunn's multiple comparisons test [mean rank = -29.63, multiplicity- 98 Figure 3.9 (cont’d) adjusted p<0.0001]) B) Male animals receiving ET-1 had greater infarct sizes relative to most Saline animals (Kruskal-Wallis test [H=28.9, p<0.0001]). GFP ET-1 animals differed from both GFP Saline (Dunn's multiple comparisons test [mean rank = -20.33, multiplicity-adjusted p=0.0006]) and OXTR Saline groups (Dunn's multiple comparisons test [mean rank = 23.94, multiplicity-adjusted p<0.0001]). OXTR ET-1 and GFP Saline animals did not significantly differ (Dunn's multiple comparisons test [mean rank = -13.39, multiplicity-adjusted p=0.0524]) but the differences to OXTR Saline remained [mean rank = -17, multiplicity-adjusted p= 0.0037]). C) All female animals receiving the ET-1 Injection had greater infarct sizes relative to the saline animals (Kruskal-Wallis test [H= 20.69, p=0.0001]) including GFP ET-1 relative to GFP Saline (Dunn's multiple comparisons test [mean rank = -16.14 , multiplicity-adjusted p=0.0104]) and OXTR Saline (Dunn's multiple comparisons test [mean rank = 15.03, multiplicity-adjusted p=0.0042]) as well as OXTR ET-1 relative to GFP Saline (Dunn's multiple comparisons test [mean rank = - 14.67, multiplicity-adjusted p= 0.0169]) and OXTR Saline (Dunn's multiple comparisons test [mean rank = -13.56, multiplicity-adjusted p= 0.0065]) D) The difference between the ET-1 animals and their respective Saline animals revealed a difference in infarct reduction between GFP ET-1 animals and OXTR ET-1 animals (Unpaired t-test [t (33) = 2.407, p=0.0218]). E) The difference in infarct reduction between GFP ET-1 and OXTR ET-1 animals held for male animals (Mann-Whitney test [n1Med=0.1206, n2Med=0.08569, U=78, p=0.0136]). F) There was no difference in infarct reduction between GFP ET-1 and OXTR ET-1 females (Unpaired t-test [t (14) = 1.04, p=0.5271]). G) Representative images separated by virus (GFP or OXTR), injury (ET-1 or saline), and sex (male or female). The difference between GFP ET- 1 and OXTR ET-1 versus their respective saline controls is plotted and used for analysis. Area of infarction was visualized with hematoxylin and eosin. Area of the lesion was determined using ImageJ area measurements and a freehand tool. Images are 6x7 4x stitched images. 99 No Difference in Myelin Index is Detected An indirect degeneration of white matter due to the infarct was a possibility and could contribute to executive dysfunction so a luxol fast blue stain for myelin was performed. Though, it revealed no difference in white matter density between the four experimental groups for both the minor corpus callosum (one-way ANOVA [F (3,65) =0.5803 , p=0.6301]) (Figure 3.10A) and the major corpus callosum (one-way ANOVA [F (3,65) = 0.6083, p=0.6120]) (Figure 3.10B). This was not entirely unexpected as the lesion was focal and localized but was necessary to consider none the less. 100 Figure 3.10. Luxol Fast Blue stain revealed no difference in white matter density between groups. A) The myelin index for minor corpus callosum was not significant (one-way ANOVA [F (3,65) =0.5803 , p=0.6301]) measured in ImageJ from 4x image. B) The myelin index for major corpus callosum was not significant (one-way ANOVA [F (3,65) = 0.6083, p=0.6120]) measured in ImageJ from 4x image. C) Representative images for minor and major corpus callosum from all four groups (4x). 101 Viral-Mediated Expression of GFP and OXTR Is Detected in The Perimeter of Damaged Tissue The presence of both GFP and OXTR in the vicinity of the infarct was detected using IHC (Figure 3.11). This confirms the presence of the viral products close to the infarct, but not completely absorbed into the lesion. A confirmation of the presence of the viral products regardless of extent was used as inclusion criteria. 102 Figure 3.11. GFP and OXTR are expressed in the proximity of the penumbra but are not absorbed in the final lesion. 103 Experiment 2 OXTR Expression in AAV6-GFAP-OXTR Treated Hemispheres Versus Contralateral Hemispheres The levels of OXTR mRNA expression derived from tissue punches from AAV6 OXTR hemispheres versus AAV6 GFP hemispheres were highly variable and ultimately not significant. This was upheld even after the removal of animals for whom the injection coordinates were found to be off target (Kruskal- Wallis test [H =6.94, p= 0.0738]) (Figure 3.12). Figure 3.12. OXTR expression in the AAV6 OXTR hemispheres versus AAV6 GFP hemispheres is highly variable as obtained by a tissue punch (Kruskal-Wallis test [H =6.94, p= 0.0738]). 104 No Difference in an Array of Cytokines mRNA was Detected Between Hemispheres at 24 Hours Using the remaining samples, a variety of cytokines linked to the resolution of an ischemic injury were chosen to be quantified. The same mRNA from the pellet used for OXTR quantification was used to better represent any change related to OXTR levels (Figure 3.13). There were no significant differences for any of the four cytokines studied, including TNFα (Kruskal-Wallis test [H =3.183, p= 0.3642]) (Figure 3.13A), TGFβ (Kruskal-Wallis test [H =4.653, p= 0.1990]) (Figure 3.13B), IL-1β (Kruskal-Wallis test [H =6.823, p= 0.0778]) (Figure 3.13C), or IL-10 (Kruskal-Wallis test [H =6.249, p= 0.1001]) (Figure 3.13D). The design of the study significantly reduced the power for analysis, and the method of tissue collection may not have best represented the ischemic environment. Furthermore, the 24 hour time point was chosen based on the time point best supported in the literature, but may not have been the ideal one to evaluate the effects of astrocytic OXTR on ischemic injury. These caveats and future directions will be discussed in greater detail below. 