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Mechanisms of neuronal swelling during spreading depolarization Liu, Yanqi 2018

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  MECHANISMS OF NEURONAL SWELLING DURING SPREADING DEPOLARIZATION by  Yanqi Liu  BSc. Queen’s University, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  January, 2018  ©Yanqi Liu, 2018 ii  Abstract Spreading depolarization (SD) is a wave of intense depolarization that propagates within the gray matter of the brain. It is implicated in migraine and acute brain injuries such as stroke, subarachnoid hemorrhage, and trauma. SD is thought to contribute to the expansions of infarct volume in energy compromised brains. Understanding SD and the associated cellular changes will provide basic knowledge to the field of neurophysiology, and will help identify potential therapeutic targets.  The experimental methodologies designed to examine SD properties in acutely prepared brain slices are described in Chapter 2. Techniques used in this study involved simultaneous two photon laser scanning microscopy, intrinsic optical signal (IOS) imaging, electrophysiology, pharmacology, and siRNA delivery by lipid nanoparticles (LNP).  In Chapter 3, experimental investigations that tested the contribution of neuronal swelling to the IOS changes in SD, as well as the potential role(s) for SLC26A11 chloride channel to neuronal swelling are described. The pharmacological agent GlyH-101 (an inhibitor of SLC26A11 and other chloride channels) reduced neuronal swelling while other properties of SD were unaltered. This suggests that neuronal swelling likely occurs downstream, or in parallel with other cellular processes. Interestingly, the temporal profiles of neuronal swelling and IOS were not correlated, suggesting that other cellular mechanisms generate IOS changes during SD. I also demonstrated that LNP-mediated delivery of siRNA was effective in gene knock-down in vitro but not in vivo. This was further supported by the lack of functional effect during SD in Slc26a11 siRNA treated samples compared to control siRNAs. Finally, I mimicked situations of mild energy failure in brain slices in parallel to SD induction; however, I observed no differences in SD electrical and optical properties compared to control conditions. These data supported the importance of cellular chloride entry in mediating neuronal swelling associated with SD. However, the role for SLC26A11 and the exact molecular mechanism(s) of swelling that are inhibited by GlyH-101 remain unidentified. My data provide insights into the molecular processes during SD, and work towards identifying potential therapeutic targets in neurological disorders associated with SD. iii  Lay Summary Spreading depolarization (SD) is a neurological phenomenon implicated in disorders such as migraine aura, stroke, cerebral hemorrhage and traumatic brain injuries. SD is thought to be harmless in otherwise healthy brains (such as in the cases of migraine aura) while it is responsible for expanding tissue damage in already compromised tissue, such as during acute brain injuries. It is interesting that one fundamental phenomenon is capable of producing two extremely different outcomes. Swelling of the nerve cells occurs during SD, which can be short-lasting or persistent depending on the tissue state, leading to recovery of function or cell death, respectively. Studying the molecular processes that contribute to swelling provide basic knowledge in SD and provide potential therapeutic targets for SD related disorders. iv  Preface All experimental protocols were approved by the animal care committee in the University of British Columbia and all procedures were followed with strict compliance in accordance to guidelines provided by the Canadian Council for Animal Care. The animal protocols of relevance are A15-0209, A15-0086, and A16-0158.  Data presented in this study are results of experiments performed in Dr. Brian MacVicar’s lab located in the Djavad Mowafaghian Center for Brain Health at the University of British Columbia. Dr.MacVicar and I designed the study. I was responsible for animal surgeries, brain slice preparation, tissue sample collection, imaging experiments, and data analysis. Lipid nanoparticles were formulated by Joslyn Quick under the supervision of Dr. Pieter Cullis. Mrs. Yuping Lu from Dr. Yutian Wang’s laboratory prepared the primary neuronal culture. Dr. Leigh Wicki-Stordeur and Dr. Hyun Beom Choi were responsible for cell culture works and performing biochemical assays. I was responsible for writing this manuscript with comments from Dr. Brian MacVicar.   v  Table of Contents  Abstract .......................................................................................................................... ii Lay Summary ................................................................................................................ iii Preface .......................................................................................................................... iv Table of Contents .......................................................................................................... v List of Tables .............................................................................................................. viii List of Figures .............................................................................................................. ix List of Abbreviations .................................................................................................... xi Acknowledgements .................................................................................................... xiii Chapter 1: Introduction ................................................................................................. 1 1.1 Mechanisms of cell volume regulation in the CNS ............................................ 1 1.1.1 Why is cell volume regulation important? ................................................... 1 1.1.2 Mechanisms of astrocyte and neuronal swelling ........................................ 2 1.2 Chloride channels and transporters in the CNS ................................................ 5 1.2.1 Ligand gated chloride channels ................................................................. 5 1.2.2 Cystic fibrosis transmembrane conductance regulator .............................. 5 1.2.3 Calcium activated chloride channels .......................................................... 6 1.2.4 Volume activated chloride channels .......................................................... 6 1.2.5 Cation chloride co-transporters .................................................................. 7 1.2.6 Solute carrier gene series .......................................................................... 7 1.3 CNS cell volume change in physiology and pathology ...................................... 8 1.3.1 Excitability .................................................................................................. 8 vi  1.3.2 Cell death ................................................................................................... 9 1.3.3 Cell swelling in pathology ......................................................................... 10 1.4 Overview of spreading depolarization (SD) ..................................................... 11 1.4.1 A brief historical view of SD ..................................................................... 12 1.4.2 Tissue responses to SD ........................................................................... 13 1.4.3 Mechanisms of SD ................................................................................... 15 1.5 The continuum of SD and ischemic depolarization ......................................... 16 1.6 Rationale, hypotheses and objectives ............................................................. 19 Chapter 2: Methods ..................................................................................................... 21 2.1 Brain slice preparation .................................................................................... 21 2.2 SD induction and pharmacology ..................................................................... 21 2.3 Electrophysiology ............................................................................................ 22 2.4 Lipid nanoparticle formulation and siRNA encapsulation ................................ 22 2.5 Stereotaxic surgery ......................................................................................... 23 2.6 Biochemistry ................................................................................................... 23 2.6.1 Primary neuronal cultures and siRNA-LNP treatment .............................. 23 2.6.2 Tissue collection ...................................................................................... 24 2.6.3 qPCR ....................................................................................................... 24 2.6.4 Western blot ............................................................................................. 25 2.7 Two photon imaging ........................................................................................ 26 2.8 Data analysis .................................................................................................. 26 Chapter 3: Results....................................................................................................... 29 3.1 SD was consistently induced by high K+ aCSF perfusion ............................... 29 vii  3.2 GlyH-101 had no effect on electrical and optical features of SD ..................... 30 3.3 GlyH-101 reduced the magnitude of the increases in neuronal cross sectional area while electrical and optical signals remain unaffected ....................................... 32 3.4 Temporal profiles of IOS and neuronal cross sectional area did not correlate 37 3.5 Lipid nanoparticle mediated siRNA uptake ..................................................... 40 3.6 Neuronal swelling was unaffected by siRNA-LNP uptake ............................... 42 3.7 siSlc26a11-LNP uptake did not alter most properties of SD ........................... 43 3.8 Mild impairment of Na+/K+ ATPase by Ouabain had no effect on SD wave front, electrical, and optical properties ................................................................................ 45 Chapter 4: Discussion and future directions ............................................................ 50 4.1 Summary of findings ....................................................................................... 50 4.2 Discussion....................................................................................................... 50 4.2.1 GlyH-101 pharmacology .......................................................................... 50 4.2.2 Potential contributors of IOS .................................................................... 51 4.2.3 Transient swelling and cytotoxic edema: hypothesis on tissue energy state    ................................................................................................................. 54 4.3 Limitations and considerations ........................................................................ 57 4.3.1 Estimates of neuronal swelling ................................................................ 57 4.3.2 Single neuron swelling and IOS ............................................................... 58 4.3.3 siRNA-LNP .............................................................................................. 58 4.4 Future directions ............................................................................................. 59 References ................................................................................................................... 63 viii  List of Tables  Table 1. List of siRNA-LNP components. ...................................................................... 28 Table 2 Sequences of SLC26A11 siRNA ...................................................................... 28 Table 3. Properties of siRNA-LNP. ................................................................................ 28 Table 4 Fisher’s exact test on SD induction by 20 minutes treatment of aCSF and aCSF with 30 nM Ouabain. ..................................................................................................... 47  ix  List of Figures  Figure 1. Proposed mechanisms of neuronal cytotoxic edema and related modulators.. 4 Figure 2. Mechanism of cytotoxic edema and cell death during ischemia. .................... 10 Figure 3. Changes in extracellular potential, glutamate and ion concentrations during cortical SD. .................................................................................................................... 15 Figure 4. A singular wave of depolarization spreads across brain tissue during ischemic stroke. ........................................................................................................................... 18 Figure 5 Electrical and optical signatures of spreading depolarization (SD). ................. 30 Figure 6 SD onset time and wave properties in control and GlyH-101 experiments. .... 31 Figure 7 Changes in neuronal cross sectional area during SD. .................................... 34 Figure 8 Comparisons between electrical recordings and optical changes of SD in control and GlyH-101 groups showed no significant differences. .............................................. 36 Figure 9 IOS changes during SD were similar in control and GlyH-101 experiments. .. 37 Figure 10 Temporal correlations between neuronal swelling and IOS changes during SD under control and GlyH-101 conditions. ........................................................................ 38 Figure 11 Representative traces of neuronal swelling and tri-phasic IOS change in control and GlyH-101 treated neurons. ..................................................................................... 39 Figure 12 Verification of SLC26A11 mRNA and protein levels treated with siRNA-LNP by qPCR and Western blot. ................................................................................................ 41 Figure 13 Colocalization of eYFP and DiIC18 as an indicator for LNP transfection. ..... 