Abstract Cessation of the blood circulation for some time during surgery of the aortic arch and repair of congenital heart defects is normally required to allow a bloodless operation field. Hypothermia is the most important mechanism for end-organ protection, particularly the brain, during such operations. Cardiopulmonary bypass is used for core cooling before total hypothermic circulatory arrest (HCA) or selective cerebral perfusion (SCP) are initiated. During hypothermic cardiopulmonary bypass, pH can be managed according to either pH- or alpha-stat principles. In the present work, the optimal pH management strategy for operations requiring HCA or SCP was explored. An experimental porcine model was used. Firstly, outcome was evaluated in a HCA model using either the α- or pH-stat perfusion strategy (I). Secondly, we sought to determine which acid-base management is more effective in attenuating ischemic brain injury during combined HCA and embolization conditions (II). In the third study, the impact of propofol anesthesia and α-stat perfusion strategy on outcome was explored (III). Finally, the acute effects of perfusion strategies in a SCP porcine were compared (IV). Hemodynamics, temperature, EEG (I-III), brain microdialysis, intracranial pressure (I-III), brain tissue oxygen partial pressure (I-III), and intravital microscopy (IV) were monitored intraoperatively. In the chronic studies, survival, postoperative neurologic recovery and brain histopathologic examination were evaluated (I-III). pH-stat strategy was associated with superior outcome compared to the α-stat strategy during a 75-minute period of deep HCA (I). In addition, despite the pH-stat strategy-related cerebral vasodilatation, this method provided better neuroprotection in a setting of cerebral particle embolization prior to a 25-minute period of deep HCA (II). Propofol anesthesia combined with α-stat perfusion strategy was observed to deteriorate the brain injury during HCA evaluated by key brain microdialysis parameters (III). Finally, when employing moderately hypothermic SCP, the differences between pH- and α-stat strategies in cerebral metabolism and microcirculation were minimal. These findings are clinically relevant since α-stat perfusion strategy is still the most commonly used acid-base perfusion strategy during hypothermic cardiopulmonary bypass in adults, and propofol one of the most used anesthetics in clinical practice. It is also noteworthy that the pH-stat strategy is not currently used in adults because of the perceived increased risk of atherosclerotic embolization. However, the advantage of pH-stat strategy over α-stat strategy could not be observed when employing SCP.

Traumatic brain injury (TBI) is a progressive disorder, in which the primary injury results in the initiation of a complex cascade of secondary biochemical and metabolic changes resulting in lasting neurological dysfunction and cognitive impairment. The heterogeneous nature of the disease has complicated the development of pharmacological agents to improve the outcomes of TBI; to date, no therapeutic treatment has been shown to be effective in clinical trials. Treatments targeting multiple secondary outcomes (cell death, axonal degeneration, and inflammation) may provide enhanced therapeutic efficacy following TBI. Small Ras family GTP-binding proteins govern diverse cellular processes by directing the relay of extracellular stimuli to the activation of select intracellular signaling pathways. Rin (RIT2) is a member of the Rit subfamily of Ras-related family of GTPases, and is expressed solely within neurons of the CNS. Early cell culture models demonstrated that Rin signaled upstream of the stress-activated protein kinase, p38, and lacked the transformative abilities displayed by other members of the Ras family, suggesting functions for Rin other than cell growth and proliferation. To begin to define the physiological function of Rin, we generated a RIT2 knockout mouse and examined the impact of Rin loss in the CNS following brain trauma. Our data demonstrates that Rin deficiency is neuroprotective following a controlled cortical impact (CCI) injury, reducing both acute hippocampal neurodegeneration and promoting sustained neuronal survival, without affecting post-CCI neurogenesis. Hippocampal neuroprotection achieved by Rin loss was accompanied by improved cognitive function in injured mice. Furthermore, we demonstrated that Rin loss led to blunting of axonal degeneration and microglial activation in the optic nerve following optic nerve stretch injury. The molecular interaction between Rin and dual leucine zipper kinase suggested a potential role for Rin in the regulation of a novel stress MAPK-dependent neuronal death cascade. Lastly, we demonstrated through diffuse closed head injury, that Rin loss mitigates cytokine release as a result of injury without altering glial activation. Together, these studies establish Rin as a novel regulator of neuronal cell death, cognitive decline, axonal degeneration, and cytokine production following traumatic brain injury.