105 Figure 3.13. mRNA Cytokine profiles in AAV6 OXTR hemispheres versus AAV6 GFP hemispheres at 24 hours post ischemic injury. There were no significant differences. A) TNF mRNA levels as determined by qPCR (Kruskal-Wallis test [H =3.183, p= 0.3642]). B) TGFβ mRNA levels as determined by qPCR (Kruskal- Wallis test [H =4.653, p= 0.1990]). C) IL-1β mRNA levels as determined by qPCR (Kruskal-Wallis test [H =6.823, p= 0.0778]). D) IL-10 mRNA levels as determined by qPCR (Kruskal-Wallis test [H =6.249, p= 0.1001]). 106 No Difference in Reactive Oxygen Species Is Detected Between Hemispheres at 24 Hours The red-channel florescence intensity of DHE as it was oxidized by tissue reactive oxygen species was compared among the four groups of tissue samples (Figure 3.14). The injury caused by ET-1 was highly effective at increasing ROS generation at 24 hours (Kruskal-Wallis test [H=12.03, p=0.0073]). However, both the GFP ET-1 hemispheres (Dunn's multiple comparisons test [mean rank =-12.21, multiplicity-adjusted p= 0.0246]) and the OXTR ET-1 hemispheres (Dunn's multiple comparisons test [mean rank =-12.55, multiplicity-adjusted p= 0.0191]) were equally affected. Implications and alternative explanations for these results when it comes to OXTR upregulation as a mediator for ischemic signaling pathways will be discussed shortly. 107 Figure 3.14. Reactive oxygen species at 24 hours post ischemic injury in AAV6 OXTR hemispheres versus AAV6 GFP hemispheres as determined with DHE. A) Mean intensity of red channel detected DHE oxidation by treatment condition. ET-1 Injury increased ROS generation at 24 hours (Kruskal-Wallis test [H=12.03, p=0.0073]). Both GFP ET-1 hemispheres (Dunn's multiple comparisons test [mean rank =- 108 Figure 3.14 (cont’d) 12.21, multiplicity-adjusted p= 0.0246]) and OXTR ET-1 hemispheres (Dunn's multiple comparisons test [mean rank =-12.55 , multiplicity-adjusted p= 0.0191]) had significantly more reactive oxygen species based on DHE oxidation fluorescence compared to GFP Saline hemispheres. 109 Discussion As an integral part of the immune response and subsequent recovery following an ischemic injury, astrocytes sit at a vital niche and their targeting could have therapeutic implications (Barreto et al., 2012; Li et al., 2008). Due to this association, our previous identification of a de novo upregulation of the OXTR on astrocytes in the vicinity of infarcts in frontal cortex tissue from VaD cases, which was replicated in white matter, warranted further investigation. The upregulation of the OXTR in the frontal cortex of SHRSPs through AAV technology was compared to SHRSPs receiving an AAV bearing control GFP in behavioral, cognitive, and tissue recovery outcomes after a prefrontal cortex ischemic injury. Further bilateral comparisons were made using the same constructs and stereotaxic coordinates to query the consequences of the upregulation of the OXTR on inflammation and reactive oxygen species in the subacute phase of ischemia. Damage to the regions have previously been associated with executive and working memory dysfunction without additional motor deficits (Cordova et al., 2014; Déziel et al., 2015). The prior targeting of astrocytes by the OXTR bearing virus resulted in a reduction of final lesion size and a recovery on a novel object task designed to reflect an object recognition and working memory impairment, but this effect was mainly attributable to the male rats. The only differences observed for female rats were differences in the elevated plus maze time in open arms that might underlie an anxiolytic effect of the lesion, which was also true for the males on a different variable of the elevated plus maze, the entries to open arms. No differences were observed for any group in spontaneous locomotor activity as measured by the open field test, or in spatial navigation memory as determined by the Barnes Maze. The lack of differences in the open field test in the last half hour allowed us to rule out any severe motility differences in the ET-1 groups. The last half hour was chosen to rule out any locomotor differences owing to the anxiety of a new environment as suggested by Gould and colleagues (Gould et 110 al., 2009). The novel object task was designed with a single encounter with the similar object and reintroduction to that object and a novel object after an hour long delay to assess the functioning of the prefrontal cortex in light of the lesion as it has been associated with the consolidation of object recognition memory (Livingston-Thomas et al., 2015; Tanimizu, Kono, & Kida, 2018). The female animals did not show impairment on the novel object recognition regardless of condition which could be attributed to the effects of gonadal steroids or the innate better performance of female rats versus male rats with similar intervals (Sutcliffe, Marshall, & Neill, 2007). It is perhaps not so surprising that the Barnes Maze did not find any deficits as it relies heavily on hippocampal function which is spared by the focal prefrontal cortex damage model chosen (Paylor, Zhao, Libbey, Westphal, & Crawley, 2001; Raber et al., 2004). The reduction in anxiety behavior in the elevated plus maze is intriguing and must be considered in the context of the novel object test results as well, as neophobia could be a confounding condition. Sex differences in this task have previously been established with females appearing less anxious in the maze than males (Xiang, Huang, Haile, & Kosten, 2011). The role of the medial prefrontal cortex in producing the anxiety behavior for the elevated plus maze via limbic reciprocal connectivity has previously been established, as lesions and inactivation have an anxiolytic effect in this task (Shah, Sjovold, & Treit, 2004; Shah & Treit, 2003). This is reflected in our own data for the GFP ET-1 group and is partially attenuated in the OXTR ET-1 group but only for the males. In the secondary experiment to examine possible effects of the OXTR upregulation in astrocytes to known