Xian, Jiawen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 201-230). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Oxidative stress causes neural damage and inhibits essential cellular processes, such as synaptic transmission. Despite this knowledge, currently available pharmaceutical agents cannot effectively protect neural cells from acute oxidative stress elicited by strokes, heart attacks, and traumatic brain injuries in a real life clinical setting. Our lab has developed an electrophysiology protocol to identify novel drugs that protect an essential cellular process (neurotransmission) from acute oxidative stress-induced damage. Through this doctoral dissertation, we have identified three new drugs, including a Big K+ (BK) K+ channel blocker (iberiotoxin), resveratrol, and a custom made resveratrol-like compound (fly2) that protect synaptic function from oxidative stress-induced insults. Further developing these drugs as neuroprotective agents may prove transformative in protecting the human brain from acute oxidative stress elicited by strokes, heart attacks, and traumatic brain injuries. Inhibiting the protein kinase G (PKG) pathway protects neurotransmission from acute oxidative stress. This dissertation has expanded upon these findings by determining that the PKG pathway and BK K+ channels function through independent biochemical pathways to protect neurotransmission from acute oxidative stress. Taken together, this dissertation has identified two classes of compounds that protect neurotransmission from acute oxidative stress, including resveratrol-like compounds (resveratrol, fly2) and a BK K+ channel inhibitor (iberiotoxin). Further developing these drugs in clinical trials may finally lead to the development of an effective neuroprotective agent. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Cone snails are carnivorous marine mollusks, utilizing their neuropeptide-rich venom for prey capture. The venom of Conus brunneus, a wide-spread Eastern Pacific vermivore, has not been extensively studied. In the current work, peptides from the dissected venom were characterized and tested using preliminary bioassays. Six peptides (A-F) were isolated and tested. Three peptide identities were determined by comparison with previously reported data: bru9a (A), bru3a (F), and an a-conotoxin (E). Preliminary screening in a stroke-related model of induced glutamate excitotoxicity in primary neuronal cells and PC12 cell cultures indicated potential neuroprotective activity of peptide fractions A, D, and F. Further testing is necessary to determine and verify structure, activity, target, and mechanism of action of the promising peptides from C. brunneus, which may prove effective neuropharmacological agents to treat stroke. / by Rebecca A. Crouch. / Thesis (M.S.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Neurodegenerative disorders constitute a class of diseases that express characteristic misfolded proteins that aggregate and induce neuronal toxicity and death. Huntington’s disease is one such fatal protein misfolding disease. Currently no therapeutic avenue can delay or stop the progression of the disease. In this context, there is a need to identify therapeutic pathways and drug targets that can prevent or delay pathogenesis in neurodegenerative diseases involving protein misfolding. This dissertation describes how our search for new drug targets have led us to identify protein disulfide isomerase and three unique small molecules that modulate its activity as a means to protect neuronal cells from neurodegenerative protein misfolding diseases, such as Huntington’s disease. Protein disulfide isomerase is a thiol-oxidoreductase in the endoplasmic reticulum that has garnered increased attention because of its implicated role in numerous human diseases, including cancer, human immunodeficiency virus pathogenesis, and thrombosis. Validating protein disulfide isomerase as target for neurodegenerative disorders may open up new therapeutic strategies to understand and treat these diseases. First, I describe the identification and validation of protein disulfide isomerase as a target of the neuroprotective small molecule, 16F16. I show that 16F16 is an irreversible inhibitor of protein disulfide isomerase that binds covalently to both cysteines in the active site. This inhibition is protective in cell and brain-slice models of Huntington’s disease, as well as in the brain-slice model of Alzheimer’s disease. Next, I describe the neuroprotective small molecule IBS141 that was originally incorrectly annotated with a chemical structure. I elucidate the correct structure of the active compound using analytical chemistry, revealing it to be the natural product securinine. Furthermore, I identify the binding site of securinine to protein disulfide isomerase and show that the inhibition of the protein is protective in cell and brain-slice models of neurodegenerative diseases. In addition to finding this unexpected activity of securinine, I provide a systematic roadmap to those who encounter compounds with incorrect structural annotation in the course of screening campaigns. Last, I describe the discovery of LOC14, a nanomolar, reversible, modulator of protein disulfide isomerase that protects cells and medium spiny neurons from the toxic mutant huntingtin protein. I find that this protection results from LOC14 binding adjacent to the active site and inducing protein disulfide isomerase to adopt an oxidized conformation. LOC14, has dramatically improved potency for protein disulfide isomerase over previously identified inhibitors and displays favorable pharmaceutical properties, making it an idea compound to evaluate the therapeutic potential of modulating protein disulfide isomerase in in vivo models of neurodegenerative diseases.

