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Wong Hei Lui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 192-226). / Abstracts in English and Chinese. / Publications Based On The Work In This Thesis --- p.i / Abstract --- p.ii / Acknowledgements --- p.vii / Abbreviations --- p.viii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Normal joint --- p.1 / Chapter 1.11 --- Biology of joint --- p.1 / Chapter 1.12 --- Structure of synovial joint --- p.1 / Chapter 1.13 --- Components of the mature synovial joint --- p.3 / Chapter 1.131 --- Articular cartilage --- p.3 / Chapter 1.1311 --- Water --- p.4 / Chapter 1.1312 --- Cartilage matrix --- p.4 / Chapter 1.1313 --- Chondrocyte --- p.5 / Chapter 1.132 --- Synovium --- p.5 / Chapter 1.1321 --- Synovium vasculature --- p.6 / Chapter 1.1322 --- Synovial blood flow --- p.7 / Chapter 1.133 --- Synovial fluid --- p.8 / Chapter 1.134 --- Bone --- p.9 / Chapter 1.2 --- Pathological processes of arthritis --- p.11 / Chapter 1.21 --- Activation of immune cells in arthritis --- p.11 / Chapter 1.22 --- Synovial proliferation --- p.13 / Chapter 1.221 --- Synovial lining cell activation --- p.13 / Chapter 1.222 --- Pannus invasion --- p.14 / Chapter 1.23 --- Cartilage and bone degradation --- p.14 / Chapter 1.231 --- Depletion of proteoglycan (GAG) --- p.15 / Chapter 1.232 --- Collagen denature --- p.15 / Chapter 1.3 --- Tachykinins (TKs) --- p.17 / Chapter 1.31 --- History --- p.17 / Chapter 1.32 --- "Synthesis, storage and release of TKs" --- p.17 / Chapter 1.33 --- Tachykinin receptors --- p.18 / Chapter 1.331 --- Characterization of NK1 receptor --- p.19 / Chapter 1.332 --- Characterization of NK2 receptor --- p.19 / Chapter 1.333 --- Characterization of NK3 receptor --- p.20 / Chapter 1.34 --- Effector systems of TKs --- p.21 / Chapter 1.35 --- Termination of TK signals --- p.21 / Chapter 1.351 --- Enzymatic breakdown --- p.21 / Chapter 1.352 --- Receptor desensitization --- p.22 / Chapter 1.353 --- Receptor endocytosis --- p.22 / Chapter 1.36 --- TK receptor antagonists --- p.23 / Chapter 1.361 --- Selective NK1 receptor antagonists --- p.23 / Chapter 1.362 --- Selective NK2 receptor antagonists --- p.24 / Chapter 1.363 --- Selective NK3 receptor antagonists --- p.25 / Chapter 1.4 --- Roles of tachykinins in arthritis --- p.28 / Chapter 1.41 --- Correlation between tachykinins and joint inflammation --- p.28 / Chapter 1.42 --- Roles of tachykinins in immune cell activation --- p.30 / Chapter 1.43 --- Roles of tachykinins in synovial proliferation --- p.31 / Chapter 1.44 --- Roles of tachykinins in cartilage degradation --- p.32 / Chapter 1.5 --- Animal model of arthritis --- p.33 / Chapter 1.51 --- Instability model --- p.33 / Chapter 1.52 --- Immobilization model --- p.34 / Chapter 1.53 --- Noxious agent-induced model --- p.34 / Chapter 1.531 --- Collagen-induced erosive arthritis --- p.34 / Chapter 1.532 --- Cartilage oligometric matrix protein-induced arthritis --- p.35 / Chapter 1.533 --- Oil-induced arthritis --- p.35 / Chapter 1.534 --- Streptococcal cell wall-induced arthritis --- p.35 / Chapter 1.535 --- Adjuvant-induced arthritis --- p.36 / Chapter 1.536 --- Pristane-induced arthritis --- p.36 / Chapter 1.6 --- Current anti-arthritic therapies --- p.39 / Chapter 1.61 --- Non steroid anti-inflammatory drugs --- p.39 / Chapter 1.62 --- Glucocorticoid --- p.44 / Chapter 1.63 --- Second-line treatment --- p.46 / Chapter 1.631 --- Sulfasalazine --- p.46 / Chapter 1.632 --- Gold salts --- p.47 / Chapter 1 633 --- D-penicillamine --- p.48 / Chapter 1.634 --- Antimalarial --- p.49 / Chapter 1 .635 --- Methotrexate --- p.51 / Chapter 1.64 --- New trends for treatment of arthritis --- p.53 / Chapter 1.641 --- Anti-cytokine therapy --- p.53 / Chapter 1.642 --- Anti-angiogenesis therapy --- p.54 / Chapter 1.7 --- Aims of study --- p.57 / Chapter Chapter 2 --- Material and drugs --- p.62 / Chapter Chapter 3 --- Methodology --- p.62 / Chapter 3.1 --- Animals used and anaesthetization --- p.62 / Chapter 3.2 --- Measurement of plasma protein extravasation --- p.63 / Chapter 3.3 --- Measurement of knee joint sizes --- p.64 / Chapter 3.4 --- Measurement of knee joint blood flow --- p.65 / Chapter 3.5 --- Measurement of histological changes --- p.65 / Chapter 3.51 --- Dissection and fixation --- p.65 / Chapter 3.52 --- Decalcification --- p.66 / Chapter 3.53 --- Processing --- p.66 / Chapter 3.54 --- Embedding --- p.67 / Chapter 3.55 --- Sectioning --- p.67 / Chapter 3.56 --- Staining --- p.69 / Chapter 3.6 --- Data analysis --- p.69 / Chapter 3.61 --- Scoring systems --- p.72 / Chapter Chapter 4 --- A model of monoarthritis in rats --- p.72 / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.2 --- Method --- p.73 / Chapter 4.3 --- Results --- p.73 / Chapter 4.31 --- Lewis rats --- p.73 / Chapter 4.32 --- Sprague-Dawley (SD) rats --- p.74 / Chapter 4.33 --- Comparison of FCA-induced changes in Lewis and SD rats --- p.74 / Chapter 4.34 --- Histological studies on arthritic SD rats --- p.75 / Chapter 4.4 --- Discussion --- p.93 / Chapter 4.5 --- Conclusions --- p.95 / Chapter Chapter 5 --- Effect of Substance P on adjuvant-induced arthritis --- p.96 / Chapter 5.1 --- Introduction --- p.96 / Chapter 5.2 --- Method --- p.98 / Chapter 5.3 --- Results --- p.99 / Chapter 5.31 --- Evans blue extravasation --- p.99 / Chapter 5.32 --- Joint size --- p.100 / Chapter 5.33 --- Knee joint blood flow --- p.101 / Chapter 5.34 --- Histology results --- p.102 / Chapter 5.341 --- Infiltration of immune cells in synovial tissue --- p.102 / Chapter 5.342 --- Synovial tissue proliferation --- p.102 / Chapter 5.343 --- Cartilage degradation --- p.103 / Chapter 5.344 --- Bone degradation --- p.103 / Chapter 5.4 --- Discussion --- p.120 / Chapter 5.5 --- Conclusions --- p.125 / Chapter Chapter 6 --- Effects of tachykinin receptor antagonists on FCA-induced arthritis / Chapter 6.1 --- Introduction --- p.126 / Chapter 6.2 --- Method --- p.128 / Chapter 6. 21 --- Intravenous NK1 receptor antagonists on FCA-induced arthritis --- p.128 / Chapter 6. 22 --- Intraperitoneal TK receptor antagonists on FCA-induced arthritis --- p.128 / Chapter 6.3 --- Results --- p.129 / Chapter 6.31 --- Intravenous NK1 227}0اreceptor antagonists on FCA-induced arthritis Evans blue extravasation and joint swelling --- p.129 / Chapter 6.32 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced arthritis Evans blue extravasation and joint swelling --- p.129 / Chapter 6.33 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced immune cell accumulation --- p.130 / Chapter 6.34 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced synovial tissue proliferation --- p.131 / Chapter 6.35 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced cartilage degration and bone erosion --- p.131 / Chapter 6.4 --- Discussion --- p.159 / Chapter 6.5 --- Conclusions --- p.162 / Chapter Chapter 7 --- Individual and combined effects of dexamethasone and TK receptor antagonists on FCA-induced arthritis --- p.163 / Chapter 7.1 --- Introduction --- p.163 / Chapter 7.2 --- Method --- p.166 / Chapter 7.3 --- Results --- p.167 / Chapter 7.31 --- Evans blue extravasation --- p.167 / Chapter 7.32 --- Knee joint size --- p.167 / Chapter 7.33 --- Body weight --- p.168 / Chapter 7.34 --- Cellular infiltration --- p.168 / Chapter 7.35 --- Synovial tissue proliferation --- p.168 / Chapter 7.36 --- Cartilage degradation --- p.169 / Chapter 7.4 --- Discussion --- p.184 / Chapter 7.5 --- Conclusions --- p.187 / Chapter Chapter 8 --- General discussions and conclusions --- p.188 / References --- p.192

<p>Conclusion of this study: These data suggest that 6 weeks of high-fat feeding induces hypertension but does not produce obesity, dyslipidemia and insulin resistance. However, this model may be useful in studying vascular reactivity in hypertension in the absence of insulin resistance.</p>

Intratympanic injection of various otoprotectants through the round window membrane (RWM) might become available in the near future as an alternative to the currently available medical and surgical methods used to treat several inner ear diseases. The most common outcome of such diseases is sensorineural hearing loss (SNHL). Two examples of  these otoprotectants are Edaravone and Brain-Derived Neurotrophic Factor (BDNF), both of which have already proved effective against  noise-induced hair cell loss, barotrauma  and ototoxicity caused by cisplatin. In four different studies we used two electrophysiological methods, auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE), to study the effects of tobramycin and Pseudomonas aeruginosa exotoxin A (PaExoA) on the inner ears of 129 male Sprague-Dawley rats. In two investigations, not only the otoprotective effects of Edaravone on tobramycin-induced ABR threshold shifts and PaExoA-induced DPOAE  threshold changes, were studied but even different application times, in order to establish in which interval it was still possible to achieve effective otoprotection.We found that Edaravone gave otoprotection from tobramycin when injected simultaneously or within 7 days, but it had only a limited effect on the changes in DPOAE thresholds caused by PaExoA when injected 1, 2, or 4 hours after the exotoxin. The effect of BDNF on PaExoA-induced ABR threshold shifts was investigated in two studies, where different doses of intratympanically injected PaExoA were used and where BDNF was applied simultaneously, 12 or 72 hours efter exotoxin instillation. We found that BDNF had an otoprotective effect on SNHL induced by different doses PaExoA when injected simultaneously or with no more than 12 hours delay.