components of ischemic cell death in the penumbra, male rats were selected due to their significant response to the OXTR virus treatment in the previous study. Bilateral pre-treatment with each virus in separate hemispheres before the induction of an injury or control injection was performed. Frozen tissue allowed for qPCR and reactive oxygen species tests that necessitate such a tissue protocol. The successful selection of the region surrounding the injection sites with the viral products proved difficult and highly variable for qPCR analysis. As might be expected based on this initial selection issue, 111 the resulting levels of the chosen cytokines IL-1β, IL-10, TGFβ, and TNFα were also highly variable. No significant differences were noted in any of these results. The in situ measurement of reactive oxygen species generation using a unique oxidation target also revealed no significant difference at the chosen 24 hour time point, but the ability to detect the consequences of the ischemic injury on reactive oxygen species generation was better preserved in this assay. The time point of 24 hours was chosen based on temporal profiles of ROS generation and cytokine generation from previous studies. While certainly capable of interaction that heightens the levels of both, in general, after ischemic injury with reperfusion in rats ROS generation peaks before many cytokines with the greatest overlap occurring at 24 hours (Abe et al., 2015; Clark et al., 1994; Perera et al., 2006; Yang et al., 2019). The observation of increased reactive oxygen species in the ET-1 animals suggests this time point was not wholly incorrect. However, the variability in the qPCR results for the cytokines and OXTR mRNA levels point to the possibility that 1) the sampling design needs adjustment, 2) the OXTR on astrocytes has little influence on the overall inflammatory environment, or 3) that another time point may make the effects of the OXTR upregulation more visible. Based on the results of Experiment 1, the tissue punch method was selected due to the relatively discreet distribution of the viruses and the desire to target penumbra tissue only. However, without a visualization of the virus location it becomes impossible to assess whether it was collected with the punch until subsequent validation. In the future more punches per animals or a gross dissection of the entire area surrounding the infarct may be more fruitful. The time point was chosen as a maximum point for the deleterious effects of the ischemic injury we hypothesized that the OXTR may counteract. However, this may also be an overwhelming point with many other factors overriding any effects of the OXTR upregulation. Earlier or later recovery phases will be worth exploring. Alternatively, it may be that the effect of OXTR upregulation are double edged. This will need to be explored in more detail to understand the mechanistic underpinnings and possible clinical relevance of OXTR’s behavioral and tissue-protective 112 effects as shown in Experiment 1. Furthermore, our selection of inflammatory and ROS generation was based on what is known of the receptor in peripheral infarction. However, it does not come close to examining the full contribution of astrocytes to the resolution of an ischemic injury and alterations to these responses and the potential beneficial or aversive influences of the OXTR should be more fully explored before any thoughts of clinical application. Finally, our sampling design targeting the immediate area of the infarction precluded the collection of enough tissue for cytokine protein analysis. Hence, we cannot rule out the possibility that OXTR expression had significant effects on cytokine protein levels at the 24 hour time point. This can also be explored further in future studies. Astrocytes are divided, based primarily on morphology and location, into two main types, protoplasmic astrocytes in the gray matter and fibrous astrocytes in the white matter (Choudhury & Ding, 2016; Sofroniew & Vinters, 2010). Debate exists as to whether protoplasmic or fibrous astrocytes are thought to be slightly more resistant to ischemia (Choudhury & Ding, 2016; Shannon, Salter, & Fern, 2007). Regardless, in culture astrocytes appear far more resistant to ischemia than neurons, able to survive up to 24 hours without oxygen or glucose (Yu, Gregory, & Chan, 1989). Moreover, in-vivo astrocytes may survive marginally longer than neurons, from 1 to 3 hours with oxygen and glucose deprivation in the core region (Liu et al., 1999; Qin et al., 2010), and from 24 hours to even weeks in surrounding regions (Giffard & Swanson, 2005; Gürer, Gursoy-Ozdemir, Erdemli, Can, & Dalkara, 2009). This increased viability is attributed to multiple factors including their natural energy stores of glycogen which allows them to maintain ATP and ion gradients longer as well as antioxidant protection via intracellular glutathione (Rossi, Brady, & Mohr, 2007). When astrocytes do succumb, it is often due to the addition of acidosis or oxidative stress in addition to hypoxia (Swanson, Ying, & Kauppinen, 2005). Ischemic stress results in a response called reactive gliosis where astrocytes take on a hypertrophic appearance and proliferate rapidly (Swanson et al., 2005). This typically becomes noticeable within two 113 days of ischemia, peaking at around 4-5 days (Cekanaviciute & Buckwalter, 2016; Kawai et al., 2010; Schmidt-Kastner, Wietasch, Weigel, & Eysel, 1993). The glial scar also contains several extracellular matrix proteins secreted by astrocytes that can inhibit axonal regrowth in the recovery phase after ischemia (Fitch & Silver, 2008). These substances include: Ephrin A, chondroitin sulfate proteoglycans, fibronectin, laminin, and tenascin (Cekanaviciute & Buckwalter, 2016; Pekny et al., 2016; Rolls, Shechter, & Schwartz, 2009). The