42 Figure 14 Neuronal cross sectional areas in siRNA-LNP transfected neurons during SD. ...................................................................................................................................... 43 x  Figure 15 Electrical and optical properties of SD in brain slice transfected with siRNA-LNP. .............................................................................................................................. 44 Figure 16 SD onset time and wave properties in control and Ouabain treated slices. .. 47 Figure 17 Electrical and optical signatures of SD in control and Ouabain groups. ........ 49 Figure 18 Fate of incident light upon hitting a scattering medium. ................................ 54 Figure 19 “Eggs in the egg carton” analogy for Gibbs free energy state proposed by Dreier et al. 2013. .................................................................................................................... 56 Figure 20 Algorithmic estimates of membrane potential (a) and cell area (b) as a function of Na+/K+ ATPase strength. ........................................................................................... 57 Figure 21 Mechanism of RNA interference involving short-hairpin RNA (shRNA) through viral mediated delivery. .................................................................................................. 62  xi  List of Abbreviations  [Ca2+]i Intracellular chloride concentration [Cl-] i Intracellular chloride concentration [K+] e Extracellular potassium concentration AAV Adeno associated virus aCSF Artificial cerebral spinal fluid ActB β- Actin ADP Adenosine diphosphate AMPAR α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor APV (2R)-amino-5-phosphonovaleric acid ATP Adenosine triphosphate CACC Calcium activated chloride channels cAMP Cyclic adenosine monophosphate Cav 2.1 Voltage gated P/Q type Ca2+ channel CFTR Cystic fibrosis transmembrane conductance regulators CNS Central nervous system DC Direct current DICER Endoribonuclease DICER DIDS 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid DiIC18 Lipophilic carbocyanine dye 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate  dsRNA Double strand RNA EAAT Glial glutamate transporter ECS Extracellular space eGFP Enhanced green fluorescent protein eYFP Enhanced yellow fluorescent protein GABA γ-aminobutyric-acid GAPDH Glyceraldehyde 3- phosphate dehydrogenase GAT GABA transporter GLUT Glucose uniporter GlyH101 Glycine Hydrazide 101 ID Ischemic depolarization IOS Intrinsic optical signal KBAT Kidney brain anion transporter/ Solute carrier family 26 member 11 KCC K+/Cl-  cotransporter LNP Lipid nanoparticles LRRC8A Leucine rich repeat containing 8A MCT Monocarboxylate transporter miRNA Micro RNA mV Millivolt NBCe Electrogenic Na+/HCO3- cotransporter xii  NKCC Na+-K+-2Cl- cotransporters NMDA N-methyl-D-aspartate NMDAR N-methyl-D-aspartate receptor PMT Photomultiplier pO2 Partial oxygen pressure PVDF Polyvinylidene fluoride RISC RNA-induced silencing complex RNAi RNA interference ROI Regions of interest ROS Reactive oxygen species S1 Primary somatosensory cortex SD Spreading depolarization SDS/PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SGLT Sodium glucose cotransporter ShRNA Short-hairpin RNA siRNA Small interfering RNA SLC Solute carrier SLC26A11 Solute carrier family 26 member 11 SLC4A3 Solute carrier family 4 member 3 TBST Tris-buffered saline containing 0.1% Tween 20 Thy1 Thymocyte antigen 1 TTX Tetrodotoxin Vm Membrane potential VRAC Voltage regulated anion channels VSOAC Volume-sensitive organic osmolyte and anion channel    xiii  Acknowledgements I express my sincerest gratitude to Dr. Brian MacVicar for his supervision and inspirations throughout my project. I am also thankful to be surrounded by passionate individuals from the MacVicar lab. In particular, I am grateful for the moral support and advices from Mrs. Rebecca Ko at times of frustration and Dr. Leigh Wicki-Stordeur for her thorough instructions and patience in training me for qPCR and Western blot techniques. I would like to thank Ms. Elisa York and Dr. Leigh Wicki-Stordeur for comments and edits on this manuscript.  I appreciate the insightful suggestions and comments from my committee members and examiners Dr. Terry Snutch, Dr. Yutian Wang, and Dr. Catharine Winstanley. I acknowledge Dr. Hyun Beom Choi, Dr. Pieter Cullis, Mrs. Yuping Lu, Ms. Joslyn Quick and Dr. Leigh Wicki-Stordeur for their contribution to the project. I also appreciate the help from Mrs. Lucy Yang for training me in rodent surgical techniques.  I am grateful for all the love and support from my friends and family. Finally, I am deeply inspired by Dr. Aristides Leão (1914-1993) for his endeavor in describing an accidental finding, spreading depolarization, which later became a classic neuroscience phenomena.        1  Chapter 1: Introduction Spreading depolarization (SD) is a phenomenon observed in many disorders in the central nervous system (CNS). It is the cellular mechanism responsible for migraine aura which occurs in an otherwise healthy brain (Leao 1987). At the same time, it is also responsible for expanding infarct volume in acute injuries such as stroke, cerebral hemorrhage, and brain trauma (Somjen 2001; Pietrobon and Moskowitz 2014; Dreier 2016; Hartings et al., 2017). One feature of SD is the presence of cellular swelling, which not only alters normal CNS function, but also increases intracranial pressure, leading to devastating outcomes such as potential death through herniation (Somjen 2001). Thus, understanding the cellular mechanisms underlying SD and cell swelling will contribute to the basic knowledge of physiology and identify potential therapeutic strategies.   1.1 Mechanisms of cell volume regulation in the CNS Cell volume is tightly regulated for optimal physiological functions. Volume regulation is especially critical in the CNS due to the presence of excitable membranes and the rigid skull. Thus, mechanisms that are sensitive to volume perturbations are of great importance.   1.1.1 Why is cell volume regulation important? Maintenance of cell volume is essential for a system to function properly. This is especially critical in the CNS for two major reasons. Firstly, the CNS encompasses excitable cells. Cell swelling decreases the extracellular space (ECS), which 1) affects the diffusion and clearance dynamics of metabolites, ions, substrates and neurotransmitters, 2) increases concentrations of substrates restricted in the ECS compartment, and 3) increases extracellular electrical resistance (Sykova 1997). Secondly, the CNS is enclosed within a fixed cavity bounded by the skull. Thus, swelling will cause rises in intracranial pressure that can result in tissue damage, restriction of blood flow, and is potentially life threatening situations through herniation and compression of brainstem vital centers.  2  Swelling in the CNS was first subdivided by Igor Klatzo in 1967, arising from either vasogenic or cytotoxic edema. Vasogenic edema describes an influx of fluid from the cerebral blood circulation to the CNS by crossing a compromised blood brain barrier (Klatzo 1967). On the other hand, cytotoxic edema refers to an influx of fluid from the ECS to the intracellular space (Klatzo 1967). Thus, although cellular swelling is present, the overall CNS volume remains uninfluenced in cytotoxic edema (Kimelberg 1995). However, in scenarios such as cerebral ischemia and intracerebral hemorrhage, vasogenic edema and cytotoxic edema often coalesce, making it difficult to distinguish the two definitively (Keep et al., 2017).   1.1.2 Mechanisms of astrocyte and neuronal swelling Numerous literature has investigated the mechanisms of astrocytic swelling.  Astrocytes swell readily in response to a variety of stimuli such as elevated extracellular potassium concentration ([K+] e) and hypotonicity (Walz 1992; Kimelberg et al., 1990; Kimelberg 2000). Orchestrations between the Cl-/HCO3- exchanger and Na+/H+ transporter mediate Na+ and Cl- intake, responsible for generating an osmotic force (Kimelberg 1991). In addition, the electrogenic Na+/HCO3- cotransporter (NBCe) also plays a role in generating osmotic gradients (Bevensee et al., 2000). The NBCe exchanges Na+ and HCO3- at a 1:2 stoichiometry (Deitmer and Schlue 1987) in response to rise in [K+]e, depolarization, and extracellular pH changes (Erlichman and Leiter 2010). Following creation of the osmotic gradient, water passively enters the cell through aquaporins (predominately AQP4) that are expressed abundantly in astrocytes (Nielsen et al., 1997). Swelling of astrocytes not only contributes to decreases in ECS, but also causes release of substrates such as L-glutamate, aspartate and taurine (Kimelberg et al., 1990; Rutledge 1996). Swelling induced glutamate release from astrocytes may contribute to excitotoxicity and neuronal death by activating neuronal glutamate receptors (Choi 1992). However, less is known with regards to swelling of neurons. The conduit for chloride currents in neurons was only identified recently (Figure 1. Proposed mechanisms of neuronal cytotoxic edema and related modulators.). Rungta and colleagues (2015) 3  described a role for the solute carrier family 26 member 11 (SLC26A11) in mediating chloride influx in a model of cytotoxic edema. Furthermore, the mediators of water entry in neuronal swelling remain a mystery. Neurons do not swell under hypotonic conditions, suggesting a lack of passive water channels (Andrew et al., 2007). This observation corresponds to immunostaining results where neurons from multiple brain regions were consistently unstained for AQPs (Nielsen et al., 1997).    4   Figure 1. Proposed mechanisms of neuronal cytotoxic edema and related modulators.  During ischemia, Na+/K+ pump function is compromised due to a lack of ATP. This results in a disrupted ion gradient causing a net inward current from Na+ and K+ currents, leading to membrane depolarization. The SLC26A11 channel is then activated and mediates Cl- influx. NaCl accumulation inside the cell creates osmotic stress. This osmotic gradient then drives water entry and consequently causes neuronal swelling. AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; NMDAR, N-methyl-D-aspartate receptor; APV, (2R)-amino-5-phosphonovaleric acid; TTX, tetrodotoxin; ATP, adenosine triphosphate; ADP, adenosine diphosphate; Vm, membrane potential; SLC26A11, solute carrier family 26 member 11; DIDS, 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid; GlyH-101, N-2-Naphthalenyl-2-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide. Figure adapted from Rungta et al., 2015.    5  1.2 Chloride channels and transporters in the CNS In the CNS, five major classes of channels and transporters are responsible for mediating Cl- flux across cell membranes. These include, 1) Ligand gated chloride channels, 2) Cystic fibrosis transmembrane conductance regulator, 3) Calcium activated chloride channels, 4) Voltage activated chloride channels, and 5) Cation-chloride transporters. The functions of these conduits include the modulation of synaptic transmission, regulation of cell volume, as well as establishment and maintenance of ion gradients.    1.2.1 Ligand gated chloride channels γ-aminobutyric-acid (GABA) receptors and glycine receptors are the two major sources of inhibitory synaptic transmissions in the mature CNS. Upon binding of the corresponding ligands, Cl- fluxes through the channel pore in accordance to its gradient. The Cl- gradient across neuronal membrane varies with developmental stage, where a steady decrease in the intracellular chloride concentration ([Cl-] i) occur as the cell matures (Jentsch et al., 2002). This reversal of Cl- gradient has been proposed to be due to the developmental changes in expression of the K+-Cl- co-transporter isoform 2 (KCC2;DeFazio et al., 2000; Hubner et al., 2001).  1.2.2 Cystic fibrosis transmembrane conductance regulator The cystic fibrosis transmembrane conductance regulators (CFTR) are activated by cyclic adenosine monophosphate (cAMP) to mediate Cl- secretion (Verkman and Galietta 2009). The CFTR is important in secretory cells and fluid transporting cells, such as epithelial cells in the airway, submucosal glands, pancreas, intestines and the testis (Jentsch et al, 2002; Amarai and Kunzelmann 2007). CFTR is expressed in many brain regions including the cortex, hippocampus, hypothalamus and midbrain (Johannesson et al., 1997; Guo et al., 2009). Mutations of the CFTR gene have been identified as a contributor for cystic fibrosis, a genetic disorder with impaired lung functions, male infertility and other secretory complications (Rommens et al., 1989).  6  1.2.3 Calcium activated chloride channels In the CNS, calcium activated chloride channels (CaCC) are involved in regulating neuronal excitation. The CaCC mediate Cl- current in respond to rises in the intracellular calcium level ([Ca2+]i). The molecular identity of CaCC is yet to be defined with several potential candidates such as bestrophins (Qu et al., 2003), calcium-activated chloride channel family proteins (Cunningham et al., 1995), ClC-3 (Huang et al., 2001), and anoctamins (Yang et al., 2008; Caputo et al., 2008; Schroeder et al., 2008). In particular, anoctamin-1 and 6 (alternatively, TMEM16A and TMEM16F) also respond to cell volume changes during apoptosis and cell swelling (Almaca et al., 2009; Kunzelmann 2015).  1.2.4 Volume activated chloride channels Swelling activated Cl- current is observed in many cells including neurons (Nillus et al., 1997; Okada 1997; Fisher et al., 2008). This current demonstrates moderate outward rectification and is associated with the passage of other anions such as NO3-, HCO3-, gluconate and taurine (Hoffman et al., 2009). Thus, the channel is denoted as volume-sensitive organic osmolyte and anion channel (VSOAC). The molecular entity of this channel is still inconclusive with P-glycoprotein (Nillus et al., 1997; Okada 1997), bestrophins (Chien and Hartzell 2007), ClC-3 (Duan et al., 1997) and phospholemman (Nillus et al., 1997) as proposed candidates.  The ClC-2 is a volume sensitive Cl- channel activated by cell swelling (Grunder et al., 1992). ClC-2 shows an inwardly rectifying current (Thiemann et al., 1992; Grunder et al., 1992) responsible for Cl- extrusion and possibly, cell volume recovery (Rinke et al., 2010).  