Introdução: A lesão da medula espinhal é uma entidade clínica grave e extremamente incapacitante. Muitos esforços estão sendo realizados para melhorar a resposta neurológica ao trauma da medula espinhal. Dentre eles, destacamos o uso de agentes farmacológicos, a descompressão e estabilização cirúrgica precoces e a hipotermia. A hipotermia pode ser induzida de forma sistêmica ou local. Várias complicações, como arritmias cardíacas, coagulopatias e infecções, foram associadas ao uso sistêmico da hipotermia. Porém, sua aplicação local demanda a necessidade de intervenção cirúrgica de emergência e manejo pós-operatório complicado. Objetivo: Avaliar o efeito neuroprotetor da hipotermia epidural em ratos. Material e método: Foram arrolados 30 ratos Wistar pesando entre 320-360 g e divididos aleatoriamente em dois grupos: o grupo da hipotermia epidural e o grupo controle, com 15 ratos cada. Uma contusão medular produzida por queda padronizada de peso de 10 g, a 25 mm de altura, usando o New York University (NYU) Impactor, foi realizada após a laminectomia em T9-10 em todos os ratos. Os ratos do grupo da hipotermia foram submetidos ao resfriamento a 9-10 °C por um período de 20 minutos, logo após a contusão medular. Os grupos foram analisados durante seis semanas quanto à função motora utilizando-se a escala BBB e o teste do plano inclinado. Ao final da sexta semana, foi realizado ainda o exame de potencial evocado motor dos ratos, cujos resultados foram comparados entre os dois grupos. Resultados: A avaliação da função motora através da aplicação da pontuação da escala BBB ao longo das seis semanas não evidenciou diferenças estatisticamente significantes entre os dois grupos. Não encontramos diferenças estatísticas na avaliação motora através da pontuação do teste do plano inclinado ao longo das seis semanas do estudo. Os valores de latência e amplitude do potencial evocado motor não mostraram diferenças estatísticas significantes entre os grupos ao final da última semana do estudo. Conclusão: A hipotermia não apresentou efeito neuroprotetor quando aplicada no sítio da lesão, logo após a contusão medular, no espaço epidural de ratos Wistar / Introduction: Spinal cord injury (SCI) is a critical and extremely disabling clinical condition. Considerable effort has been made to improve the neurological response to the spinal cord lesion. We must highlight pharmacological agents, early surgical decompression and stabilization and hypothermia. Therapeutic hypothermia can be achieved systemically or locally. Many complications have been associated to the systemic hypothermia, such as cardiac arrhythmias, coagulopathies and infection. However, local application demands surgical intervention and difficult post operative care. Objetive: To evaluate the neuroprotective effect of epidural hypothermia in rats. Methods: Wistar rats (n = 30; weighting 320-360 g) were randomized in two groups: the hypothermia and the control group, with 15 rats in each. A spinal cord lesion was produced by the standardized drop of a 10 g-weight from a height of 2,5 cm, using the New York University Impactor, after the laminectomy at the T9-10 level. Rats of the hypothermia group underwent epidural hypothermia for 20 minutes immediately after spinal cord injury. Motor function was assessed during six weeks using the BBB motor scores and inclined plane test. At the end of the last week, neurologic status was monitored by the motor evoked potential exam and the results were compared between the two groups. Results: Analysis of the BBB scores during the six-weeks period did not show any statistically significant difference between the two groups. We did not find any significant difference between the groups in the scores of the inclined plane test during the six-weeks period. Latency and amplitude values of the motor evoked potential exam did not show any statistically significant difference between the two groups at the end of the study. Conclusion: Hypothermia did not produce any neuroprotective effect when applied immediately after spinal cord contusion, at the injury level and in epidural space of Wistar rats

Stroke is the 3rd leading cause of death and the leading cause of major disability worldwide. Currently, there are no neuroprotective drugs approved for the acute treatment of ischaemic stroke. The vast majority of stroke therapeutics failed in clinical trials due to toxic side effects and/or a clinically irrelevant therapeutic window. This thesis is focused on exploiting the delayed neurodegeneration that occurs in the compromised penumbra, as these cells may be capable of being saved by therapeutic intervention in a clinically obtainable window. In order to investigate the ischaemic cascade and be able to draw conclusions that are applicable to humans, the international gold standard animal model for cerebral ischaemia, the filament insertion middle cerebral artery occlusion (MCAO) model, was established at the University of Otago. This model was validated under new laboratory conditions and employed adult male Sprague Dawley rats. After testing multiple occlusion lengths, it was concluded that a 2hr ischaemic period was sufficient to produce a consistent infarct of optimal size. It has been well documented that neuroinflammation contributes to much of the delayed progression of neural injury post-stroke. Therefore, the catechin (-)-epigallocatechin gallate (EGCG), which is an anti-inflammatory, anti-oxidant and free-radical scavenging agent was investigated in the MCAO model of stroke. 