Neuropathic pain is becoming a disease of global burden with growing prevalence worldwide. It is difficult to treat and there is no assured cure for it so far. Patients have to live with it relying on combinations of drug treatments to ameliorate the effect. The cause of neuropathic pain is often considered as a dysfunction of the nervous system which involves both peripheral and central nervous system sensitization. However, the mechanism behind it remains poorly understood at the molecular level. In my thesis, I have investigated Endothelin-1 (ET-1), a potent vasoconstrictor and neuro-modulator, in the central pain-process in neuropathic pain. ET-1 induction has been reported in a variety of pathological states such as cancer, ischaemia, and neuropathic pain. The effect of central endogenous ET-1 in development of neuropathic pain has not been adequately studied. Therefore, in my first study, I evaluated the influence of endogenous ET-1 in neuropathic pain by antagonizing Endothelin Receptor type A (ETAR), which is documented as the receptor that ET-1 acts on to induce pain, in the CNS. BQ-123, an ETAR selective antagonist, was administered intrathecally to study the effect of central ET-1 in neuropathic pain induced by Peripheral Sciatic Ligation (PSL) in Sprague Dawley (SD) rat. I found that repeated administration of BQ-123 alleviated mechanical allodynia, which developed after PSL as a typical symptom of neuropathic pain. Thus, ET-1 and ETAR may involve in PSL- induced neuropathic pain. To the best of our knowledge, exogenous administration of ET-1 in the CNS has been shown to exert a pain-inhibiting effect in thermal and inflammatory pain, but the mechanism and the cellular origin of such pain-inhibiting nature remains elusive. Hence, I further investigated the effect of central ET-1 in neuropathic pain. Here, transgenic mouse overexpressing ET-1 in astrocytes (GET-1) was used as a model. I found that GET-1 mice exhibited 15-fold of ET-1 mRNA induction in the spinal cord by real-time PCR. However, GET-1 transgenic mice did not show altered mechanical threshold compared to non-transgenic (Ntg) mice at basal level under physiological condition. After PSL, I found that GET-1 transgenic mice did not show signs of neuropathic pain while age-matched Ntg mice had mechanical allodynia. High expression of ETAR observed only in Ntg mice after PSL supports the pain-alleviating effect of ETAR antagonist shown in our first study. Moreover, I explored the relationship between ET-1 and glial glutamate transporter EAAT2 which is responsible for major clearance of glutamate in the CNS. Interestingly, high expression level of EAAT2 was observed distinctively in GET-1 transgenic mice which did not develop PSL-induced neuropathic pain. Thus, EAAT2 may be the key factor in neuron protection from central sensitization by enhanced glutamate release in induction of neuropathic pain. These results suggest that endogenous central ET-1 may mediate neuropathic pain partially via ETAR. Taken together with the evidence that GET-1 did not develop neuropathic pain and showed high expression of EAAT2, I concluded that overexpressed astrocytic ET-1 in GET-1 mice exerted an analgesic effect in neuropathic pain by modulating expression of EAAT2. / published_or_final_version / Anaesthesiology / Master / Master of Philosophy

 Endothelin-1 (ET-1), a 21-amino acid peptide, exerts multiple functions including vasoconstriction, neurotransmission and neuromodulation. The correlation between ET-1 and pain was examined by localized injection into the animal showing that pain response was induced in the peripheral nervous system, but reduced in the central nervous system. In addition, the pathogenic effect of ET-1 in the nervous system was found to be cell-type specific as ET-1 expression was significantly induced in the astrocytes under certain pathological conditions such as stroke and Alzheimer’s disease. Therefore, in this study, it aims to determine the role of astrocytic ET-1 in inflammatory pain. To better illustrate such notion, transgenic mouse line over-expressing ET-1 in the astrocytes (GET-1) was generated. Under physiological conditions in GET-1 mice, it was revealed that its gross anatomy, in addition with thermal heat and mechanical pain-thresholds, were indistinguishable between GET-1 and its age-matched non-transgenic control (NTg). Inflammatory pain was then induced in the GET-1 mice and NTg mice by subcutaneous formalin injection. Spontaneous and heat-induced pain response were evaluated and analyzed based on the two phases of pain response (i.e. acute and tonic responses). Our results indicated that GET-1 mice exhibited less inflammatory pain-like response when compared to the NTg control, implicating that astrocytic ET-1 over-expression modulates inflammatory pain response. The molecular mechanism substantiating the reduction in this response may be due to the basal expression of pain-mediators in GET-1 mice. Further results showed that over-expression in astrocytic ET-1 evoke an up-regulation in calcitonin gene-related peptide (CGRP) as well as the glutamate transporter-1 (GLT-1), also known as excitatory amino-acid transporters (EAAT2). In addition, under formalin-induced pathological pain, over-expression in astrocytic ET-1 was found to negatively regulate GLT-1 transcription. These findings elucidate the extent that ET-1 contributes to the inflammatory pain pathway via ET-1 and GLT-1 interactions. Overall, this study not only pointed out the involvement of astrocytic ET-1 in inflammatory pain processing, but it also warrants further investigation into the molecular mechanism and potential therapeutic intervention of ET-1-mediated pain. / published_or_final_version / Anaesthesiology / Master / Master of Philosophy