The general aim of the thesis was to deduce whether selected naturally occurring flavonoids (genistein, epicatechin gallate (EC), epigallocatechin gallate (EGCG), baicalin) attenuate various secondary insults that may cause death of ganglion cells in primary open angle glaucoma (POAG). An ischemic insult to the rat retina significantly causes the inner retina to degenerate indexed by changes of various antigens, proteins and mRNAs located to amacrine and ganglion cells. These changes are blunted in animals treated with genistein as has been shown for ECGC. Studies conducted on cells (RGC-5 cells) in culture showed that hydrogen peroxide, L-buthionine sulfoximine (BSO)/glutamate and serum deprivation (mimicking oxidative stress), rotenone, sodium azide (affecting mitochondria function in specific ways) and light (where the mitochondria are generally affected) all generated reactive oxygen species and caused death of RGC-5 cells. EGCG was able to attenuate cell death caused by hydrogen peroxide, sodium azide and rotenone. Only EC was able to attenuate BSO/glutamate-induced cell death, in addition to cell death caused by hydrogen peroxide and rotenone. Genistein had no positive effect on cell death in experiments carried out on RGC-5 cells. Exposure of RGC-5 cells to flavonoids showed that EC and EGCG increased the mRNA expression of endogenous antioxidants such as HO-1 (heme oxygenase 1) and Nrf-2 (nuclear erythroid factor-2-related factor 2). Light insult, rotenone and sodium azide activate the p38 (protein kinase 38) pathway, while only light and rotenone activate the JNK (c-Jun amino-terminal kinase) pathway. Serum deprivation affects mitochondrial apoptotic proteins causing an increase in the ratio of Bax/Bcl2 (Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2). An insult of light to RGC-5 cells, unlike that induced by sodium azide, is inhibited by necrostatin-1 and causes an activation of AIF (apoptosis-inducing factor) with alpha-fodrin being unaffected. These studies suggest that ganglion cell death caused by insults as may occur in POAG involves various cellular signaling pathways. The selected flavonoids have diverse actions in increasing cellular defense mechanisms, and in negating the effects of ischemia and specific types of oxidative stress. The results argue for the possible use of flavonoids in the treatment of POAG to slow down ganglion cell death.

Abstract In the repair of complex congenital heart defects or in surgery of the aortic arch, normal circulation may be temporarily halted to ensure a clean, bloodless operation field. The brain is the organ most vulnerable to ischemic injury during this no-flow period, and the mortality and morbidity of these procedures today consists mostly of neurological complications. Hypothermia decreases the need for oxygen and other metabolites, and cooling the patient with an extracorporeal heart-lung machine can provide enough time to perform the necessary surgical procedures during a circulatory standstill. This procedure is referred to as hypothermic circulatory arrest (HCA). Sometimes the cerebral circulation can be maintained even if the rest of the body undergoes circulatory arrest, and this strategy, involving separate catheterization of brain-destined vessels, is referred to as selective cerebral perfusion (SCP). In this work, four separate brain protection strategies were evaluated. Two studies were performed on a surviving porcine model (I, II) to evaluate neurological recovery as well as cerebral metabolism and histopathology, and two were acute in design (III, IV), employing the modern technology of intravital microscopy to examine cerebral microcirculation. The first study (I) showed that the administration of hypertonic saline dextran (HSD) led to a decrease in intracranial pressure, improved brain metabolism, better neurological recovery and less histopathological injury of the brain tissue in association with HCA. In the second study (II) a novel pharmacological molecule, levosimendan, reduced the intracranial pressure during the operation, but no improvement in terms of cerebral metabolism, neurological recovery or histopathological brain injury was observed after HCA. In the third study (III), real-time intravital microscopy showed that in association with HCA, a leukocyte depleting filter (LDF) attached to the cardiopulmonary bypass circuit reduces the number of activated leukocytes in cerebral microcirculation. In the fourth study (IV), cerebral metabolism and microcirculation were similar during SCP independent of the acid-base management strategy. The results of this work suggest that HSD could be assessed in human trials, that levosimendan needs further studies to optimize its potential, that the LDF functions as designed and that the differences between the α- and the pH-stat acid-base management strategies with SCP did not differ in moderate hypothermia.