Recently, two independent groups identified a new protein, Leucine rich repeat containing 8A (LRRC8A/SWELL-1), is essential for volume activated anion channel activity (Voss et al., 2014; Qui et al., 2014). The expression profile shows wide tissue distribution, including the brain, and on the plasma membrane (Qiu et al., 20014). LRRC8A mediates swelling induced aspartate, glutamate and taurine efflux (Voss et al., 2014; Qui et al., 2014). 7  1.2.5 Cation chloride co-transporters Under physiological conditions, Cl- uptake is mediated by the Na+-K+-2Cl- cotransporters (NKCC), while the K+-Cl- cotransporters (KCC) mediate Cl- extrusion (Payne et al., 2003). The cation-Cl- cotransporters are involved in establishing Cl- gradients (Kaila 1994) and regulating cell volume (Hoffmann and Dunham 1995). These transporters are secondary active and electrically neutral (Payne et al., 2003).  Expressions of NKCC1 and KCC isoforms 1 to 4 have been detected in the CNS. NKCC is ubiquitously expressed in both neurons and glia as well as other cells in the CNS (Plotkin et al., 1997; Yan et al., 2001; Mikawa et al., 2002). The KCC isoforms are also present in the CNS although expression levels of KCC1, 3, and 4 are limited (Payne et al. 1996; Rivera et la., 1999; Li et al., 2002). It is important to note that KCC2 is exclusively expressed in mature neurons, responsible for establishing a low [Cl-]i (Kanaka et al., 2001; Li et al.2002). The mechanisms of activation for NKCC and KCC involves, respectively, protein phosphorylation (Hass et al., 1995; Lytle and Forbush 1996) and dephosphorylation (Jennings and Schulz 1991), which are sensitive to [Cl-]i. During development, immature neurons express NKCC1 (Mikawa et al., 2002; Li et al., 2002; Clayton et al., 1998), while KCC2 levels are low (Rivera et al., 1991; Vu et al., 2000; Clayton et al., 1998,). A steady increase in KCC2 expression level is observed as CNS development progresses and is responsible for the low [Cl- ]i present in mature neurons (Rivera et al., 1991; Vu et al., 2000; Payne et al., 2003). NKCC1 also responds to cell shrinkage and mediates regulatory volume increase to restore cell volume (Russel 2000; Fuster et al., 2004). Likewise, KCC2 is activated by cell swelling and mediates regulatory volume decrease (Payne et al., 2003).  1.2.6 Solute carrier gene series The solute carrier (SLC) gene series encompasses 395 transporters belonging to 52 families (Hall et al., 2016). As the name suggests, SLCs are responsible for transporting ions and small molecules, such as neurotransmitters. Specifically, members of the SLC family 4 and 11 are capable of mediating chloride currents. However, only SLC4 member 3 (SLC4A3) show CNS expression. Under physiological conditions, 8  SLC4A3 exchanges extracellular Cl- for intracellular HCO3- at a 1:1 ratio. SLC4A3 activity is increased by a rise in intracellular pH, and is thus potentially involved in pH regulation by exporting HCO3- (Brien et al., 2014). Similarly, two members of the SLC26 family, SLC26A9 and SLC26A11, also qualify for the purpose of this report in their specificity for Cl-, and CNS expression. SLC26A9 and SLC26A11 are also Cl-/HCO3- transporters that function in a pH dependent manner (Bee et al., 2009). Importantly, SLC26A11 was identified to mediate Cl- influx that resulted in neuronal swelling (Rungta et al., 2015).   SLC26A11 (also known as the kidney brain anion transporter; KBAT) is expressed in the kidney, brain and placenta. This 606 amino acid protein forms an 11 transmembrane domain transporter that is found on both plasma membrane and intracellular membranes (Vincourt et al., 2003). The transporter shows permeability to Cl-, and is sensitive to GlyH-101 and other CFTR inhibitors (Rahmati et al., 2013). Rungta et al. (2015) demonstrated that both GlyH-101 and siRNA to Slc26a11 resulted in a significant block of neuronal swelling during cytotoxic edema (Figure 1.).   1.3 CNS cell volume change in physiology and pathology Volume changes in cells of the CNS are present in both physiology and pathophysiology. Normal CNS functions such as synaptic activity and apoptosis are correlated with cell volume increase and decrease, respectively. Cell swelling also occurs in acute injuries, such as stroke and traumatic brain injury.    1.3.1 Excitability Changes in cell volume can have impacts on neuronal excitability. Studies have demonstrated that hypotonicity increased the excitability of hippocampal CA3 neurons and pyramidal neurons in the neocortex (Rosen and Andrew, 1990; Saly and Andrew, 1993). In addition, excitatory postsynaptic potentials were potentiated along with an increased likelihood of evoked epileptiform activity (Saly and Andrew, 1993).  It has been suggested that cell swelling may modulate excitability by swelling-induced release of excitatory neurotransmitters and/or by shrinkage of the ECS. Swelling-activated release of neurotransmitters, such as glutamate, has been well documented in 9  brain slices (Saransaari and Oja, 1999; Franco et al., 2001). Likewise, dendritic swelling has also been observed during intense neuronal activity (McBain et al., 1990; Dietzel et al., 1980). The shrinkage of the ECS likely facilitates ephaptic transmissions, and influences the diffusion dynamics of neurotransmitters and other solutes (Schwartzkroin et al., 1998). The potentiated ephaptic interaction likely contribute to phenomena involving synchronous activities, such as seizures (Pasantes-Morales and Tuz, 2006).  1.3.2 Cell death The current view on cell death categorizes the process into three groups: necrosis, apoptosis, and autophagy (Fink and Cookson 2005). Among these processes, necrosis and apoptosis are accompanied by changes in cell volume. Oncosis (“onkos”, Greek for swelling), an early stage of necrosis, is a process involving organelle swelling, cellular swelling, and disruptions of membrane integrity (Majno and Joris, 1995). Imbalances between ATP generation and energy consumption result in oncosis, which often is a consequence of external interference (Majno and Joris, 1995). Elevated [Ca2+]i contributes to necrosis through activations of cellular proteases, which in turn mediate breakdown of cytoskeleton, plasma membrane, and plasma membrane associated proteins (Liu and Schenellmann 2003; Liu et al., 2004). For instance, energy failure as a result of ischemia initiates necrosis by disrupting Na+/K+ ATPase function, leading to failure of ion gradients. This disruption causes depolarization, Cl- and water entry, glutamate release and activations of glutamate receptors. Activation of ionotropic glutamate receptors, such as the N-acetyl-D-methyl (NMDA) receptor, leads to Ca2+ influx. Rises in [Ca2+]i activate proteases and generate reactive oxygen species, ultimately leading to cell death and the release of cellular contents (Figure 2). These contents are deleterious to nearby cells, creating a series of necrosis and cell death in the proximity (Pasantes-Morales and Tuz 2006; Liang et al., 2007). In contrast to necrosis, apoptotic neuronal death is a precisely controlled process that involves cell shrinkage mechanisms. The shrinkage process involves coordinated K+, Cl- , water and organic solute efflux through various volume and/or voltage sensitive channels and transporters (Pasantes-Morales and Tuz, 2006; Fink and Cookson, 2005). 10  The series of orchestrated events during apoptosis correspond to the term’s elegant definition in Greek, which describes leaves “falling off” a tree, identified and named by Kerr and colleagues (1972).   Figure 2. Mechanism of cytotoxic edema and cell death during ischemia.  Due to the energy failure associated with ischemia, ATP supply is no longer sufficient for the Na+/K+ ATPase activity. This leads to an increase in [Na+]i, depolarization, cell swelling, and glutamate release through exocytosis, voltage regulated anion channels (VRAC) and reversal of glutamate transporters. Excess glutamate in the ECS triggers further Ca2+ influx, leading to the generation of reactive oxygen species (ROS) and membrane lipid peroxidation. The compromised cell membrane in combination with more swelling ultimately results in cell rupture and release of cell contents, which is deleterious to nearby cells. Figure adapted from Pasantes-Morales and Tuz, 2006.   1.3.3 Cell swelling in pathology During cytotoxic edema, there is a shift in fluid from the ECS into the intracellular space (Klatzo 1967). This shunting process is driven by the redistribution of ion gradients, which is often a result of energy failure (Pasantes-Morales and Tuz, 2006). Such energy 11  shortages occur in cases such as stroke and traumatic brain injury. The mechanisms of cell swelling and cell death follow the processes described in necrosis. Again, this involves depolarization, cell swelling, glutamate release, NMDAR activation, calcium activated proteases, and cell death (Pasantes-Morales and Tuz, 2006; Liang et al., 2007). Alternatively, cell death independent of Ca2+ influx following edema has also been demonstrated. This was first demonstrated by Rothman in 1985 in hippocampal cultures, where cells still exhibited swelling and lysis upon exposure to excitatory amino acids when Ca2+ was removed from the media (Rothman, 1985). This aspect of cell death was also observed in the experiments of Rungta et al., (2015) where pharmacological inhibition of chloride transporters abolished Ca2+-independent but not Ca2+-dependent cell death.   However, it is interesting that cell swelling is also observed in spreading depolarization, a phenomenon observed in gray matter that does not involve energy shortages. Experimentally, spreading depolarizations are consistently induced by elevating [K+]e in vitro and in vivo. This rise in [K+]e introduces a disruption to the ion gradients across the membrane, resulting in depolarization. High K+ solutions alone, without methods that interfere with cell metabolism, are sufficient to cause transient cell swelling (Gido et al., 1993; Gido and Siesjo, 1994). Thus, spreading depolarization is viewed as an innocuous, self-limiting process occurring in an otherwise healthy brain. On the other hand, imposing challenges on cell metabolism (such as oxygen-glucose deprivation and anoxia), result in irreversible cell swelling and death.   1.4 Overview of spreading depolarization (SD) Spreading depolarization (SD) was first described by Dr. Aristides Leão in 1944 as an accidental finding while studying experimental models of epilepsy. SD produced an intense, transient wave of depolarization and silencing of cortical activity that propagated across the grey matter. Additional features of SD include disturbances in ion gradients, membrane potentials and optical signals. Throughout the decades, perspectives on the implications of SD has shifted from purely innocuous, to both harmless and detrimental depending on the tissue status.   12  1.4.1 A brief historical view of SD In 1944, Brazilian neurophysiologist, Aristides A. P. Leão described an unexpected observation in an attempt to study experimental epilepsy. In Leão’s seminal publication, he described a silencing wave of spontaneous brain activity measured upon electrical stimulation in the cortex of an anesthetized rabbit (Leão, 1944). Following high frequency stimulation, a complete silencing of cortical activity was observed; first at the recording electrode in close vicinity to the stimulation, then traveling to the next recording electrode in proximity (hence, SD was previously termed “spreading depression”). Later works from Leão concluded that SD is associated with: (1) a slow negative voltage change of up to -15mV (Leão 1947), (2) dilation of pial arteries (Leão 1947), (3) self-sustained spread at a speed of 3 mm/minute (Leão 1987), and (4) a refractory time of at least 1 minute (Leão 1987). SD is consistently observed in numerous brain regions, including the hippocampus, cerebral cortex, striatum, and cerebellar cortex (Somjen, 2001).   The implications of SD were unveiled when Karl Lashley (1941) analyzed his own scintillating scotoma (a visual form of migraine aura) and attributed it to a wave of depression across the visual cortex at an approximate speed of 3 mm/minute. Linking the speed as a commonality, Leão and Morison (1945) proposed that SD may be the underlying cause of scintillating scotoma. For the next few decades, there was a lack of electrophysiological evidence on the presence of SD in humans. Technical advancement of subdural-electrocorticography enabled the detection of SD in patients with acute brain insults such as stroke and trauma (Mayevsky et al., 1996; Strong et al., 2002; Dreier et al., 2006, Fabricius et al., 2006; Dohmen et al., 2008). Initiating at the injury core, SD propagates into the penumbra where it imposes additional metabolic burdens (Bere et al., 2014, Kao et al., 2014). There is now ample evidence demonstrating the role of SD in causing secondary damage and expanding infarct volume in acute brain injuries (Iijima et al., 1992; 1993; Chen et al., 1993; Takano et al., 1996; Mayevsky et al 1996; Mies et al., Rawanduzy et al., 1997; Dijkhuizen et al., 1999). Therefore, it has been proposed that monitoring SD parameters could help in predicting surgery and recovery outcomes (Hartings et al., 2009). In addition, therapeutic approaches targeting SD (such as 13  hypothermia and NMDA receptor antagonists) could produce promising effects (Sakowitz et al., 2009; Hartings et al., 2009; Dreier, 2011).   1.4.2 Tissue responses to SD As described originally by Leão, SD is associated with a slow negative shift in the extracellular direct current (DC) potential of -7 to -15 mV (Leão 1944). It was confirmed that inward current flow predominates during this negative phase of the extracellular DC potential (Wadman et al., 1992; Kraig et al., 1999).  SD is also associated with massive ion redistributions. K+ levels increase while Cl-, Na+ and Ca2+ levels decrease in the ECS (Somjen 2001; Pietrobon and Moskowitz 2014) (Figure 3). Consistent with these observations, complete depolarizations of neurons are present (Collewijn and Van Harreveld 1966; Higashida et al., 1974; Sugaya et al., 1975).  In his later works, Leão also described an increase in tissue electrical resistance associated with SD (Leão 1953). This has been attributed to cell swelling (Van Harreveld and Schade 1959; Van Harreveld and Khattab 1967; Kow and Vanharreveld, 1972) and shrinkage of ECS (Hansen and Olsen, 1980). It is interesting to note that the ECS also shrinks (to a lesser extent) during normal neuronal activity (Dietzel et al., 1980).   