50mg/kg i.p. of EGCG or saline was administered immediately post-MCAO and animals were sacrificed at 72hr post-filament insertion. The results confirmed that treatment with EGCG was neuroprotective and non-toxic. However, EGCG also induced an over 50% increase in the risk of haemorrhagic conversions. The anti-platelet effects of EGCG and lack of toxicity suggests that the catechin may prove to be an efficacious prophylactic for stroke. The contrary findings for EGCG treatment led to the re-evaluation of the neuroinflammatory pathway for alternate mechanisms to target therapeutic interventions. The temporal profile of the primary inducible enzymes nitric oxide synthase (NOS), cyclooxygenase (COX) and arginase (and their isoforms) were quantified 0, 3 and 7 days post-stroke. In both hemispheres, total NOS activity exhibited a significant and sustained up-regulation to 7 days post-occlusion. In the ipsilateral hemisphere at least half of the total increase was accounted for by inducible NOS (iNOS) expression. Arginase, which competes with NOS for L-arginine, demonstrated a delayed but significant increase in activity by day 7 in the infarcted hemisphere, thereby correlating well with the downward slope of NOS activity (illustrating the switch in the conversion pathway). COX activity was observably increased in the ipsilateral hemisphere, but the up-regulation did not reach significance by day 7. Alternately, the contralateral hemisphere displayed a significant decrease in activity by day 3. These results give conclusive evidence that the contralateral hemisphere is NOT an appropriate internal control and imply that NOS and COX inhibitors may prove to be efficacious for a much longer therapeutic window than current treatments. However, the delayed induction of COX activity may also indicate that this enzyme has a finite therapeutic window, as it may also stimulate remodelling of surviving neural networks. The prolonged up-regulation of inflammatory mediators implies that there may be an induction of an autoimmune component to the response. Therefore, the thymus (T) lymphocyte activation was quantified up to 14 days post-stroke. Cluster of differentiation (CD) 3⁺ T lymphocytes (equally contributed to by CD4⁺ and CD8⁺ T cells) exhibited a significant and sustained up-regulation in the infarcted region from day 3 up to at least day 14 post-ischaemia. Quantitative analysis of all cells present post-stroke determined that immune cells make up an average of 73% of all cells present in the 'peak' ischaemic areas. The CD4⁺ T helper cell response was delineated by double immunohistochemical labelling. Interferon-γ positively labelled with CD4⁺ T cells at days 3, 7 and 14 post-insult detailing a Th1-driven pro-inflammatory response. This evidence indicates that the autoimmune response is critical post-ischaemia and that it may be highly susceptible to modification by anti-inflammatory therapeutic intervention. The primary downstream effect of the pro-inflammatory/immune cascade is apoptosis. The main organelle responsible for the 'go, no go' response to apoptotic factors is the mitochondria. In order to distinguish whether mitochondrial dysfunction was initiated shortly after ischaemia induction or if it was delayed, like the inflammatory/immune response, to a clinically relevant window, the temporal profile of mitochondrial complex inactivation was studied. It was found that mitochondrial membrane viability was impaired by day 3, followed by a significant decrease in respiratory complex activation and an increase in tissue injury by oxidative stress by 7 days post-ischaemia. These results indicate that targeting the early decrease in membrane viability or mitochondrial permeability transition pore opening combined with anti-apoptotic therapeutics, may attenuate the proceeding mitochondrial impairment in oxidative phosphorylation, reactive oxygen species generation and subsequent cell death cascades. The current investigations into the temporal profile and quantitative contributions of the inflammatory, immune and apoptotic mechanisms post-stroke highlight potential strategies for modulation by acute stroke therapeutics. Furthermore, the general knowledge amassed from these studies dictates the necessity of a new approach to therapeutic intervention. The acknowledgement of so many contributing systems suggests that in addition to a thrombolytic, a combination therapy involving multiple neuroprotectants should be employed to account for the multifaceted nature of the sequelae of ischaemic stroke.