Alzheimer’s disease and vascular dementia are the leading causes of cognitive disorders in the elderly worldwide. Increasing cases with overlaps in neuropathology between both disorders are becoming evident, giving rise to the concept of “mixed dementia”, which is characterized by cerebrovascular dysregulations along with tauopathies and β-amyloid (Aβ)-associated neurotoxicity. While the exact mechanisms leading to the exhibited vascular-neurodegenerative pathophysiology are still unclear, it is often found that ischemic-stroke contributes to amyloid pathogenesis, thus exacerbating cognitive impairment. Astrocytes, the most abundant cell type in the brain, appear to be important mediators in the central nervous system homeostasis and pathophysiology. This study proposes that upon ischemic stress, endothelin-1 (ET-1) overexpression in astrocytes leads to β-site APP cleaving enzyme 1 (BACE1) upregulation, hence contributing to enhanced amyloid pathology. ET-1 is a potent vasoconstrictor associated to Aβ pathogenesis in the brain, and BACE1 is the rate-limiting enzyme for Aβ synthesis. In order to assess astrocytic responses to ischemic stress, two previously modified astrocytic cell lines, mock-transfected control astrocytes (WT) and ET-1 overexpressing astrocytes (C6), were used. The exposures of WT and C6 cells to oxygen and glucose deprivation (OGD) – to mimic ischemic stress in vitro – evoked no abrupt differences between both cell lines. After OGD, astrocytes were characterized by cellular swelling, detachment from neighboring cells, increased cell death, decreased cell proliferation, and reduced BACE1 expressions during reperfusion. In addition, the attempt to modulate BACE1 protein levels, by blocking the receptor for advanced glycation end products, induced no significant differences. This study also investigated astrocytic ET-1 influences in the neuropathology of the transgenic mouse models APPGET-1 (amyloid precursor protein and astrocytic ET-1 overexpression) and GET-1 through a proteomics approach. The abnormal expressions of tropomyosin-3, transgelin-3, ATP synthase α chain, 3-hydroxyisobutyrate dehydrogenase, superoxide dismutase 1, glutathione S-transferase P1, and peroxiredoxin-6 in the mice hippocampi were identified. It is most likely that these proteins participate in cytoskeleton structural changes, energy metabolism impairment, and oxidant-antioxidant system imbalances that contribute to the observed increased brain atrophy displayed in these two animal models. In summary, this study has identified the possible participation of several proteins in the accelerated declination of cognitive functions exhibited by GET-1 and APPGET-1 mice through a proteomics approach. However, our in vitro results suggest that ET-1 did not play any pivotal role in C6 response to the hypoxic conditions evaluated. Furthermore, results showed no correlation between astrocytic ET-1 and BACE1. Further investigations examining alternative BACE1-independent pathways are required to elucidate the intricate relationship between ET-1 and Aβ in astrocytes. / published_or_final_version / Psychiatry / Master / Master of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 92-107).

Thesis (Ph. D.)--University of Hong Kong, 1998. / Includes bibliographical references (leaves 217-244).

Thesis (Ph.D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on March 12, 2008) Includes bibliographical references.

Thesis (Ph. D.)--University of Oregon, 2005. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 102-108). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (M.S.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; containsvi, 64 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 51-64).

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