In 1958, Gouras reported a propagation of both electrical signatures and a visible “milky wave” during SD in frog retinas. This observation has been confirmed and characterized in the neocortex as an initial brief increase in light reflectance (also termed the intrinsic optical signal, IOS) followed by a brief decrease and finally a slow increase (Martins-Fereira and de Oliveira Castro, 1966). The causes of these alterations in IOS is attributed to changes in tissue light scattering properties due to cell swelling (Fayuk et al., 1999; Lipton 1999), organelle swelling (Bahar et al., 2000), and dendritic beading (swelling of dendrites that resemble “beads on a string”; Andrew et al., 1998).  During SD there is an increase in the extracellular glutamate concentration (Van Harreveld and Kooman, 1965; Somjen 1987; Fabricius et al., 1993; Davies et al., 1995). This was explained by glutamate and organic anion efflux as compensation for the decrease in extracellular Cl- level (Nicholson, 1984). In addition, swelling-induced 14  glutamate release from astrocytes also contributes to this rise (Snow et al., 1983; Kimelberg et al., 1990; Kimelberg, 2000).  Extracellular pH changes are observed during SD as an initial alkalization followed by acidosis. Production of ammonium ions (Kraig and Cooper 1987), extrusion of HCO3- (Kaila 1994), and proton uptake (Smith et al., 1994; Grichtchenko and Chesler 1996; Smith and Chesler, 1999) is thought to contribute to the pH increase. On the other hand, acidic metabolites, from the increased metabolic activity needed to reestablish ion gradients, contribute to the acidosis (Cruz et al., 1999; Křivánek, 1961).   Finally, SD is associated with changes in local blood flow, tissue partial oxygen levels (pO2) and haemoglobin desaturation. Local blood flow increases initially, but a prolonged reduction follows (Piilgaard and Lauritzen, 2009). The reduction in blood flow extends long after spontaneous brain activity has resumed, suggesting compromises in neurovascular coupling (Wahl et al., 1987; Seitz and Lindauer, 2004). Increased mitochondrial activity, with high oxygen demand for oxidative phosphorylation and ATP production, contribute to both pO2 (partial oxygen pressure) decrease and haemoglobin desaturation (Takano et al., 2007, Piilgaard and Lauritzen, 2009; Chang et al., 2010). Collectively, these changes result in local tissue hypoxia or even anoxia (Takano et al., 2007, Piilgaard and Lauritzen 2009; Chang et al., 2010).  To summarize, tissues respond to SD by 1) slow shift in the extracellular direct current potential, 2) massive ion redistributions, 3) glutamate release, 4) reduction of extracellular space due to cell swelling, 5) changes in the intrinsic optical signals, 6) changes in tissue pH, and 7) local hypoxia. 15   Figure 3. Changes in extracellular potential, glutamate and ion concentrations during cortical SD. SD wave front (arrow and dotted line) propagates across (toward the left on figure) the cerebral cortex resulting in a negative shift in extracellular potential (Vo), rise in extracellular K+ and glutamate, and decrease in Na+, Cl- and Ca2+.  Figure adapted from Pietrobon and Moskowitz 2014.  1.4.3 Mechanisms of SD The definitive mechanisms for SD initiation and propagation are still controversial. One major challenge is that SD evoked by different stimuli are pharmacologically heterogeneous. Furthermore, there are no current experimental models suitable for reflecting the spontaneous SD that appears in migraine aura (Pietroborn and Moskowitz, 2014).  SD induced by brief local puffs of high K+ solution or persistent exposure of high K+ solution are better studied among different induction protocols. Initiation and 16  propagation of SD evoked by K+ pulses are dependent on a net inward K+ current that leads to depolarization (Kager et al., 2002), release of more K+ into the ECS, voltage gated P/Q type Ca2+ channel (Cav 2.1)-dependent glutamate release (Tottene et al., 2011), activations of NMDA receptors (Kruger and Luhmann, 1999; Hernandez-Caceres et al., 1987), and the glutamate and K+ clearance capacity of astrocytes (Cholet et al., 2002; D’ambrosio et al., 2002). On the other hand, persistent exposure to elevated extracellular K+ induces SD that is not blocked by Cav 2.1 channel inhibitors (Petzold et al., 2008) or Ca2+-free solution (Zhou et al., 2010). Presynaptic NMDA receptors and mitochondrial release of Ca2+ through the mitochondrial Na+/Ca2+ exchanger may be relevant in [Ca2+]e independent SD (Zhou et al., 2013). Although researchers are puzzled by such pharmacological contradictions, evoking SD with different protocols may be of relevance for SD in distinct tissue conditions. For example, persistent high [K+]e might be relevant for scenarios during ischemia while brief high K+ puffs may be more reflective of conditions during migraine (Pietroborn and Moskowitz, 2014).   1.5 The continuum of SD and ischemic depolarization There has been a separation between SD and the persistent depolarization observed in an injured brain (ischemic depolarization, ID). The involvement of SD in migraine aura (Lashley, 1941; Leao and Morison, 1945) and the absence of tissue damage even from multiple SD in a healthy brain (Nedergaard and Hansen, 1988) supported this notion. However, indirect early observations suggested that depolarization of neuronal membrane potential in SD and ID were of “similar nature” (Collewijn and van Harreveld, 1966). Later on, key observations supporting the commonality between SD and ID arose from multiple electrical recordings on the cortical surface of brains that had suffered from middle cerebral artery occlusion. Depolarization first occurred from the ischemic core and propagated into the surrounding tissue as a single wave with changing properties (Koroleva and Bures, 1996; Dijkhuizen et al., 1999; Nallet et al., 1999; Buresh et al., 1999). This single continuous wave that suppressed activity in both the injury core and the penumbra may be the “common denominator” between SD and ID (Hansen, 1985) (Figure 4). 17   It is now generally accepted that SD and ID span across a continuum of a common phenomenon with cellular changes occurring at varying magnitudes (Hartings et al., 2017). These changes include 1) slow extracellular DC shift, 2) cellular depolarization, 3) altered ion concentrations, 4) glutamate release, 5) cell swelling and dendritic beading, 6) intrinsic optical change, and 7) the rate of wave propagation (Hartings et al., 2017). How is same principal mechanism capable of generating two extremely distinct outcomes (no tissue damage versus cell death)? The results of my thesis work, along with other literature, will be discussed in the final chapter in an attempt to provide clues to this question.    18   Figure 4. A singular wave of depolarization spreads across brain tissue during ischemic stroke.  SD as a single wave initiating (circle with cross) and propagating outward (arrows) from the ischemic core at an arbitrary time point (time A) post stroke. (Top) Depiction of regional cerebral blood flow (rCBF, measured in ml/100g/min), and spontaneous neuronal activity in the ischemic core, classical (inner) penumbra, outer penumbra, and healthy regions of the gray matter. (Bottom) Extracellular direct current (DC) potential shift (black trace) as a function of time from middle cerebral artery occlusion (MCAO) to time A at three recording sites (white stars). rCBF in the ischemic core (red star) is also depicted (red trace). Figure adapted from Hartings et al., 2017.    19  1.6 Rationale, hypotheses and objectives Rationale:  SD involves a transient wave of breakdown in ion concentrations in the grey matter, silencing of brain activity, neuronal depolarization, changes in tissue light reflectance, and cellular swelling. However, current literature focuses heavily on the causal events for these changes (for example, Cav 2.1 channel, glutamate release, and NMDA receptor activations), yet the relationship between these characteristics still requires clarification. Thus, it is possible that these events characterizing SD may be the cause of, or in parallel to each other.  Recently, Rungta and colleagues identified the SLC26A11 channel as the mediator of Cl- influx in a model of cytotoxic edema (Rungta et al., 2015). The investigators hypothesized that depolarization triggers SLC26A11 activation, leading to Cl- entry and cell swelling. While the exact cellular mechanism of neuronal swelling during SD is still lacking, it is possible that the SLC26A11 channel is involved given the presence of both depolarization and cellular swelling during SD. Finally, cellular processes underlying the intrinsic optical signal in SD remain unresolved since its discovery in 1958 (Gouras, 1958). As pointed out earlier, cellular swelling was proposed as one possible contributor (Fayuk et al., 1999; Lipton, 1999). Evaluating any temporal correlation between neuronal swelling and IOS change will inform upon this hypothesis.  Research hypotheses: 1) Neuronal swelling during SD is associated with SLC26A11 channel activation, and SLC26A11 inhibition will result in a reduction of swelling. 2) The intense depolarization associated with SD precedes neuronal swelling, and blocking neuronal swelling will not affect the extracellular potential shift. 3) Neuronal swelling contributes to changes in tissue light intensity during SD, and decreased swelling will alter this signal.   20  Research aims: 1) To investigate the involvement of the SLC26A11 chloride channel in neuronal swelling associated with SD. 2) To explore the relationship between neuronal swelling and extracellular potential shift during SD. 3) To examine the relationship between neuronal swelling and the IOS changes during SD.    21  Chapter 2: Methods 2.1  Brain slice preparation All experimental protocols were approved by the Animal Care Committee at the University of British Columbia, and all procedures were conducted in accordance to the guidelines provided by the Canadian Council for Animal Care. Brain slices were prepared from mice between the age of 21-35 days expressing enhanced green fluorescence protein under the Thy1 promotor (Thy1-eGFP) and 22-26 days old Sprague Dawley rats.   Animals were anesthetized deeply by isoflurane and decapitated. The brains were extracted and submerged into cold slicing solutions containing (in mM) 120 N-Methyl-D-glucamine, 2.5 KCl, 1.2 NaH2PO4, 25 NaHCO3, 1 CaCl2, 7 MgCl2, 2.4 Sodium pyruvate, 1.3 Sodium ascorbate, and 20 D-glucose, at pH 7.35. The brains were then sliced into 350 µm (mice) or 400 µm (rats) coronal sections containing the primary somatosensory cortex (S1) using a vibratome (VT 1200s; Leica Biosystems, Wetzlar, Germany). Subsequently, slices were transferred into a recovery chamber filled with artificial cerebral spinal fluid (aCSF) containing (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, 2 MgCl2, and 10 D-glucose, with pH 7.35. All solutions were saturated with 95% O2/5% CO2. Prior to experiments, slices were allowed to recover at room temperature for >30 minutes.  2.2 SD induction and pharmacology Brain slices were transferred to a recording chamber and perfused with oxygenated low magnesium aCSF at a rate of 3 mL/min. The low magnesium aCSF contained (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, 0.7 MgCl2, and 10 D-glucose, at pH 7.35. SD was induced with perfusion of high K+ aCSF at 3 mL/min for 1.5 minutes in mouse tissue, and 3 minutes in rat tissue. The high K+ aCSF contained (in mM) 88.5 NaCl, 40 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, 0.7 MgCl2, and 10 D-glucose, at pH 7.35. Following high K+ solution exposure, the perfusions were switched back to low magnesium aCSF for recovery. All experiments were conducted at room temperature. 22  For GlyH-101 experiments, slices were incubated in oxygenated aCSF containing GlyH101 (50 µM, MilliporeSigma) at room temperature for 30 minutes prior to imaging. Throughout the imaging session, all solutions contained 50 µM of GlyH-101.   From animals injected with AAV9-CamKIIα-eYFP and siRNA-LNPs, only slices within 400 µm of the injection sites were used for experiments. The anterior/posterior orientation of the slices were kept track of such that the top surface during imaging was closer to the injection site. These measures were taken to ensure maximum siRNA-LNP concentration in the imaging regions.  In Ouabain experiments, slices were imaged in low magnesium aCSF containing 30 nM Ouabain (Tocris Bioscience) for 20 minutes prior to the induction of SD and recovery.  2.3 Electrophysiology Extracellular recordings were obtained by two recording electrodes filled with low magnesium aCSF. The electrodes (~1.5 MΩ) were prepared with a Flaming/Brown micropipette puller (Model P-97; Sutter instruments, Novato, CA) from thin-walled glass capillaries (1.5 mm outer diameter, World precision instruments, Sarasota, FL). One electrode (slice electrode) was placed 90-100 µm into the S1 cortical layer IV while the other (bath electrode) was positioned in the recording bath away from tissue. For experiments monitoring neuronal cross sectional area changes, the slice electrodes were place within 100 µm of the cell of interest. Field potentials were amplified (MultiClamp 700B amplifier; Molecular Devices, Sunnyvale, CA), digitized (Digidata 1322A; Molecular Devices, Sunnyvale, CA), and directed to a computer. The field signals were sampled at 100 Hz, filtered at 10 kHz (low pass, Bessel) and monitored by the Clampex 9.0 software (Molecular devices). The final extracellular potentials were calculated by subtracting bath signals from the slice signals.   2.4 Lipid nanoparticle formulation and siRNA encapsulation Lipid nanoparticles (LNP) were prepared based on methodologies described in Rungta et al. 2013. In brief, constituents (Table 1. List of siRNA-LNP components.) were 23  dissolved in ethanol and mixed with small interfering RNA (siRNA) targeting Luciferase (non-targeting control) or Slc26a11 mRNA (Integrated DNA Technologies, Table 2) using a microfluidic micromixer (Precision NanoSystems, Vancouver, BC). A lipophilic dye (DiIC18) was incorporated to track LNP uptake. After mixing, ethanol was removed by dialysis against phosphate-buffered saline. Finally, the LNPs were concentrated to obtain a final siRNA concentration of 5 mg/mL. Properties of the resultant siRNA-LNPs are summarized in Table 3. Properties of siRNA-LNP.  2.5 Stereotaxic surgery Each animal was subjected to two injections, 1) neonatal intraventricular injection for neuronal eYFP induction and 2) intracerebral delivery of siRNA-LNP.  