Thesis (M.Sc.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science, Centre for Neuroscience. Title from pdf file main screen (viewed on January 27, 2010). Includes bibliographical references.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

In order to understand the possible mechanisms in the glaucoma-related retinopathy, the role of the vasoconstrictor, endothelin-1 (ET-1), receptor for advanced glycation end-products (RAGE) as well as its ligand, Aβ in the degeneration of retinal ganglion cells (RGCs) were studied in experimental models. In addition, the relationship of ET-1, RAGE and Aβ for the RGC protective mechanism of Lycium Barbarum (LB) was also investigated. In the first part, ET-1 together with its receptors, ETA and ETB, were studied to understand their possible roles in chronic ocular hypertension (COH). The neuronal protective mechanism of LB was also determined by using a well established COH rat model. In normal rats, ET-1 and its receptors, ETA and ETB, were distributed in the retina, vasculature and optic nerve. Interestingly, ET-1 expression was up-regulated after COH. LB could decrease the expression of ET-1 and regulate its receptors (up-regulation of ETB and down-regulation of ETA in vasculature; up-regulation of ETA and down-regulation of ETB in RGCs) under the condition of COH. These data suggested that the RGC protective mechanism of LB might be related to its ability to regulate the biological effects of ET-1. To investigate the pathogenic effect of ET-1 in glaucoma, in the second part, we used transgenic mice with over-expression of ET-1 on endothelial cells (TET-1 mice). We found that beginning at 10-12 months, TET-1 mice showed a progressive retinal degeneration (loss of RGCs associated with neurons in the inner nuclear layer and outer nuclear layer of the retina) without elevation of the intraocular pressure (IOP). The data demonstrated that TET-1 mice may serve as a potential model to investigate the role of endothelial ET-1 in the pathogenesis of normal tension glaucoma and other degenerative retinopathy. To investigate whether LB plays a role on neuronal protection other than in COH, in the third part, we used an acute ocular hypertension (AOH)-induced ischemia mouse model. We found that LB could rescue RGCs under AOH insult, associating with blood vessel protection (decreasing the damage of blood-retinal-barriers and rescuing the survival of endothelial cells and pericytes) and inhibiting retinal gliosis. We also found the protective mechanism of LB was closely correlated with down-regulation of the expression of RAGE, ET-1, APP (amyloid precursor protein), AGE (advanced glycation end-product) as well as Aβ; therefore to reduce the damage effects of these RAGE-mediated reactions to the retinal neurons, blood vessels and glial cells involved in the ischemic insult. Taken together, the present study demonstrated that TET-1 mice may be a potential model for investigating the role of ET-1 in degenerative retinopathies, such as normal tension glaucoma. We also showed the neuronal protective mechanism of LB in vivo was associated with inhibiting the biological effect of ET-1 and down-regulating the damage signaling pathways mediated by the activation of RAGE and its ligands (AGE and Aβ). These results provided further understandings in the mechanism of the glaucoma-related retinopathy. In addition, LB could be a neuroprotective agent to the retina following both chronic and acute injuries. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Ischemic stroke is a devastating cerebrovascular disease resulting in high mortality rate and distressing sequelae such as hemiplegia, ataxia and even visual impairment. Retinal ischemia refers to a common pathological feature shared by many blinding diseases including retinal vascular occlusions, diabetic retinopathy, glaucoma, and retinopathy of prematurity. Ischemia/reperfusion injury is implicated in both of these pathological conditions, which greatly impact on one’s daily life. The eventual consequence of the insult is irreversible neuronal cell death and functional deterioration. Apart from current symptomatic treatment for these diseases, researchers and clinicians are dedicated to look for ideal neuroprotectant to meet the clinical needs. Traditional Chinese medicine has been received accumulating attention in recent years, and Lycium barbarum is one of them. The polysaccharides (LBP) utilized in the present study are the rich extracts of the fruit of Lycium barbarum that has been shown to exert many biological effects. This study aims to evaluate its protective effects in cerebral and retinal ischemia, which has not yet been fully investigated. A well-established rodent model, middle cerebral artery occlusion, was utilized in the present study to mimic cerebral and retinal ischemia/reperfusion injury. In the study of cerebral ischemia, both pre-treatment and post-treatment of LBP were explored. Seven-day LBP pre-treatment revealed significant protection against neurological deficits and cerebral infarction. Besides, it attenuated cerebral edema and glial activation, as well as preserved blood-brain barrier integrity. Further study showed that these beneficial effects of LBP pre-treatment might act via anti-apoptosis, antioxidation and anti-inflammation. However, similar findings were not noted in LBP post-treatment experiments, possibly due to the timing of intervention. In the investigation of retinal ischemia, the observation time was prolonged to 7 days after the insult. Electroretinogram was used to evaluate the functional alternation of retinal neurons. Sustained retinal dysfunction was induced by two-hour ischemia. LBP pre-treatment with continuous daily supplementation effectively alleviated visual dysfunction and protected the retina from morphological impairment including neuronal death, glial activation and blood-retinal barrier disruption. Similarly, these protective effects might be associated with the involvement of attenuation of apoptosis and oxidative stress. In conclusion, LBP pre-treatment with continuous daily supplementation protected the brain and retina, both functionally and morphologically, from ischemia/reperfusion injury. This dosing regimen hold great promise in serving as a prophylactic neuroprotectant in patients at high risk for ischemic stroke, as well as preserving normal visual function and reducing irreversible neuronal death in ischemic retinopathies. Further studies on the active ingredients and underlying mechanisms would be informative for better application of LBP in clinical situation. / published_or_final_version / Ophthalmology / Doctoral / Doctor of Philosophy