activation of these filaments appears to be dependent on STAT3 (Herrmann et al., 2008), and the initiating signals varies from cytokines, neurotransmitter, hormones purines (Pekny et al., 2016). Their proliferation can be activated by similarly multiple signals including epidermal growth factors, fibroblast growth factor, endothelin-1 and ATP (Sofroniew, 2009). As mentioned in the previous paragraph, oxidative stress is cytotoxic to astrocytes in ischemia due to the peroxidation of lipids (Yu et al., 1989), which augments damage due to rising intracellular ion levels causing cytotoxic swelling (Khanna, Kahle, Walcott, Gerzanich, & Simard, 2014; Song & Yu, 2014). Reactive oxygen species play an important role in the post-ischemic brain, including those driven by astrocytic effects. Since astrocytes have an inherent antioxidant protection due to their high concentration of glutathione (Raps, Lai, Hertz, & Cooper, 1989), neurons and astrocytes can support each other’s antioxidant protection through the redox cycling of either glutathione, ascorbate, or their substrates (Dringen, Gutterer, & Hirrlinger, 2000; Makar et al., 1994). When reactive oxygen species overwhelm astrocytes’ defense mechanisms, they can reduce glutamate transporter function contributing to excitotoxicity (Sorg, Horn, Yu, Gruol, & Bloom, 1997). Moreover, astrocytic end feet of the BBB contain an important channel call aquaporin 4 that helps buffer water in the brain, but in the case of ischemia can contribute to cytotoxic edema (Fu, Li, Feng, & Mu, 2007; Zador, Stiver, Wang, & Manley, 2006). 114 A unique niche filled by astrocytes is that of a ‘sink’, or the ability to buffer and redistribute harmful substances. In the case of ischemia this is a double-edged sword, on the one hand lessening the burden of struggling cells, but also exposing more remote cells to potential danger. Astrocytes can act as a ‘sink’ for multiple ions including calcium, hydrogen, and potassium (Kraig, Pulsinelli, & Plum, 1985; Walz, 2011; Westenbroek et al., 1998). Astrocytes are connected by connexins, particularly connexin 43 but also connexin 30, which make up gap junctions (Lin et al., 2008). These junctions remain partially open during ischemia, but endothelins activating endothelin-B receptors seem to contribute to gap junction closing in addition to rises in calcium and acidification (Li et al., 2008; Lipton & Budd, 1998). Astrocytes communicate under healthy conditions through calcium waves through gap junctions, but in ischemic conditions rises in intracellular calcium can facilitate the distribution of pro-apoptotic waves (Lipton & Budd, 1998; Wang, Tymianski, Jones, & Nedergaard, 2018). The spreading calcium waves can also influence toxic spreading neuronal depolarizations (Anderson et al., 2003; Duffy & MacVicar, 1996). Supporting this toxic wave idea is the observation that calcium signaling in astrocytes has a synchronized rhythm (Choudhury & Ding, 2016). In fact, Lin and colleagues (Lin et al., 1998), found that in the presence of an abundance of Ca2+ ionophore, the density of gap junctions predicted the number of connected cells that died through apoptosis. This death by diffusion through gap junctions to otherwise salvageable cells is called ‘bystander death’ (Lin et al., 1998; Lipton & Budd, 1998). The same connexins make up outward facing hemichannels, allowing distribution into the extracellular space (Lin et al., 2008). These hemichannels remain open during ischemia (Rossi et al., 2007). The positive aspect of the leak from these hemichannels is the release of ATP which can be converted to adenosine that has neuroprotective affects at A1 receptors, suppressing over excitability (Lin et al., 2008; Rossi et al., 2007). Chemokines released by astrocytes can attract immune cells to the site of damage to localize and target the inflammatory response, preventing global inflammation (Panenka et al., 2018; Zhang et al., 1999). With respect to revascularization, some of the proangiogenic and trophic factors that are 115 released by astrocytes include thrombospondin-1, IGF-1, and VEGF (Lu & Kipnis, 2010; Rolls et al., 2009; Shetty, Hattiangady, & Shetty, 2005). Synaptogenesis in the wake of the injury can also be aided by astrocytes through the release of other trophic factors like erythropoietin, neurotrophin-3, thrombospondins, nerve growth factor, platelet derived growth factor, and brain derived neurotrophic factor (Rudge et al., 1992; Ruscher et al., 2018; Saha, Liu, & Pahan, 2006; Swanson et al., 2005). Erythropoietin specifically inhibits pro-apoptotic caspases and upregulates the survival signal Bcl-2 in neurons (Swanson et al., 2005). Astrocytes can induce other cells to adopt either a pro- or anti- inflammatory phenotype through the release of corresponding cytokines (Choi, Lee, Lim, Satoh, & Kim, 2014; Kim et al., 2004; Knuckey et al., 1996; Kuboyama et al., 2011). The pro-inflammatory cytokines are released as the end results of p38 MAPK, gp130 and NFkβ pathways (Choi, Lee, Lim, Satoh, & Kim, 2014; Kuboyama et al., 2011). The anti-inflammatory phenotype is driven by SOC3/gp130 and TGFβ pathways (Choi et al., 2008; Kim et al., 2004; Knuckey et al., 1996). Finally, they also release a couple matrix metalloproteinases, MMP-2 and MMP-9, which aid remodeling, but also contributes to BBB leakage (Gottschall & Yu, 1995; Lee et al., 2007). Astrocytes can also provide energy for surrounding neurons. They release the powerful energy substrates lactate and glutamine (Walz & Mukerji, 1988; Waniewski & Martin, 1986). These glial cells are the main reservoir of glycogen in the brain and can convert it to lactate to supply to other cells (Benarroch, 2010). Astrocytes are able to take up the potentially excitotoxic neurotransmitter glutamate via two sodium dependent transporters called EAAT1 and EAAT2 in humans (Landeghem, Weiss, Oehmichen, & Deimling, 2006; Miralles et al., 2001), and detoxify it to glutamine, via glutamine synthase (Chisholm & Sohrabji, 2016; Waniewski & Martin, 1986), thus buffering the synapse. Astrocytes have lower typical levels of glutamate, so they can continue to take it up safely for a time, but with persistent ischemia causes eventual glutamate efflux through the transporters (Rossi et al., 2007). This glutamate efflux leads to activation of NMDA glutamate receptors and further toxic influx of calcium (Frandsen, 116 Drejer, & Schousboe, 1989). Excitotoxicity at NMDA receptors can also be exacerbated by the additional release of the co-modulator D-serine (Katsuki, Nonaka, Shirakawa, Kume, & Akaike, 2004). Recently, an additional role for astrocytes as phagocytes in the penumbra region in later points of ischemic injury has been reported (Morizawa et al., 2017). Astrocytes are not normally phagocytic, but perhaps in states of ischemia engulfment occurs via an ABCA1 mediated pathway in response to apoptotic cells (Morizawa et al., 2017). The broad range of both protective and deleterious effect of astrocytes in ischemic injury is certainly much broader than the mechanisms we examined. This also leaves many options for potential intersections of OXTR signaling with these responses to ultimately benefit or harm tissue. Our results for Experiment 1 certainly suggest that there is a benefit to preserving tissue and cognitive function that is worth pursuing both in alternative models and in the continued examination of the downstream response dictating the behavioral and pathological results. A preservation of cognitive function in the long-term should not be pursued if it means increasing mortality in the short term. Further work should be focused on identifying the full intracellular consequences of OXTR signaling in astrocytes during ischemic brain injury. Several studies have found evidence of a better functional outcomes following stroke with oxytocin treatment (Moghadam et al., 2018; Kaneko et al., 2016; Karelina et al., 2012; Seo et al., 2018), but none to our knowledge have pursued the receptor itself absent the ligand. The strengths of our study are its basis in recreating an observation of a local change to ischemia damaged tissue and the animal model of cognitive damage without motor impairments along with a common pre-morbidity. The limits are not just those applying to all studies of experimental stroke like differences in acute versus subacute phases (Kozuka et al., 2002; Malone, Amu, Moore, & Waeber, 2019) the difficulty in applying findings in a single model to a heterogeneous condition (Muir, 2002), and sex differences (Haast, Gustafson, & Kiliaan, 117 2012). We have the added limitation of clinical application, though OXT does not readily cross the blood brain barrier (Ermisch, Rühle, Landgraf, & Hess, 1985), alternative methods for delivery of the ligand are constantly evolving (Lee & Jayant, 2019; MacDonald et al., 2011; Modi, Connor-Stroud, Landgraf, Young, & Parr, 2014). A pre-emptive astrocyte-specific upregulation of the receptor beyond a small loci is not a likely avenue, but the full characterization of what the peri-infarct OXTR bearing astrocytes we observed may be doing using in vivo methods will surely provide vital, novel mechanistic insights into the therapeutic rationale for targeting this signaling pathway was a disease-modifying treatment for VaD/VCI. 118 CHAPTER 4: Final discussion Conclusions Cognitive impairment following a stroke, called vascular dementia when memory is involved and called vascular cognitive impairment when memory is spared (O’Brien, 2006), is a common and debilitating complication to the quality of life of survivors (Kalaria, Akinyemi, & Ihara, 2016). This work was aimed at identifying and testing the efficacy of a potential new avenue for salvaging tissue and preventing associated cognitive decline. This design combined the results of a profile of gene expression changes in VaD postmortem cortex to postmortem cortex from non-cognitively impaired and AD cases with a survey of the existing literature. This led to the identification of a novel increase in OXTR expression in VaD with the potential to nullify aspects of ischemic damage (Gonzalez-Reyes et al., 2015; Jankowski et al., 2010; Szeto et al., 2008). The remaining work presented in this document was focused on the validation of this finding (McKay et al., 2019), the application to an animal model, and attempts to clarify the mechanisms that might underlie the observation. Our animal model was generated to mimic the observations in the human tissue. While a few studies have examined the effects of giving oxytocin to animals undergoing experimentally induced stroke (Moghadam et al., 2018; Karelina et al., 2012), this would not be a replication of the findings in the human tissue that was limited to the receptor. Therefore, the decision was made to use a viral vector strategy to target astrocytes in the vicinity of a future infarct to model the implications of our human tissue findings. To further parallel the condition of human ischemic stroke and subsequent VaD, a rat model with a common hypertension comorbidity found in human stroke studies, the SHRSP (Okamoto & Aoki, 1963), and an ET-1 injury model in prefrontal cortex that has been established to lead to cognitive impairment in executive functions such as discerning novelty similar to initial VaD symptoms, but without motor impairments (Cordova et al., 2014; Déziel et al., 2015). Separate 119 experiments were devised to test the behavior and tissue loss following experimental injury in these animals and the inflammatory and oxidative stress response to this upregulation under ischemic conditions The injury to the PFC was successful in replicating the deficit in object recognition