Neonatal Sprague Dawley rats (postnatal day 0-2) were deeply anesthetized using isoflurane. Bilateral injections of AAV9-CamKIIα-eYFP (Canadian Neurophotonic Platform Viral Vector Core, 3x1013 genome copy/mL, 1 µL per site at a rate of 0.3 µL/minute) into the lateral ventricles were performed using a glass micropipette fitted on a Hamilton syringe in combination with a stereotaxic frame. Intraventricular injection sites were approximated at the 2/5th distance on an imaginary line extending from lambda to each eye. A two week period of expression and recovery time was allowed before the second set of injection. Animals previously injected with AAV9-CamKIIα-eYFP were anesthetized with isoflurane and injected bilaterally with siRNA-LNP (5 mg/mL, 0.5 µL/site at a rate of 0.3 µL/minute) in the S1 cortex. The injection sites were determined by stereotaxic coordinates: ML ± 3.0 mm, AP -2.0 mm and DV -0.8 mm. Animals were then given 8-9 days for recovery and siRNA- LNP transfection before tissue harvesting.    2.6 Biochemistry 2.6.1 Primary neuronal cultures and siRNA-LNP treatment Primary neuronal cultures were prepared as described in Taghibiglou et al., 2009. In brief, rat embryos were collected from sacrificed Sprague Dawley mothers 18 days after fertilization. Embryo cortices were extracted and incubated with 0.25% trypsin-EDTA for 30 minutes. The subsequent homogenates were dissociated by trituration and plated 24  on poly-D-lysine coated plates (Sigma, P7280) containing plating medium for two days. The plating medium contained Neurobasal medium (Invitrogen, 21103-049), B27 (Invitrogen, 17504044), glutamic acid (Sigma, G8415) and GlutaMax (Invitrogen, 35050-061). Two thirds of the medium was then replaced with Neurobasal feeding medium containing Neurobasal medium, B27, and GlutaMax. Neuronal cultures were maintained at 37°C in incubators with a 5% CO2 atmosphere. Mature neurons (18 days in vitro) were supplemented with 5 µg/mL Apolipoprotein E and treated with 0.4 µg/mL LNP containing siLuciferase or siSlc26a11. Neuronal cultures were collected at 8 days post LNP treatment for qPCR analysis.   2.6.2 Tissue collection Brains were quickly extracted from animals (P22-26) previously injected with eYFP and siRNA-LNP. Brain slices were prepared as described in section 2.1. Thin strips (<1 mm) of cortical tissue lateral to the LNP injection sites were removed for biochemical verification of SLC26A11 knockdown. Tissue samples from the insular cortex were collected as controls from regions distant to the injection sites  2.6.3 qPCR Tissue samples and neuronal cultures were homogenized in TRIGent (Biomatik) and incubated at room temperature for 5 minutes. The homogenates were mixed vigorously with 1-bromo-3-chloropropane (Sigma- Aldrich, 10% v/v) and incubated for 10 minutes at room temperature. Subsequently, the mixtures were centrifuged at 12 000 x g for 10 minutes at 4°C. The upper aqueous phase was collected and mixed with isopropanol (50% v/v) and magnetic RNA binding beads (NanoMag-D beads, MicroMod).  The beads were trapped with magnets and the supernatants were discarded. The beads were then rinsed repeatedly with wash solutions and air dried. The wash solution contained (in mM): 80% ethanol, 10 KCl, 2 Tris (pH 7.00), and 0.2 EDTA. Finally, RNA was eluted from the dried beads using elution buffer (10 Mm Tris, pH 8.0 and 1 mM EDTA) and collected for cDNA synthesis. RNA concentrations were determined by a spectrophotometer (NanoPhotometer P300; IMPLEN, Munich, Germany). The cDNA was 25  synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and a thermal cycler (2720 Thermal cycler; Applied Biosystems, Foster city, CA). The resultant cDNA was then replicated with the KAPA probe universal PCR mix (KAPA biosystems) and primers/probes (FAM/ZEN/IBFQ double quench probes; Integrated DNA Technologies) for GAPDH, ActB sequences, and Slc26a11 primer sequences (rSLC26A11-2, Rn.PT.53a.13211186) in a thermal cycler (C1000 Touch Thermal cycler, Bio-Rad, Hercules CA). All primers were purchased from Integrated DNA Technology. The program for the PCR cycles was 95°C for 3 minutes, 40 rounds of 95°C for 15 seconds, 60°C for 45 seconds. RNA levels were detected and analyzed by the CFX Maestro Software (Bio-Rad).  2.6.4 Western blot Tissue samples were homogenized in lysis buffer containing (in mM): 100 Tris (pH 7.0), 2 EGTA, 5 EDTA, 30 NaF, 20 sodium pyrophosphate, 0.5% NP40 with phosphatase and protease inhibitor cocktail (Roche, Basel, Switzerland). The homogenates were centrifuged (13 000 x g for 20 minutes at 4°C) and the supernatants were collected. Samples containing equal amounts of proteins were diluted with 4 x Laemmli sample buffer (Bio-Rad) and mixed overnight at room temperature. Subsequently, the samples ran through SDS/PAGE and were transferred to polyvinylidene fluoride (PVDF) membranes through a transfer system (Trans-Blot Turbo, Bio-Rad, Hercules CA). Membranes were then blocked by 2% non-fat milk and rinsed with Tris-buffered saline containing 0.1% Tween 20 (TBST). The membranes were treated with rabbit anti-SLC26A11 polyclonal antibody (Abnova, 1:200 dilution) or goat anti-GAPDH polyclonal antibody (Santa Cruz, 1:500 dilution). After incubation, the membranes were washed with TBST and incubated with anti-rabbit or anti-goat secondary antibodies conjugated with horseradish peroxide. The membranes were washed again with TBST following secondary antibody treatment. Finally, protein bands were visualized using enhanced chemiluminescence (Clarity Western ECL substrate, Bio-Rad) through a blot scanner (C-DiGit Blot scanner 3600, Li-Cor Biociences, Lincoln, NE). The intensities of protein bands 26  were collected and analyzed through ImageJ 1.4 9 (NIH). Protein levels of SLC26A11 were normalized to GAPDH levels for analysis.   2.7 Two photon imaging A two-photon laser scanning microscope (Zeiss LSM 510 Axioskop 2, Oberkochen, Germany) in combination with a Titanum:sapphire laser (Coherent Mira 900, Santa Clara, CA) was used to image brain slices. For experiments involving rat brain slices, changes in the IOS was monitored with a 2.5X/ 0.075 numerical aperture (NA) objective lens and two-photon infrared laser light at 800 nm. The IOS was directed through the transmitted optical path and acquired with a photomultiplier (PMT). In experiments involving single neuron imaging (Thy1-eGFP and AAV9-CamKIIα-eYFP + siRNA-LNP animals), both the IOS and fluorescent signals were monitored with a 40X/1.0 NA objective and detected by separate PMTs. The laser was tuned to 870 nm for eGFP excitation and the low pass filter (LP680) was used to block the excitation laser light from the non-descanned detectors. eYFP was excited at 890 nm and a BP605/55 emission filter was used. DiIC18 was excited at 720 nm and a KP685 emission filter was used for detection. Cells were selected based on 1) location in layer IV of the cerebral cortex, 2) at a depth of > 50 µm, 3) shape of a pyramidal neuron, 4) spatial separation from other cell bodies with a well-defined boundary, and for siRNA-LNP experiments only, 5) co-localization of DiIC18 and eYFP. The excitation focal plane was manually adjusted throughout the experiment to account for focus changes due to tissue swelling. A z-stack with 10 planes of 1 µm increments was taken at the end of imaging sessions to ensure the focal plane was maintained with reasonable consistency.   2.8 Data analysis Regions of interest (ROI) surrounding the recording electrode were selected to collect tissue light intensity values for IOS analysis. Changes in IOS were indicated by differences (ΔI) between the light intensity at every time point and the average of those during baseline (I) and normalized to baseline as a percentage (IOS = ΔI/I). The rates of IOS change were calculated by taking the first order derivative of IOS and time. 27  Recordings were processed in ImageJ 1.49 and analyzed in MATLAB R2016a (MathWorks).   Single neuron morphology recordings were aligned in ImageJ to correct cell displacement in the x-y plane due to tissue swelling. Neurons were isolated and their cross sectional areas were obtained by custom MATLAB algorithms. Area values at each time point were normalized to baseline values. The IOS were calculated with ImageJ for the entire image.  Values were statistically compared using two tailed unpaired Student’s t-test or Two-way ANOVA with a significance value of p < 0.05. All values are reported as mean ± standard error of the mean (SEM).28   Table 1. List of siRNA-LNP components. Components Manufacturer Molar ratio in LNP  Volumetric ratio in siRNA-LNP  heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA) Biofine International, Vancouver, BC 50 1 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) Avanti Polar Lipids, Alabaster, AL 10 Cholesterol  Sigma-Aldrich, St. Louis, MO 37.5 (R)-2,3-bis(octadecyloxy)propyl-1-(methoxy polyethylene glycol 2000) carbamate (PEG-DMG) Synthesized as described in Akinc et al., 2008 1.5 lipophilic carbocyanine dye 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiIC18)* Invitrogen 1 siRNA (Luciferase or SLC26A11) Integrated DNA Technologies  3  * DiIC18 were incorporated to allow visualizations of LNP uptake.  Table 2 Sequences of SLC26A11 siRNA Sense rGrCrArUrGrUrCrArGrCrArArUrArUrArGrA Antisense rGrGrUrGrUrArGrUrCrUmArUmArUrUrGrC  Table 3. Properties of siRNA-LNP.  Size(nm) Polydispersity index % Encapsulated siSLC26A11 46.15 ± 0.84 0.077 ± 0.012 94.60 ± 0.21 siLuciferase 47.54 ± 2.51 0.085 ± 0.016 95.97 ± 1.10 29  Chapter 3: Results 3.1 SD was consistently induced by high K+ aCSF perfusion SD was consistently evoked by the perfusion of aCSF containing 40 mM KCl at a speed of 3 mL/min. Simultaneous recordings of extracellular field potential and transmitted light signals served to confirm the occurrences of SD by the presence of characteristic IOS response and the extracellular DC shift. Images of a representative SD wave front and plots of IOS and extracellular recordings are shown in Figure 5. Following SD induction, the SD wave front first appeared as a focal point that showed an abrupt increase in light intensity and then propagated throughout the cortex in all directions (Figure 5a). SD wave fronts were defined as the continuous expansion of the focal point. IOS changes were measured and quantified from a region of interest adjacent to the field recording electrode. The SD IOS had a characteristic tri-phasic pattern with an initial rapid rise in light intensity, followed by a smaller decrease and finally a gradual increase (Figure 5b). The initial rapid phase was further quantified by plotting the rate of IOS change (rIOS, the first order derivative of the IOS, Figure 5c) as described by Zhou et al (2010).  Zhou and colleagues (2010) proposed that this initial rapid IOS component is generated by neuronal swelling whereas astrocyte swelling accounts for the later slower rise. Simultaneously, there is a transient negative shift in the extracellular potential indicating the time of the SD induction (Figure 5d). The time at maximal extracellular shift amplitude coincided with the onset of the initial IOS component (Figure 5c) as well as the maximal rIOS (Figure 5b). 30   Figure 5 Electrical and optical signatures of spreading depolarization (SD).  a) Images of SD wave front propagation in the cortical brain slice. A region of interest was selected (square) for the measurement of intrinsic optical signal (IOS). Upon SD induction, a focal point appeared (asterisk) which was followed by a propagating wave front across the entire neocortex (curved outline and arrows). b) The characteristic IOS profile during SD demonstrating a tri-phasic response with an initial rapid rise, then a decrease and finally gradual increase in light intensity. The initial peak coincided with the maximal extracellular DC shift (blue dotted line). c) The rates of IOS increase calculated by taking the first order derivative of IOS plot, highlighting the abrupt initial IOS component. d) Extracellular shift in direct current (DC) potential during SD.  3.2 GlyH-101 had no effect on electrical and optical features of SD In the presence of GlyH-101 (50 µM), SD was still consistently evoked by the SD induction protocol involving high K+ aCSF perfusion. Results from 13 experiments under control conditions and 10 with GlyH-101 treatment were analyzed. The SD onset time, defined as the first appearance of a focal point post arrival of high K+ solution, was 1.522± 0.159 minutes in controls and 1.587± 0.173 minutes in GlyH-101 experiments (Figure 6a), p= 0.786; t=0.275 df=21. SD wave front traveled at a speed of 0.106± 0.010 mm2/s in controls and 0.082± 0.007 mm2/s in GlyH-101 treated slices (Figure 6b), p= 0.114; t=1.883 df=21. The wave front appeared for a total of 96.0± 9.54 seconds in 31  controls and 101.4± 9.092 seconds in GlyH-101 experiments (Figure 6c), p= 0.695; t=0.3981 df=21. The total area occupied by the wave front was 9.826± 0.718 mm2 in control slices and 8.629± 0.417 mm2 in GlyH-101 slices (Figure 6d), p= 0.198; t=1.33 df=21. All values are represented as mean ± standard error of the mean (SEM). All comparisons were statistically analyzed by Unpaired Student’s t test with a significance value p< 0.05. These data demonstrate that channels inhibited by GlyH-101 were not required for SD initiation and propagation. In addition, these channels are not involved in determining SD trigger threshold as suggested by comparing the onset time.    Figure 6 SD onset time and wave properties in control and GlyH-101 experiments.  A) SD onset time defined as the time at the first appearance of a focal point post arrival of K+ solution. B) The average speed of SD wave front expressed as mm2/s. C) Duration for which the wave front is present. D) Total area on slice that has been occupied by the SD wave front. For analysis, control n=13 and GlyH-101, 50 µM, n=10.  