NMDARs are ligand-gated cation channels which are activated by the neurotransmitter glutamate. NMDARs are essential in coupling electrical activity to biochemical signalling as a consequence of their high Ca2+ permeability. This Ca2+ influx acts as a secondary messenger to mediate neurodevelopment, synaptic plasticity, neuroprotection and neurodegeneration. The biological outcome of NMDAR activation is determined by a complicated interrelationship between the concentration of Ca2+ influx, NMDAR location (synaptic vs. extrasynaptic) as well as the subtype of the GluN2 subunit. Despite the recognition that NMDAR mediated physiology is multifaceted, tools used to study subunit and location dependent signalling are poorly characterized and in other cases, non-existent. Therefore, the aim of this thesis is to address this issue. Firstly, I assessed the current pharmacological approach used to selectively activate extrasynaptic NMDARs. Here, synaptic NMDARs are first blocked with MK-801 during phasic activation and then extrasynaptic NMDARs are tonically activated. This approach relies on the continual irreversible blockade of synaptic NMDARs by MK-801 yet contrary to the current dogma, I demonstrate this blockade is unstable during tonic agonist exposure and even more so when physiologically relevant concentrations of Mg2+ are present. This confines a temporal limit in which selective activation of extrasynaptic NMDARs can occur with significant consequences for studying synaptic vs. extrasynaptic NMDAR signalling. Dissecting subunit-dependent signalling mediated by the two major GluN2 subunits in the forebrain, GluN2A and GluN2B, has been advanced significantly by selective GluN2B antagonism yet a reciprocal GluN2A selective antagonist has been lacking. Utilizing novel GluN2A-specific antagonists, I demonstrate a developmental upregulation of GluN2A-mediated NMDA currents which concurrently dilutes the contribution of GluN2B-mediated currents. Moreover, I tested the hypothesis that the Cterminus of GluN2A and GluN2B are essential in controlling the developmental switch of GluN2 subunits utilizing knock-in mice whereby the C-terminus of GluN2A is replaced with that of GluN2B. Surprisingly, the exchange of the C-terminus does not impede the developmental switch in subunits nor the proportion of NMDARs at synaptic vs extrasynaptic sites. However, replacing the C-terminus of GluN2A with that of GluN2B induces a greater neuronal vulnerability to NMDA-dependent excitotoxicity. Collectively, this work enhances our understanding of the complex physiology mediated by the NMDAR by determining how pharmacological tools are best utilized to study the roles of NMDAR location and subunit composition in addition to revealing the importance of the GluN2 C-terminus in development and excitotoxicity.

Brain injury, either surgically induced or as a result of trauma or stroke, is one of the leading causes of death and disability worldwide. Since transplantable stem cell sources are showing a great deal of promise and are actively being pursued to provide neuroprotection post-injury, in this body of work, we set out to characterize and examine the therapeutic potential of amniotic fluid derived (AF) cells as a potential cell source for cell-based therapies in mediating neuroprotection post-injury. Despite their heterogeneity, we found that AF cells are mainly epithelial in origin and express various genes involved in stem cell maintenance and neural commitment. A very small subset of AF cells also express pluripotency markers OCT4a, SOX2 and NANOG, which can be enriched for by single cell cloning. SOX2 positive clones have the capacity to give rise to a neuronal phenotype, in neural induction conditions, which can be used to examine the neural differentiation capabilities of AF cells. Subsequently, we examined the ability of AF cells to mediate a neuroprotective effect in a surgically induced brain injury model through gap junctional-mediated direct cell-cell communication and as a vehicle for GDNF delivery post-injury. AF cells express high levels of CX43 and are able to establish functional gap junctional intercellular communication (GJIC) with cortical astrocytes. We report an induction of Cx43 expression in astrocytes following injury and demonstrate, for the first time, CX43 expression at the interface between implanted AF cells and host astrocytes. In an effort to boost host endogenous neuroprotective mechanisms post-injury, via neurotrophic factor delivery, we engineered AF cells to secrete GDNF (AF-GDNF). GDNF pre-treatment significantly increased AF cell and cortical neuron survival rates following exposure to hydrogen peroxide. AF-GDNF cells, seeded on polyglycolic acid (PGA) scaffolds, survived longer in serum-free conditions and continued to secrete GDNF post-implantation activating the MAPK/ERK signaling pathway in host neural cells in the peri-lesion area. Despite some promising trends, we did not observe significant behavioural improvements following AF-GDNF/PGA implantation nor reduced lesion volume during the 7 day time-frame. In conclusion, through GJIC with cortical astrocytes and delivery of exogenous neurotrophic factors, AF cells hold great promise in mediating neuroprotection post-injury.