memory and anxiolytic activity as reported for previous lesions or inactivation of the targeted region (Déziel et al., 2015; Shah et al., 2004; Shah & Treit, 2003). Other subtypes of memory such as spatial navigation memory were preserved. Taken together, this animal model for VAD replicates the human condition to the extent possible with preclinical translational impact. The lesion was focal and limited to the grey matter of the PFC without any conclusive evidence of white matter degeneration. The size and location of the lesion was reproducible. When a significant difference in lesion volume was detected a matching behavior deficit followed, more often for the males than for the females. This homogeneity and predictable correlation to subsequent cognitive deficits were vital for testing the hypotheses of our study. These deficits were rescued only for male animals and not for female animals. This is an interesting finding in light of the fact that many of the previous oxytocin and stroke studies used only male animals (Moghadam et al., 2018; Karelina et al., 2012). While their results in males are generally in agreement with ours, the lack of response in the female animals raises concerns that may translate to clinical endeavors. Sex differences in trials for females versus males concerning treatment for ischemic stroke have been well established (Roof & Hall, 2000; Spychala, Honarpisheh, & McCullough, 2017). Much of this work has concerned gonadal steroid hormone differences and specifically pre versus post menopause (Lisabeth & Bushnell, 2012; Lisabeth et al., 2009). All of our female animals were given the experimental stroke at the same time of the estrous cycle in an attempt to reduce variability. However, it is possible that this still played into the lack of effect noted, especially considering work pointing to 120 the effects of gonadal steroids on astrocytic responses to ischemia (Chisholm & Sohrabji, 2016). This should be explored further in future experiments. Despite these encouraging results, a mechanism remains undefined despite the exploration of inflammatory molecules and reactive oxygen species generation. As discussed in the previous chapter this could be due to the method, time point, or a true lack of effect on these pathways. Certainly, a mix of more than one could be possible. It could be that the height of engagement of the astrocytic OXTR with these pathways could occur earlier or later in the scope of ischemic injury response. Nonetheless, the data and reporting of the complications using this method and time point will be vital in steering the direction of further work. The signaling cascades of both ischemia activated astrocytes (Li et al., 2008; Swanson et al., 2005) and GPCRs like the OXTR (Chatterjee et al., 2016) are vast, this is to say nothing of the divergence of acute and subacute response to ischemic injury. The identification of a mechanism of this potential protective effect suggested by the animal behavior data is just as important as further validation studies to ultimately pursuing or discarding the astrocytic OXTR as a potential VaD preventative option. Regarding the overall implications of these experiments, any application of this work is not meant to replace the gold standard of rapid reperfusion of infarcted tissue, but rather to augment this therapy to protect more salvageable penumbra region. While the efficacy of reperfusion therapies are limited to a 4 to 6 hour window (Wardlaw et al., 2012), the ischemic penumbra is well established to survive for hours to days following an ischemic injury (Bastianello et al., 2006; Fisher & Garcia, 1996). As the total volume of final infarction is closely tied to the emergence of VaD symptoms in the cortex (Tomlinson et al., 1970), strategies to preserve this tissue could prevent VaD and extend the therapeutic window. The ultimate loss of the ischemic penumbra is often a mix of apoptosis and some necrosis (Uzdensky, 2019). This apoptotic loss is tied to the balance of a closely entwined cascade of 121 inflammatory, oxidative stress, and excitotoxic agents and protective counters like anti-inflammatory, antioxidant and buffering agents (Horváth et al., 2018; Lambertsen et al., 2012; Ramos-Cabrer et al., 2011). These protective signaling cascades have been linked to the OXTR in ischemic responsiveness (Jankowski et al., 2010; Szeto et al., 2008) and are capabilities of astrocytes (Cekanaviciute et al., 2014; Kim et al., 2010; Li et al., 2008). Astrocytes also fill other unique niches in tissue preservation during ischemia, such as the formation of a glial scar and as energy substrate suppliers (Herrmann et al., 2008; Ransom & Fern, 1997; Rossi, Brady, & Mohr, 2007; Stoll, Jander, & Schroeter, 1998). The possibility of targeting astrocytes to mediate ischemic damage is not a new idea (Anderson et al., 2003; Barreto et al., 2012). The possibility of OXT and in turn its receptor having a protective role during ischemic challenge is not a new idea (Düşünceli et al., 2008; Jankowski et al., 2010; Karelina et al., 2012; Tuǧtepe et al., 2007). The novelty of this work is the combination of the two and the link to our discovery of the de novo upregulation of the OXTR in VaD using clinically and pathologically well- characterized human tissue, and the cognitive preservation referable to lesion site and size in the animal model. This research provides further specifics on the therapeutic potential for astrocytes and the OXTR, strengthened by the careful connection between evidence from human tissue and proof of concept in an animal model. It should serve as a basis for future research while considering its limitations, both of which will be discussed below. Future Directions The use of both sexes in this study allowed for the characterization of a sex difference in the proposed effectiveness