32  3.3 GlyH-101 reduced the magnitude of the increases in neuronal cross sectional area while electrical and optical signals remain unaffected Next, we investigated whether GlyH-101 alters swelling by monitoring neuronal cross sectional area using two-photon imaging. Brain slices from Thy1-eGFP mice allowed visualization of neuronal morphology. Field potential and the IOS changes were also monitored in parallel as indicators of SD induction. Upon SD induction, neurons displayed a transient increase in cross sectional area followed by recovery in both control and GlyH-101 experiments (Figure 7). There was a significant reduction (p<0.0001, ANOVA) of neuronal cross sectional area (Figure 7) in the GlyH101 group (n=5) in comparison to controls (n=7).   In contrast to neuronal swelling, electrical and optical signals associated with SD were unaffected in the presence of GlyH-101. Data were collected from 12 experiments under control conditions and 10 with GlyH-101 treatment for these analysis. The magnitude of extracellular DC shift was 1.356± 0.174 mV for controls and 1.368± 0.137 mV for GlyH-101 treated slices (Figure 8a), p=0.960; t=0.051 df=19. Likewise, IOS evoked by SD was also similar between controls and GlyH-101 experiments. The maximum IOS change was 1.319± 0.037 fold increase for controls and 1.303 ± 0.036 for drug treated (Figure 8b), p=0.766; t=0.302 df=20. The time period required to achieve ½ maximum of the IOS initial peak was 31.03± 10.26 s in control and 36.32± 11.59 s in GlyH-101 group (Figure 8c), p= 0.735; t=0.343 df=20. This was define as the time at ½ max initial peak IOS minus the onset of IOS change identified by the time point for which tissue light intensity is ≥ 105% of baseline. In control experiments, the maximum rate of IOS change was 1.187± 0.139, which is similar to 1.022± 0.176 observed in GlyH-101 experiments (Figure 8d), p=0.465; t=0.746 df=20. These observations suggested that neuronal swelling is secondary to field potential DC shifts and the rapid tissue IOS transients during SD.  The slower IOS component was thought to be principally contributed by astrocytic swelling. Zhou et al (2010) demonstrated that slow perfusion of high K+ solution did not evoke SD and neuronal swelling but did produce astrocyte swelling and a gradual rise in IOS similar to the slower component seen in SD. Because astrocytes 33  also express targets blocked by GlyH-101, analysis of the slower component may reveal an effect. The maximum for the gradual rise in IOS was 1.342 ± 0.05 in controls and 1.432 ± 0.690 in GlyH-101 experiments, p= 0.297; t=1.076 df =17 (Figure 8e). This occurred after 6.733 ± 0.457 minutes after the arrival of high K+ solution in controls and 7.705 ± 0.577 minutes in GlyH-101 experiments, p= 0.199; t=1.334 df=17 (Figure 8f). Given that neuronal swelling precedes astrocyte swelling during SD (Somjen 2001; Zhou et al., 2010), suppressing neuronal swelling could potentially remove this neuronal IOS superimposition and selectively reveal the astrocytic contribution. This relationship is indirectly analyzed by measuring the time period between the onset of the rapid IOS phase (which coincides with neuronal swelling) and the gradual IOS phase (which corresponds to astrocyte swelling as suggested by Zhou et al., 2010). Time delay between the maximum IOS of the rapid component and the maximum IOS of the gradual component in control was 4.378 ± 0.506 minutes and 4.891 ± 0.639 minutes in GlyH-101 condition, p= 0.534; t=0.635 df=17 (Figure 8).  Finally, the IOS changes during SD under control and GlyH-101 conditions show similar profiles as analyzed using two-way ANOVA,  p>0.999 (Figure 9). Overall, these data show that GlyH-101 was unable to alter both the rapid and gradual phases of the IOS parameters during SD as well as their temporal relationship.      34    Figure 7 Changes in neuronal cross sectional area during SD.  While both control (gray) and GlyH-101 (red) groups showed a transient increase in neuronal cross sectional area during SD, GlyH-101 treated neurons exhibited significantly smaller changes. Neuronal cross sectional areas at each time point were normalized to baseline values. For controls, n=7; GlyH-101, 50 µM, n=5. Values are shown as mean ± SEM. Two-way ANOVA analysis demonstrated a significant difference between the two conditions, p< 0.0001.  35   36  Figure 8 Comparisons between electrical recordings and optical changes of SD in control and GlyH-101 groups showed no significant differences.  A) Magnitude of extracellular field potential shift, p= 0.960. B) Maximum IOS of the initial peak, p= 0.766. C) Maximum rate of IOS change, p=0.735. D) Time required to achieve ½ of max IOS of the initial peak, p=0.465. Control, n=12; GlyH-101, 50 µM, n=10. E) Maximum IOS of the slower peak, p= 0.297. F) Time at the maximum slower IOS peak post arrival of high K+ solution, p= 0.199. G) Time delay between maximum IOS of the initial rapid component and the slower component, p= 0.534. Control, n=10; GlyH-101, 50 µM, n=9.    37   Figure 9 IOS changes during SD were similar in control and GlyH-101 experiments.  Tissue light intensities were normalized and expressed as percentages of baseline. There were no significant differences between the IOS profiles between treatments, p> 0.999 (two-way ANOVA). Control, n=12; GlyH-101, 50 µM, n=10.  3.4 Temporal profiles of IOS and neuronal cross sectional area did not correlate The changes in tissue light intensity associated with SD have been attributed to various physiological processes such as cellular swelling (Fayuk et al., 1999; Lipton, 1999), organelle swelling (Bahar et al., 2000) and/or dendritic beading (Andrew et al., 1998). Averaged plots of temporal profiles of neuronal swelling and IOS changes are shown in Figure 10. The onsets of neuronal swelling, initial IOS rise, and the maximum DC shift potential coincided in both controls (Figure 10a) and GlyH-101 slices (Figure 10b). Representative plots are shown in Figure 11 with snapshots of neuronal morphologies corresponding to each IOS stage. However, the time at maximal neuronal cross sectional area did not correspond with any of the IOS phases. These plots suggested that neuronal swelling alone could not account for any of the distinct phases in the IOS associated with SD although it could contribute to the slower later increase in IOS.  38   Figure 10 Temporal correlations between neuronal swelling and IOS changes during SD under control and GlyH-101 conditions.  The time at maximal neuronal cross sectional area (dashed line) did not correlate with any of the IOS phases (solid lines) in both control (a) and GlyH-101 (b) experiments.  Values are normalized to baseline and expressed as mean ± SEM. Control, n=7; GlyH-101, n=5.  39   Figure 11 Representative traces of neuronal swelling and tri-phasic IOS change in control and GlyH-101 treated neurons. a) Control neuron; b) GlyH-101 neuron. Snap shots of neuronal cell body visualized by GFP (a and b, top panel) during (1) baseline, (2) initial IOS peak, (3) secondary IOS decrease, (4) maximum cross sectional area, (5) maximum of the IOS final component and (6) recovery of swelling. Temporal profile of neuronal cross sectional area and IOS change (a & b, bottom panel) with labels 1-6 corresponding to the snap shots in the top panel.  40  3.5 Lipid nanoparticle mediated siRNA uptake To test for the involvement of the SLC26A11 channel in neuronal swelling during SD, we attempted an siRNA mediated knockdown approach. Lipid nanoparticles carrying small interfering RNA (siRNA-LNP) have previously been effective in knocking down target genes in the central nervous system both in vivo and in vitro (Rungta et al., 2013). Slc26a11 mRNA expression levels were determined in both neuronal cultures and brain tissue with siRNA-LNP using qPCR (Figure 12a &b). Neuronal cultures were used to ensure that the siRNA was effective against neuronal expression and were used to validate the subsequent in vivo approach.  In cultured neurons treated with siSlc26a11-LNP (0.4 mg/mL, Figure 12a), SLC26A11 mRNA levels were significantly reduced (27.33 % ± 2.246 relative to untreated controls, n=6) compared to those treated with 0.4 mg/mL siLuciferase-LNP (112 % ± 5.073 relative to controls, n=6), p <0.000; t=15.26 df=10 (Unpaired Student’s t test).  In tissue samples collected from in vivo siRNA-LNP injections (Figure 12b), SLC26A11 mRNA was significantly reduced in siSlc26a11-LNP injected brains (54.79 % ± 3.291 relative to controls, n=6) compared to tissue transfected with siLuciferase-LNP (86.12 % ± 6.425 relative to controls, n=5), p <0.0013; t=4.578 df=9. Next, we checked the SLC26A11 protein levels in siRNA-LNP injected brains. Western blot analysis revealed similar levels of the SLC26A11 protein (p=0.834; t=0.2183 df=6, Figure 12c) in tissues injected with either siLuciferase-LNP (108.2 % ± 31.3 relative to tissue distant from injection site, n=4) or siSlc26a11-LNP (100.2 % ± 19.18, n=4).  An example of the Western blot is shown in Figure 12c. These data show that the siRNA-LNP system was insufficient to suppress the SLC26A11 protein and suggests that the in vivo LNP protocol was inadequate.  41   Figure 12 Verification of SLC26A11 mRNA and protein levels treated with siRNA-LNP by qPCR and Western blot.  Mature neuronal cultures were treated with LNPs (0.4 µg/mL) containing either siLuciferase or siSlc26a11. Tissue samples were collected from animals injected with AAV9-CamKIIα-eYFP (3*1013 genome copies/mL) and siRNA-LNP (5 mg/mL, siLuciferase or siSlc26a11).  Both culture and tissue samples were collected 8 days post LNP treatment. qPCR quantifications of SLC26A11 mRNA expression in neuronal cultures (a) and injected tissue (b). (c) Representative Western blot indicating protein levels of SLC26A11 and Glyceraldeyde-3-phosphate (GAPDH) in tissue injected with siRNA-LNP. C, tissue injected with siLuciferase-LNP as control; S, tissue injected with siSLC26A11-LNP. D, tissue collected from the insular cortex (distant to the LNP injection site); I, tissue collected within 1 mm from the LNP injection site. GAPDH levels were measured as the loading control. (d) Group data for Western blot analysis of SLC26A11 protein levels in tissue samples from the siRNA-LNP injected cortex. All values are expressed as mean ± SEM and compared by Unpaired Student’s t test with a significance value p< 0.05.         42  3.6 Neuronal swelling was unaffected by siRNA-LNP uptake Imaging experiments and biochemical assays described above were done in parallel since slice preparations and tissue samples came from the same brain. SD was induced in brain slices of the siRNA-LNP injected animals to investigate whether siRNA-LNP transfection had any functional impacts. Brain slices were prepared from animals injected with AAV9-CamKIIα-eYFP and LNP containing either siLuciferase or siSlc26a11. Neurons showing colocalizations of eYFP and DiIC18 were selected for imaging (Figure 13). SD were induced and cross sectional areas for the neurons of interest were obtained. Transfections by siSlc26a11-LNP did not alter SD induced neuronal swelling (p> 0.999, ANOVA) compared to those transfected with siLuciferase-LNP (Figure 14).   Figure 13 Colocalization of eYFP and DiIC18 as an indicator for LNP transfection.  Brain slices were prepared from animals injected with AAV9-CamKIIα-eYFP and siRNA-LNP. Top Left, eYFP fluorescent; Bottom Left, DiIC18 fluorescent; Right, composite image of eYFP and DiIC18 signals with arrows indicating colocalizations. The two-photon wavelength was 890 nm for eYFP and 720 nm for DiIC18 excitation. 43    Figure 14 Neuronal cross sectional areas in siRNA-LNP transfected neurons during SD. Neuronal cross sectional areas at each time point were normalized to baseline values. For siLuciferase-LNP, n=8; siSlc26a11-LNP, n=9. All values are expressed as mean ± SEM and compared with two-way ANOVA. There are significant difference between the two conditions, p>0.999.  3.7 siSlc26a11-LNP uptake did not alter most properties of SD Similar to neuronal cross sectional area, siRNA-LNP had no effect on most properties of SD. The amplitude of extracellular DC shift was 0.915 ± 0.141 mV in siLuciferase-LNP group and 1.085 ± 0.138 mV in siSlc26a11-LNP group (Figure 15a), p= 0.406; t=0.8595 df=13. The maximum IOS was 1.237 ± 0.024 fold change relative to baseline in siLuciferase-LNP slices and 1.288 ± 0.034 for siSlc26a11-LNP slices (Figure 15b), p= 0.252; t=1.2 df=13. Maximum rIOS was 1.256 ± 0.260 relative to baseline in siLuciferase-LNP group and 0.906 ± 0.071 in siSlc26a11-LNP group (Figure 15c), p= 0.191; t=1.381 df=13. Finally, there was a significant increase in time required to achieve ½ max IOS in tissues transfected with siSlc26a11-LNP (32.46 ± 9.438 s) in comparison to those injected with siLuciferase-LNP (62.9 ± 5.466 s), p= 0.013; t=2.882 df=13 (Figure 15d). This parameter was defined as the time required from the onset of 44  IOS change to the time point at half of the maximal IOS of the initial peak. This indicates that siSlc26a11-LNP tissue showed a slower rise during the initial IOS component. Data from 7 brain slices were analyzed for the siLuciferase-LNP group and 8 for the siSlc26a11-LNP group.   Figure 15 Electrical and optical properties of SD in brain slice transfected with siRNA-LNP.  a) Magnitude of extracellular field potential shift. b) Maximum IOS of the initial peak. c) Maximum rate of IOS change. d) Time period required to achieve ½ of max IOS. For siLuciferase-LNP group, n=7; siSlc26a11-LNP slices, n= 8. All values are expressed as mean ± SEM and compared with Unpaired Student’s t test with a significance value p< 0.05.  45  3.8 Mild impairment of Na+/K+ ATPase by Ouabain had no effect on SD wave front, electrical, and optical properties In the final section of the current study, we investigated the concept that the characteristics of the SD and ID vary as a function of Na+/K+ ATPase activity which is dependent on tissue energy state. Specifically, what are the substrates that determine whether the cell will undergo transient swelling or progress toward sustained irreversible swelling? Through computational modeling, Dijkstra et al (2016) proposed that the progression toward cytotoxic swelling is dependent on the level of Na+/K+ ATPase activity.  We attempted to delineate the two forms of swelling and test this hypothesis by inhibiting the Na+/K+ ATPase using Ouabain (an Na+/K+ ATPase inhibitor) which is proposed to have similar impacts as imposing a mild metabolic challenge in parallel to SD induction.   