Lactate, a metabolic end product of glycolysis in mammals, has emerged as an important energy substrate for the brain. In addition to its energetic role, lactate was shown to modulate the excitability of neurons, to have a neuroprotective role and to participate in long-term memory formation. One previous investigation from our group reported that lactate modulates 4 synaptic plasticity-associated genes and potentiates the activity of the N-Methyl-D-aspartic acid (NMDA) receptor, a major receptor type involved in glutamatergic neurotransmission. The current thesis aimed at first to extend these findings by examining genome-wide transcriptional responses to this metabolite in cortical neurons. Using ribonucleic acid(RNA) sequencing to evaluate expression changes in protein-coding genes, we found that lactate modulates robustly after 1h, 20 genes involved in the mitogen-activated protein kinase (MAPK) signaling pathway and in synaptic plasticity in a NMDA receptor activitydependent manner and that nicotinamide adenine dinucleotide, reduced (NADH), but not pyruvate, reproduces the modulatory effects of lactate on 70% of all differentially expressed genes. In a time course experiment, genes modulated after lactate treatment for 6h and 24h were also identified; these are involved in 9 signaling pathways including circadian rhythm, drug addiction, and retrograde endocannabinoid signaling. Bioinformatics analyses indicated CREB1 and CREM as candidate master regulators of gene expression and the modulatory effect of lactate was prevented by inhibitors of Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, indicating a role for this kinase in mediating lactate signaling. An examination of changes in dendritic spines’ morphology and density - a morphologicalcorrelate of synaptic plasticity – has shown that lactate modulated spine density changes induced by potassium chloride (KCl) and carbachol. An additional investigation described in this thesis indicated that different gut microbiota manipulations (germ-free, prebiotics, high-fat diet) regulated mRNA expression of genes involved in the Astrocyte-Neuron Lactate Shuttle (ANLS) - a metabolic cooperation mechanism between astrocytes and glutamatergic neurons. Overall, the results of this thesis help to establish a role for lactate as a signaling molecule in the brain, highlight mechanisms implicated in its signaling, and open new avenues for investigation of links between the gut microbiota and brain energy metabolism.

Magister Pharmaceuticae - MPharm / Neurodegenerative disorders (NDs) are a range of chronic brain disorders that includes amongst others motor function loss. Parkinson’s disease (PD) is one of the common NDs that has an insidious onset and diagnosed when dopaminergic neurons in the substantia nigra are already lost. The loss creates a deficiency of the dopamine (neurotransmitter) thereby causing neurochemical imbalance resulting in the signs and symptoms of PD. NDs overlap at multiple levels so some of the symptoms overlap as well. NDs currently have no cure yet and current drug therapies only improve the quality of life of the patients by targeting the symptoms mainly. Treatment of PD currently involves different classes of drugs and depending on the stages of the disease, some drugs can be only used as an adjunct therapy. Anti-oxidants and monoamine oxidase inhibitors (MAO-I) are part of the treatment options.

The naked mole-rat possesses several unique physiological and molecular features that underlie their remarkably and exceptional resistance to tissue hypoxia. Elevated pattern of Epo, an erythropoietin (Epo) factor; c-fos; vascular endothelial growth factor (VEGF); and hypoxia-inducible factors (HIF-1α) contribute to the adaptive strategy to cope with hypoxic stress. Moreover, the naked mole-rat has a lower metabolic rate than any other eutherian mammal of comparable size that has been studied. The ability to actively reduce metabolic rate represents a strategy widely used in the face of decreased tissue oxygen availability. Understanding the different molecular and physiological factors that induce metabolic suppression could guide the development of pharmacological agents for the clinical management of stroke patient.

Since several different pathways are involved in cerebral ischemia/reperfusion injury, combination therapy rather than monotherapy may be required for efficient neuroprotection. In this study, we examined the protective effects of an apoptosis inhibitor Gly 14-humanin (HNG) and a necroptosis inhibitor necrostatin-1 (Nec-1) on hypoxia/ischemia/reperfusion injury. Cultured mouse primary cortical neurons were incubated with Nec-1, HNG or both in a hypoxia chamber for 60 min. Cell viability was determined by MTS assay at 24 h after oxygen-glucose deprivation (OGD) treatment. Mice underwent middle cerebral artery occlusion for 75 min followed by 24 h reperfusion. Mice were administered HNG and/or Nec-1 (i.c.v.) at 4 h after reperfusion. Neurological deficits were evaluated and the cerebral infarct volume was determined by TTC staining. Nec-1 or HNG alone had protective effects on OGD-induced cell death. Combined treatment with Nec-1 and HNG resulted in more neuroprotection than Nec-1 or HNG alone. Treatment with HNG or Nec-1 reduced cerebral infarct volume from 59.3 ± 2.6% to 47.0 ± 2.3% and 47.1 ± 1.5%, respectively. Combined treatment with HNG and Nec-1 improved neurological scores and decreased infarct volume to 38.6 ± 1.5%. In summary, we demonstrated that the combination treatment of HNG and Nec-1 conferred synergistic neuroprotection on hypoxia/ischemia/reperfusion injury in vitro and in vivo. These findings provide a novel therapeutic strategy for the treatment of stroke by combining anti-apoptosis and anti-necroptosis therapy.