of the OXTR upregulation in astrocytes. Sex differences are a rich field of study in stroke research (Haast et al., 2012; Roof & Hall, 2000; Spychala et al., 2017), and to an extent VaD (Gannon, Robison, Custozzo, & Zuloaga, 2019), as well as in OXTR function (Dumais, Bredewold, Mayer, & Veenema, 2013; Rice, Hobbs, Wallace, & Ophir, 2017). It follows that perhaps this difference is not 122 unexpected but has gone widely unexplored in previous studies on the OXT/OXTR system in cardiovascular and cerebrovascular disease. In this regard, the effect of sex, particularly gonadal steroids, should be explored further. In a clinical application ischemic stroke will not always occur at the same cycle phase. Strategies to safely combat this potential confounding effect or prescreen may become necessary. The spread of the virus was quite limited and strategies to enhance its penetration into the tissue should be pursued. One such option is the use of mannitol to assist the diffusion of the virus (Mastakov, Baer, Xu, Fitzsimons, & During, 2001). The focal and predictable nature of the injury chosen for the animal studies allowed for direct placement of the virus near the site of eventual infarction. However, in practice the unpredictable nature of ischemic injury in both size and location makes such an enhancement necessary. Along with this addition, different methods of injury, particularly larger ones might become possible and would better evaluate the potential for the targeting of the receptor. The mechanistic set of experiments were perhaps the most convoluted and would benefit greatly from amendments in the future. The collection of tissue from the around the injection site was a difficult task without guidance cues other than the needle tracks or lesions, moreover the small region possessing the GFP or OXTR virus products was difficult to capture. The addition of a diffusion enhancing agent would likely remedy some of this variability, but gross dissection of cryostat sections might also be a viable option to enhance collection of the region of interest. Although we selected a time point for molecular analysis in our studies based on the rigor of prior research, multiple time points are warranted to explore the consequences of receptor engagement in different phases to inform treatment timing, and to rule out the potential for competing beneficial and deleterious effects on a temporal basis. In order to reduce the number of animals used these experiments, these studies could be briefly 123 moved to cell culture, perhaps a mixed primary culture of astrocytes and neurons, to identify promising and concerning time points for future exploration. Along these lines, cell sorting from collected frozen tissue is one option, as would be laser capture microdissection of selected peri-infarct astrocytes and neurons. The difficulty with this is that immunohistochemistry methods can impair RNA integrity. However, enhanced GFP (EGFP) has been reported to not require immunohistochemistry and has been used with LCM before (Bhattacherjee et al., 2004). Targeting astrocytes with this protein and excising ones far from and around the site of infarct to correlate OXTR levels with downstream responses is an alternative approach. Another would be a quick lectin-based visualization of these same populations of astrocytes. Lectin-based LCM methods have been deployed successful before (Mojsilovic-Petrovic, Nesic, Pen, Zhang, & Stanimirovic, 2004), and astrocyte specific lectins are characterized as well (Derouiche, Härtig, Brauer, & Brückner, 1996). A final question for future direction is perhaps the most important one, the functionality of the receptor. The usability and application of these results hinge on the ability of the receptor deposited by the virus to bind its ligand and trigger intracellularly signaling cascades as the endogenous one does. The ligand binding should be confirmed with either radioligand binding, of which several options, iodinated OXTR antagonist or tritiated OXT, exist (Klein, Jurzak, Gerstberger, & Fahrenholz, 1995; Loup, Tribollet, Dubois-Dauphin, & Dreifuss, 1991). We are currently pursuing this question using tritiated OXT in frozen tissue sections from the AAV-OXTR treated animals. Another option is the frequently reported but commercially unavailable florescent ligands (Mouillac, Manning, & Durroux, 2008). The benefits of this ligand binding test over the previous is the shorter duration and the lack of radiation making it a safer and quicker option if it is available. The intracellular signaling could be determined in culture using the virus to express OXTR in cells where it is not endogenously found and careful assay of downstream signaling products in the presence and absence of antagonists. It is feasible that the addition of 124 antagonist treatment groups to the LCM study described above could allow a variation of this study to be performed in vivo. Alternatively, co-labelling with intracellular signaling partners that bind directly to the receptor could be used as evidence for intracellular functionality. Limitations Any study suggesting a clinical application should be carefully weighed in light of its limitations both in regard to caveats to what it has found as well as how feasible it is as a treatment. For instance, we have presented evidence for the receptor’s role in cortical infarction, but the response of the cortex to an ischemic injury is not the same as that of subcortical regions. In particular, when it comes to the preservation of behavior there is evidence from studies of motor recovery that the cortex has more opportunity for remapping to areas of redundancy than subcortical regions (Karthikeyan, Jeffers, Carter, & Corbett, 2019; Shelton & Reding, 2001). This could mean that while some benefit of OXTR upregulation is suggested