Among the SD literature, Ouabain at 100 µM has been a stimulus for evoking SD-like depolarizations (Balestrino et al., 1999, Andersen et al., 2005, Steffensen et al., 2015). In the current study, tissue light intensities were monitored for 20 minutes in aCSF or in aCSF containing 30nM Ouabain before the induction of SD to ensure that Ouabain, at this concentration, was not capable of evoking SD. The concentration was chosen based on the Ki (31 ± 3 to 47 ± 4 nM) for Ouabain inhibition in neuronal Na+/K+ ATPase isoform containing the α3 subunit (Blanco and Mercer, 1998). The Ki for α3 containing ATPase is much lower relative to α1 (ubiquitously expressed, 43 ± 19 µM) and α2 (astrocyte specific, 150 ± 20 nM to 170 ± 10 nM) containing ATPase (Blanco and Mercer, 1998; McGrail et al., 1991; Cameron et al., 1994; Cholet et al., 2002). The occurrences of SD were identified by the presence of electrical and optical signals. Indeed, SD was not triggered throughout the 20 minute incubation period in all control as well as Ouabain treatment group (Table 4, p >0.999, Fisher’s exact test). Following incubation, SD was induced by perfusion of high K+ aCSF for 3 minutes. The onset time of SD was 1.765 ± 0.264 minutes for controls and 1.563 ± 0.150 minutes for the Ouabain group (Figure 16a, p =0.505; t=0.702 df=7). The average wave front spread rate was 0.112 ± 0.015 mm2/s in controls and 0.103 ± 0.006 mm2/s in Ouabain experiments (Figure 16b, p=0.587; t=0.569 df=7). Under the control condition, the 46  duration of wave front was 97.31 ± 21.46 s and 110.1 ± 10.99 s in presence of Ouabain (Figure 16c), p= 0.589. The total area for which SD wave front occupied was 11.47 ± 1.05 mm2 for controls and 11.75 ± 0.99 mm2 for Ouabain experiments, p= 0.853; t=0.5654 df=7 (Figure 16d).  Similar to SD wave front properties, electrical and optical properties of SD were also unaffected by Ouabain treatment. The amplitude of extracellular DC shift was 0.945 ± 0.313 mV in controls and 0.928 ± 0.185 mV for Ouabain experiments (Figure 17a, p= 0.962; t=0.04908 df=7). Maximum IOS was 1.083 ± 0.266 in controls relative to baseline and 1.234 ± 0.207 in treated groups (Figure 17b, p=0.663; t=0.5753 df=7). Maximum rIOS was also indistinguishable among control (0.853 ± 0.140 fold increase relative to baseline) and treated groups (1.166 ± 0.254), (Figure 17c p= 0.351; t=1 df=7). Time to ½ maximum IOS was 26.09 ± 5.635s in control and 51.22 ± 15.67s in Ouabain slices (Figure 17d, p=0.215; t=1.363 df=7). The maximum for the gradual rise in IOS was 1.332 ± 0.066 in controls and 1.289 ± 0.096 in Ouabain experiments (Figure 17e, p= 0.726; t=0.376 df=4). This occurred after 6.95 ± 0.636 minutes after the arrival of high K+ solution in controls and 5.485 ± 0.325 minutes in Ouabain treated slices, (Figure 17f, p= 0.207; t=1.504 df=4). Time delay between the maximum IOS of the rapid component and the maximum IOS of the gradual component in control was 3.542 ± 0.394 minutes and 3.115 ± 0.108 minutes in Ouabain experiments (Figure 17g, p= 0.513; t=0.7167 df=4). These observations suggest that at Ouabain concentrations of 30 nM, SD induced by 40 mM KCl aCSF was unaffected both electrically and optically compared to controls. For DC shift and initial peak analysis control, n=4; Ouabain experiments, n=5. For slower IOS component analysis, control n=4, Ouabain n=2. Since data from the GlyH-101 experiments suggested that IOS changes are not an indicator for neuronal swelling, it remains possible that Ouabain alters neuronal volume response to SD. This question will be addressed in future experiments using AAV9-CamKIIα-eYFP rats and we expect to observe greater or prolonged swelling after Ouabain treatment relative to control conditions.   47  Table 4 Fisher’s exact test on SD induction by 20 minutes treatment of aCSF and aCSF with 30 nM Ouabain.  Control 30 nM Ouabain Total No SD 4 5 9 SD 0 0 0 Total 4 5 9   p value >0.999   Figure 16 SD onset time and wave properties in control and Ouabain treated slices.  A) SD onset time defined as the time at the first appearance of a focal point post arrival of high K+ aCSF. B) The average speed of SD wave front expressed as mm2/s. C) Duration for which the wave front is present. D) Total area on slice that has been occupied by the SD wave front. For analysis, control n=4; Ouabain, 30 nM, n=5. 48   49  Figure 17 Electrical and optical signatures of SD in control and Ouabain groups.  A) Magnitude of extracellular field potential shift. B) Maximum IOS of the initial peak. C) Maximum rate of IOS change. D) Time period required to achieve ½ of max IOS. Control, n=4; Ouabain, 30 nM, n=5. E) Maximum IOS of the slower peak, p= 0.726. F) Time at the maximum slower IOS peak post arrival of high K+ solution, p= 0.207. G) Time delay between maximum IOS of the initial rapid component and the later slower component, p= 0.513. Control, n=4; Ouabain, 30nM, n=2.  50  Chapter 4: Discussion and future directions 4.1 Summary of findings In the present study, SD was induced in cortical brain slices containing by perfusion of high K+ aCSF. SD wave front characteristics, extracellular DC potential shift, IOS, and neuronal cross sectional area were obtained and analyzed under different conditions. First, we investigated the effect of GlyH-101, a blocker of SLC26A11 mediated Cl- currents. While neuronal swelling induced by SD was significantly reduced by GlyH-101, other properties of SD remained unaltered. The onset of swelling coincided with the DC potential shift and the initial component of IOS. Surprisingly, the maximum neuronal cross sectional area did not coincide temporally with any distinct IOS phases.  To dissect the exact mechanism of GlyH-101’s effect, we delivered siRNA via LNP to reduce expressions of SLC26A11 channels. Unfortunately, SLC26A11 mRNA was only slightly reduced in vivo as detected by qPCR and protein levels in brains injected with siSlc26a11-LNP were unaltered compared to siLuciferase-LNP injected controls. This was further supported by functional analyses demonstrating that siSlc26a11-LNP uptake did not alter neuronal swelling, extracellular DC shift, nor IOS properties during SD.  Finally, mild Na+/K+ ATPase inhibition by Ouabain was imposed in parallel to SD induction to determine if SD could be prolonged by preferentially inhibiting ATPase isoforms containing the α3 subunit, which are expressed in neurons. Ouabain in the nano-molar range was unable to alter features of SD, including wave front characteristics, DC potential shift magnitude, and IOS profiles.  4.2 Discussion 4.2.1 GlyH-101 pharmacology In the current study, brain slices treated with 50 µM GlyH-101 displayed significantly reduced neuronal swelling associated with SD. Glycine hydrazides (GlyH) were first synthesized by Muanprasat et al. (2014) to generate CFTR inhibitors with 51  greater water solubility. Through screening, GlyH-101 was identified as a potent reversible CFTR inhibitor with a Ki of 4.3 µM and a high water solubility of 1 mM (Muanprasat et al., 2014). It has been shown that GlyH-101 also inhibits SLC26A11 mediated current at 10 µM in recombinant expression systems (Stewart et al., 2011; Rhamati et al., 2013) and blocks the SLC26A11 Cl- current in cortical pyramidal neurons (Rungta et al., 2015). Beyond CFTR and SLC26A11 channel inhibition, GlyH-101 also inhibits Ca2+ dependent Cl-  conductances (CACC) and volume sensitive outwardly rectifying Cl- conductances (VSORC) in both CFTR expressing and non-expressing cell lines at 20 µM (Melis et al., 2014).  Furthermore, Barman et al. (2011) reported that GlyH-101 suppressed Na+ currents and L-type Ca2+ currents at 30 µM in cardiac myocytes. Moreover, GlyH-101 also caused cytotoxicity at 50 µM (Melis et al., 2014) as well as mitochondrial depolarization, decreased mitochondria oxygen consumption rate, and generation of reactive oxygen species at 20 µM (Kelley et al., 2010).  Therefore, the effect of GlyH-101 in reducing neuronal swelling during SD can potentially be attributed to mechanisms including 1) reduction of Cl- influx (via CFTR, SLC26A11 and/or CACC inhibition), 2) reduction in neuronal depolarization (via inhibition of Na+ current), or 3) indirectly through unknown mechanisms (via L-type calcium channel and/or mitochondria pathways). Given that attempted channel knockdown via siRNA-LNP delivery was unsuccessful in the current study, SLC26A11 mediated Cl- currents remain a potential mechanism of GlyH-101 mediated effect. On the other hand, neuronal depolarization does occur during SD, and SLC26A11 was observed to show voltage-dependent activation (Rungta et al., 2015). While the magnitude of extracellular potentials, as an indicator of depolarization, did not differ among control and GlyH-101 groups, direct measurement through single cell patching may provide further evidence on GlyH-101’s effect on depolarization.    4.2.2 Potential contributors of IOS  The IOS describes the intrinsic property of tissue to reflect and transmit white light. As the incident light reaches a media (such as brain tissue), light can be reflected, refracted, transmitted, or absorbed (Figure 18). Reflected light (LR) is defined as light 52  scattered in random directions while light being transmitted (LT) is detected at the opposite side of the tissue relative to the incident path. Scattering of the media decreases the amount of LT. On the other hand, light absorbed cannot be reflected nor transmitted.  IOS changes associated with SD were first described as an expanding “milky area” that correlated with electrical SD signals in isolated frog retinas (Gouras, 1958). It is now commonly accepted that changes in tissue scattering, rather than absorption contribute to the IOS (Martins-Ferreira and Oliveira Castro, 1965, Oliveira et al., 1985, Aitken et al., 1999). For instance, Aitken and colleagues (1999) demonstrated that brain tissue changes in LR and LT are always in opposite directions and in similar magnitudes during synaptic activity, hypotonicity, and SD. As demonstrated in the current study, SD IOS profile show a tri-phasic response beginning with an abrupt increase, followed by a decrease, and finally a gradual increase in LT. This observation is in agreement with those described previously where the main response associated with SD passage is an increase of LT (Andersen and Andrew 2002; Zhou et al., 2010; Zhou et al. 2013).  Three processes were proposed as determinants of brain tissue scattering: 1) cellular swelling, 2) organelle swelling, and 3) dendritic beading. Indeed, all three are present during SD. Cell volume increases are associated with decreases in light scattering through dilution of cytosolic contents. This has been consistently demonstrated in a variety of preparations including erythrocytes, leukocytes, and tumor cells (Orskov, 1935; Shapiro and Parpart, 1935; Lucke and Parpat, 1937). This was confirmed in brain slice preparations where mild hypotonic solution also caused an increase in light transmittance (Andrew and MacVicar 1994). The role of mitochondria has been examined during induced SD by Zhou and colleagues (2010). Transient depolarization of the mitochondrial membrane occurred immediately after the onset of SD. This effect was abolished in the absence of extracellular Ca2+, or by applying RU350 to block mitochondrial Ca2+ uptake via the mitochondrial calcium uniporter. These data suggest that mitochondrial uptake of cytosolic Ca2+ occurs after SD. Increased mitochondrial inner membrane permeability allows solutes (such as Ca2+) to 53  enter the mitochondrial matrix, which then osmotically causes water influx and leading to mitochondrial swelling (Hunter et al., 1976; Kobayashi et al., 2003). This process has been proposed to be highly dependent on the amount of Ca2+ in the mitochondrial matrix (Eisenhofer et al., 2010). Finally, evoking SD through hypoxia or oxygen-glucose deprivation (OGD) in hippocampal slices points to the role of dendritic beading in tissue scattering. Obeidat and Andrew (1998) observed a decrease in LT in the CA1 dendritic regions (statum radiatum and stratum oriens) while a LT increase in the CA1 cell body region (stratum pyramidale) was observed under OGD. In line with this evidence, Muller and Somjen (1999) reported that SD-related IOS change were more prominent in the stratum radiatum and stratum oriens.  In experiments described by MacVicar and Hochman (1991) and MacVicar and Andrew (1994), hypotonicity and neuronal activity both induced an increase in LT in both the cell body and dendritic layers of hippocampal slices. Zhou and colleagues (2010) noted that the initial IOS component was no longer present when high K+ aCSF was perfused slowly at a rate that does not induce SD and neuronal swelling. This treatment paradigm still caused astrocyte swelling and a gradual rise in LT, similar to those during the slower phase of SD IOS changes (Zhou et al., 2010). Incorporating these two observations, it is rational to predict that the maximal neuronal swelling should overlap with the maximum IOS of the rapid component during SD. However, the present study demonstrated that maximal neuronal swelling is temporally located in the transition between the second and third phase of the SD IOS profile. This observation supports the notion that IOS response reflects multiple, overlapping mechanisms that alter tissue scattering, and occur with distinct temporal profiles. It is possible that other cellular processes that decrease LT dominate and mask the effect of neuronal swelling. Data from the current study contribute to the continuing endeavor in delineating the mechanisms that give rise to the signature IOS response.   54   Figure 18 Fate of incident light upon hitting a scattering medium.  