in our animal work, this may not apply equally to subcortical injury that contributes a great deal to the presence of VaD or VCI symptoms. There is also the possibility that the observed reduction in infarct size with OXTR administration and subsequent salvaging of damaged tissue may not be the cause of improved behavioral outcomes, since mechanism is still unclear. That is, it is worth considering that creating a plasticity-benefiting microenvironment might be the real reason for the beneficial behavioral improvement noted versus salvaging endangered tissue (Kriz & Lalancette-Hébert, 2009), but again this will depend on the results of future mechanistic and behavioral studies in our model. More limits come from the basis of studying cognition, particularly prefrontal cortex in a rat model. A lively debate exists as to whether rats have a true prefrontal cortex (Uylings, Groenewegen, & Kolb, 2003). The region was chosen for these animals to best replicate the impairments found in VaD patients. However, the exact role of this area compared to that linked to the most common VaD 125 symptoms in humans is up for debate. Additionally, the prefrontal cortex has many cortical and subcortical afferents, efferent, and reciprocal connections (e.g., the amygdala and locus coeruleus) that could be affected by the lesion of this area (Leonard, 1969; Reppucci & Petrovich, 2016). These indirect effects and the fact that they may differ from human patients to this rat model should be taken into account. The timing and response to an ischemic injury is also beyond our ability to fully explore in this single study. It is well known that reperfusion injury differs from a permanent occlusion (Collino et al., 2006; Zhou et al., 2013). However, as we were focused on the rescue of the penumbra region we chose a reperfusion model necessary to spare the penumbra. However, very few patients actually undergo a reperfusion therapy (Katzan et al., 2000). While a majority of patients, particularly with cortical obstructions, do ultimately undergo spontaneous reperfusion (Jørgensen, 1994), the potential lack of applicability to all chronic obstructions should be noted. Additionally, like most injuries time is an important factor in when cellular responses are beneficial or destructive. As we are presenting a potential, but still unproven, role for the receptor in antioxidant and anti-inflammatory pathways, it should be noted that these immune responses can benefit the clearance of debris and prevent the loss of neighboring cells (Lambertsen, Finsen, & Clausen, 2019). It is also worth noting that the infarct- reducing pathways suggested in the literature to be tied to oxytocin might impair the response to a stroke in the acute phase. Indeed, our human tissue studies were cross sectional and based on autopsied tissue, so we cannot know for certain when during the ischemic cascade the OXTR upregulation occurs. This could mean that our preventative upregulation could differ in encouraging a protective or reductive response based on the time frame of induction post insult. As noted above, stroke is a heterogeneous condition. As such, a major complication with a distinct symptomology, like VaD or VCI, belies the true number of underlying injuries that contribute to 126 it (Kalaria et al., 2016; McAleese et al., 2016). We have only focused our study on small, cortical, and fully occluded ischemic injury. However, large cortical strokes, such as those occurring from middle cerebral artery occlusion, can lead to cognitive impairments including the frontal syndromes mentioned as well as domains such as limbic memory, aphasia, and visual processing deficits (Hoffmann, Schmitt, & Bromley, 2009). Subcortical strokes lead to a distinct and fairly common type of VaD tied to small vessel disease (Wallin et al., 2017). Global hypoperfusion and transient ischemic attacks also contribute to the emergence of cognitive dysfunction, particularly executive syndromes, due largely to the loss of vulnerable white matter (Duncombe et al., 2017; Inaba et al., 2019; Pendlebury, Cuthbertson, Welch, Mehta, & Rothwell, 2010). Finally, hemorrhagic stroke and microbleeds, even independent of CAA, also contribute to CVD related cognitive dysfunction (Seo et al., 2007). It is important to note that even though we are targeting a remedy for the cognitive deficits characterizing VaD or VCI, there is no way to divorce this from responding to the underlying cause of this condition. We have studied the results of intervening in a single example of this condition, although it should be noted that we discovered OXTR upregulation in the peri-infarct region of white matter infarctions in addition to those in the grey matter (McKay 2019). As mentioned above this should be at the forefront of future research along with mechanism directed studies. Finally, a consideration of the role of covert stroke in the onset of VaD is warranted. Frank strokes are actually far rarer than covert strokes and the damage these silent attacks contribute to cognitive functioning goes unnoticed until a frank episode or the seeming spontaneous appearance of impairment (Durrani, Hill, & Smith, 2020; Smith, 2017). As it is being suggested here as a preventative therapy for VaD in the wake of an ischemic injury, the targeting of the OXTR should be weighed based on how effective reducing damage from a reported stroke would be in reducing the subsequent rate of VaD with the co-occurrence of covert strokes. 127 Even with the limitations described above the research presented in this document, our discovery provides a starting point and evidence for the necessity to further explore the role of astrocytic OXTR signaling in the emergence or non-emergence of VaD or other cognitive impairments following a stroke. 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