Light can be transmitted, reflected or absorbed by the tissue (blue box). Transmission is defined as light detected at the opposite side of the tissue relative to the direction of the incident light. Reflection is the light dispersed at random directions away from the incident light. Scattering (yellow dashed line) affects both reflection and transmission such that it affects the first positively and the latter negatively. Light being absorbed cannot be reflected nor transmitted. Light absorbed by the tissue can be converted to heat, fluorescence, or chemical energy such as ATP. Yellow arrows indicate the direction of light path.    4.2.3 Transient swelling and cytotoxic edema: hypothesis on tissue energy state The current project was designed to examine the contribution of the SLC26A11 Cl- channel in neuronal swelling during SD. The rationale for investigating this channel was based on its critical role in mediating Cl- influx during cytotoxic edema (Rungta et al., 2015). In the previous study, cytotoxic edema was induced through either inhibiting the inactivation of voltage gated sodium channels (via veratridine treatment in the presence of glutamate receptor antagonists CNQX and APV), or by selective activation of NMDA receptor (by applying NMDA along with TTX and CNQX). These applications evoked irreversible neuronal swelling and cell death, which were both prevented by removal of extracellular Cl- (Rungta et al., 2015). These responses closely resemble those observed during ischemic depolarization. It should be emphasized again, that SD is associated with transient swelling and in healthy tissue, is not thought to cause cell death. Although SD and ID may be manifestations of the same fundamental denominator (Hansen 1985; Hartings et al., 2017), they still lie on the extremes of the spectrum.  55  As described in Section 1.5, the key difference between SD and ID reside in the health of the tissue. For instance, evoking multiple SD events in a healthy brain does not cause tissue damage (Nedergaard and Hansen, 1988) while cell death is evident in ID during acute brain injuries. Major determinants of cell damage are the energy supply of the tissue, the magnitude of depolarization and durations of the two events (Hartings et al., 2016). Although persistent depolarization will eventually damage the healthy brain, it can caused tissue infarct within one hour only when severe ischemia was present (Nedergaard and Hansen 1988; Dreier et al., 2013).  An interesting perspective raised by Dreier and colleagues (2013) conceptualizes SD as a massive release of the Gibbs free energy stored in the transmembrane ion gradients (Figure 19). The simple Gibbs-Donnan equilibrium is described as follows: impermeable anions in the cytoplasm attract and retain membrane permeable cations such as K+ and Na+. Cl- also enters the cell to maintain electro-neutrality. Consequently, the number of charged molecule is greater in the ICS relative to the ECS causing water to flow inwards, leading to cytotoxic edema, cell lysis and death. However, mechanisms opposing this effect must exist given that cell volumes maintain relatively stable under physiological conditions. The Na+/K+ ATPase actively extrudes Na+ using ATP, and generates electrochemical energy in the form of a transmembrane potential. The work of the Na+/K+ ATPase creates a double Gibbs-Donnan steady state. Based on the laws of thermodynamics, Gibbs free energy of the current system is defined by G = U – TS, where U is the internal energy, T is the temperature, and S (entropy) is the disorder of the system. Thus, G is the lowest when ions are distributed equally across the membrane. Indeed, near-complete loss of ion gradient is observed in SD, suggesting a need for the Na+/K+ ATPase to reestablish the system after SD. It is noteworthy that Dreier and collaborators (2013) regarded SD as the “Twilight state” in deciding whether the tissue will continue to live (double Gibbs-Donnan steady state) or progress toward death (simple Gibbs-Donnan equilibrium). The importance of the Na+/K+ ATPase in regulating cell volume is further supported through computational modeling. Dijkstra and colleagues (2016) described an algorithm that predicts cell volume based on parameters such as Na+ current, K+ 56  current, SLC26A11 Cl- current, KCC co-transporter activity, leak currents, ion concentrations, membrane potential, and Na+/K+ ATPase activity. The predicted neuronal volume closely matched experimental data from Rungta et al. (2015) in modeling veratridine-induced cytotoxic edema. Using Na+/K+ ATPase strength as a variable, the algorithm revealed a “tipping point” of 65% pump function, after which membrane potential (Figure 20a) and cell volume (Figure 20b) respond dramatically to small changes in the remaining pump activity (Dijkstra et al., 2016). Thus, I imposed mild impairment of Na+/K+ ATPase activity on SD wave front and IOS changes by Ouabain. My data demonstrated that Ouabain at 30 nM (Ki of the neuronal ATPase isoform α3; Blanco and Mercer 1998) had no effect on both the wave characteristics and IOS initial spike parameters. However, given that the IOS is not a reflector of edema, further experiments involving direct measurements of tissue and cellular swelling are required to test this hypothesis. In addition, the concentration of Ouabain was predicted to inhibit only 50% of the neuronal ATPase isoform α3 which may not be sufficient.   Figure 19 “Eggs in the egg carton” analogy for Gibbs free energy state proposed by Dreier et al. 2013.  Ictal epileptic activity (IEA) and spreading depolarization (SD) with corresponding extracellular K+ concentration ([K+]e), field recording and proposed “eggs in the carton” analogy for Gibbs free energy at a given time point. IEA and SD were triggered through NMDA receptor over-activation, resulting in IEA followed by SD (Mody et al., 1987). Under physiological conditions, the system is at it’s highest energy state and [K+]e is relatively low. During IEA, [K+]e rises to a “ceiling level” of 12 mM and triggers SD, in which [K+]e increases to 60 mM. Dreier and colleagues proposed four possible energy states exist in a region of tissue representing physiological condition (highest energy), IEA (moderate), SD (low), and death (no energy). Each state is separated by an energy barrier, like the way eggs sit in pockets of an egg carton. Figure adapted from Dreier et al., 2013.  57   Figure 20 Algorithmic estimates of membrane potential (a) and cell area (b) as a function of Na+/K+ ATPase strength.  Solid line denotes stable equilibria, dashed line unstable equilibria. H, hopf bifurcation, where the stability of the system shifts. SN, saddle node, where small changes in one variable that result in a dramatic change to another. a) When pump strength falls below 65% of default, stable physiological equilibrium disappears. The pathological equilibrium remains stable until the pump strength is increased beyond physiological levels (185%). b) Under physiological conditions, cell volume remains constant. In the pathological state, cell area remains relatively stable until ATPase strength falls below 65%. After this point, the cell area responds greatly to minor changes in pump strength. Overall, the model suggests that a “tipping point” of 65% pump function regulates cell membrane potential and volume. Pump strength of 185% can potentially rescue the system from pathology back to physiology. Algorithm and figure developed by Dijkstra et al., 2016.  4.3 Limitations and considerations 4.3.1 Estimates of neuronal swelling Neuronal cross sectional areas at the optimal focal plane were obtained throughout the experiment as an indicator of swelling. However, cross sectional area (x, y plane) is not the best indicator because neurons are 3D structures and swelling expands the cell volume in all three axes (x, y, z). In addition, the optimal focal planes were determined and adjusted manually. Therefore, swelling induced changes in focal plane inevitably introduced variations in cross sectional area by occasional out of focus images. Previous literature examined the shrinkage of extracellular space by monitoring the concentrations of quaternary ammonium salts applied to the ECS (Perez-Pinzon et al., 1995; Mueller 58  and Somjen 1999; Mazel et al., 2002). Salts, such as tetramethylammonium, are restricted in the ECS and become concentrated as the ECS shrinks. While this method may be more indicative of volumetric changes, it is impossible to differentiate contributions from neuronal swelling, astrocyte swelling and dendritic beading. Alternatively, swelling in the z axis can be estimated by taking multiple x-y images at several z intervals (z-stack) at each time point. However, this approach sacrifices temporal resolution and increases the chances of mishandled focus adjustment since a single z-stack requires significantly longer acquisition time.  4.3.2 Single neuron swelling and IOS The most intriguing finding from the current study demonstrated the distinct temporal profiles of neuronal cell body swelling and IOS changes. These results must be interpreted with caution since IOS encompasses changes from the entire tissue within the region of interest. This region consists of numerous astrocytes, neuronal processes and other neurons. Thus, morphological markers that label a group of neurons in the field of interest, such as bulk dye loading or image regions with more abundant Thy1-eGFP expression, may be advantageous for this purpose.   4.3.3 siRNA-LNP siRNAs are a part of endogenous mRNA degradation pathway involving the assembly of RNA-induced silencing complex (RISC). Double stranded RNA is processed into siRNA, which loads onto and activates the RISC. In the cytoplasm, activated RISC seeks and degrades RNA with complementary sequences. In theory, perfectly processed synthetic siRNA can mediate gene silencing without the need for initial cleavage (Rao et al., 2009). However, such siRNAs require delivery vehicles, as naked siRNAs do not readily diffuse across membranes (Silva and Schnierle, 2010). Furthermore, naked siRNAs are vulnerable to degradation by tissue RNases (Silva and Schnierle, 2010). LNPs function to stabilize siRNA, confer protection, and facilitate cellular uptake through an apolipoprotein E4-dependent pathway (Akinc et al., 2010; Leung et al., 2014). Upon cell entry, only 1-2% of the siRNA are released into the cytoplasm within a limited time 59  window (Gilleron et al., 2013). Therefore, higher concentrations are required, which can potentially causes off-target effects. Because of the low siRNA release efficacy, the presence of DiIC18 is not an accurate indicator for siRNA location or activity. Most of the internalized particles are released back into the extracellular space through endosomal recycling processes (Sahay et al., 2013). DiIC18, as a lipophilic dye, likely interacts with intracellular membranes, or follows the lipid components to be recycled and released. There may be unintended variations in the LNP formulations and stability that created the ineffective in vivo delivery of siRNA in these experiments.  4.4 Future directions As discussed above, the SLC26A11-mediated Cl- currents remain a potential explanation for the efficacy of GlyH-101 to reduce neuronal swelling during SD. Results from biochemical assays demonstrated that the siRNA-LNP mediated knockdown strategy was effective in cell culture, but not in vivo. RNA interference (RNAi) is an evolutionarily conserved mechanism to degrade mRNA. RNAi has been regarded as a potent tool for gene silencing with specificity since its discovery in 1998 (Fire and Mello, 1998). Two identified processes responsible for RNAi are siRNA and short-hairpin RNA (shRNA). siRNAs are products of exogenous double-strand RNA (dsRNA) through processing by DICER endonuclease activity (Rao et al., 2009; Silva and Schnierle, 2010). The resultant siRNAs are incorporated into RNA-interfering silencing complex (RISC) which actively seeks and degrades RNA with complimentary sequence. In contrast to siRNA, shRNAs contains a hairpin like stem-loop structure and is synthesized in the nucleus (Figure 21). shRNAs are processed into microRNA (miRNA) by Drosha and DICER endonucleases and then incorporated to the RISC (Rao et al., 2009; Silva and Schnierle, 2010). The miRNA behaves similarly to siRNA to mediate gene silencing. Once integrated into the host genome, shRNA can be continually produced by host RNA polymerases and thus concentrations required for effective knockdown are much lower in comparison to siRNA. Therefore, viral delivery of shRNA represents an alternative with greater transfection and genome integration, conferring superior knockdown efficacy and duration. This approach could be a promising alternative to mediate SLC26A11 60  knockdown. In vivo delivery of shRNA through adeno-associated virus has been tested to display efficacious CNS protein knockdown (Cao et al., 2004; Xia et al., 2004; Harper et al., 2005; Badcock et al., 2005; Garza et al., 2008).  As noted in section 4.3.2, the tissue is composed of more than a single neuron being monitored. Therefore, approaches that allow morphological visualization of cell populations will provide better indications of the molecular processes underlying the IOS. Potential targets include neuronal processes, mitochondria morphology, astrocyte cell body and again, neuronal cell body. Visualization methodologies such as dye loading (non-specific) and virally mediated fluorescent protein expression (thus allowing cell/ organelle specificity) may be advantageous.    The final section of the current study examined the effect of mild impairment of Na+/K+ ATPase function on SD wave front and IOS characteristics. This was done through the use of an inhibitor, Ouabain, at the Ki of the neuronal ATPase isoform α3 (Blanco and Mercer 1998). Direct measurements of tissue and cellular swelling will provide further insight towards the hypothesis proposed by Dijkstra and colleagues (2016) in which ATPase activity is a critical determinant in the magnitude of swelling. Wide field imaging (that captures the entire area of tissue sections) during SD experiments, with or without Ouabain, will provide clues with respect to tissue swelling. Performing such protocols on brain slices of Thy1-eGFP mice would allow swelling analyses at the single cell level. In addition, it would be interesting to investigate the magnitude of swelling at various concentrations of Ouabain (up to 10 µM where it triggers SD) for a dose-dependent relationship.   Finally, the molecular conduits mediating water flux in neurons remain an enigma. Neurons do not express the passive water channels, aquaporins (Nielsen et al., 1997; Andrew et al 2007). Alternatively, some co-transporters and uniporters are capable of transporting water alongside solutes. These include the Na+/Cl- cotransporter (KCC4), Na+/K+/2Cl- cotransporter (NKCC1), Monocarboxylate transporter (MCT-1), GABA transporter (GAT-1), glucose cotransporter (SGLT-1), glial glutamate transporter (EAAT1), and glucose uniporter (GLUT1 and GLUT2) (MacAulay and Zeuthen, 2010). 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