Background and Purpose: The endothelium is a single-cell layer positioned at the blood-vascular wall interface, where in response to blood-borne signals and hemodynamic forces, endothelial cells act as central regulators of vascular homeostatic processes including vascular tone, growth and remodeling, inflammation and adhesion, and blood fluidity and coagulation. Agonist- or flow-stimulated endothelium-dependent vasorelaxation becomes impaired in states of cardiovascular disease (CVD) risk and has been identified as a possible biomarker of overall endothelial dysfunction leading to vascular dysregulation and disease pathogenesis. Accordingly, it is important to elucidate the mechanisms accounting for this endothelial vasomotor dysfunction. Upon stimulation, endothelial cells can synthesize and release a variety of endothelium-derived relaxing factors (EDRFs), the most prominent of which is nitric oxide (NO) derived from NO synthase (NOS). In addition, under certain CVD risk conditions including hypertension and aging, stimulated endothelial cells can become a prominent source of endothelium-derived contracting factors (EDCFs) produced in a cyclooxygenase (COX)-dependent manner. Consequently, endothelial dysfunction may be caused by under-active EDRF signaling and/or competitive over-active EDCF signaling. Much attention has been given to elucidating the mechanisms of under-active EDRF signaling and its role in causing endothelial dysfunction, wherein excess reactive oxygen species (ROS) accumulation and oxidative stress under CVD risk conditions have been recognized as major factors in reducing NO bioavailability thus causing under-active EDRF signaling and endothelial dysfunction. Less attention however, has been given to elucidating the mechanisms of over-active COX-mediated EDCF signaling and its role in causing endothelial dysfunction. Moreover, while COX-mediated EDCF signaling activity has been investigated in some segments of the vasculature, most notably the aorta, it has not been well-investigated in the common carotid artery (CCA), a highly accessible cerebral blood flow conduit particularly advantageous in exploring the roles of the endothelium in vascular pathogenesis. It was the global purpose of this thesis to gain a better understanding of the cellular-molecular mechanisms accounting for endothelial dysfunction in the CCA of animal models known to exhibit COX-mediated EDCF signaling activity, in particular essential (spontaneous) hypertension and aging. Experimental Objective and Approach: This thesis comprises three studies. Study I and Study II investigated the CCA of young-adult (16-24wk old) normotensive Wistar Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats. Study III investigated the CCA of Adult (25-36wks old) and Aging (60-75wks old) Sprague Dawley (SD) rats treated in vivo (or not; CON) with L-buthionine sulfoximine (BSO) to chronically deplete the cellular anti-oxidant glutathione (GSH) and increase ROS accumulation and oxidative stress. The global objective and approach across these studies was to systematically examine the relative contributions of NOS and COX signaling pathways in mediating the acetylcholine (ACh)-stimulated endothelium-dependent relaxation (EDRF) and contractile (EDCF) activities of isometrically-mounted CCA in tissue baths in vitro, with a particular focus on elucidating the mechanisms of COX-mediated EDCF signaling activity. An added objective was to examine the in vivo hemodynamic characteristics of the CCA in each animal model investigated, serving both to identify the pressure-flow environment that the CCA is exposed to in vivo and to provide assessment of potential hypertension, aging, and oxidative stress effects on large artery hemodynamics. Key Findings: Study I hemodynamic analysis confirmed a hypertensive state in young adult SHR while also exposing a reduction in mean CCA blood flow in SHR compared to WKY accompanied by a multi-faceted pressure-flow interaction across the cardiac cycle relating to flow and pressure augmentation. Study III hemodynamic analysis found that neither aging nor chronic BSO-induced GSH depletion affected CCA blood pressure or blood flow parameters in SD rats. Study I and II demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from young adult SHR, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to WKY. Examining ACh-stimulated contractile function specifically from a quiescent (non pre-contracted) state revealed that EDCF activity did exist in WKY CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in SHR CCA could not fully suppress its >2-fold augmented EDCF activity vs. WKY CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the SHR-WKY CCA model revealed that the EDCF signaling activity was completely dependent on the COX-1 (but not COX-2) isoform of COX and was almost exclusively mediated by the thromboxane-prostanoid (TP) sub-type of the prostaglandin (PG) G-protein coupled receptor family and by Rho-associated kinase (ROCK), a down-stream effector of the molecular switch RhoA. Furthermore, it was found that while exogenous ROS-stimulated CCA contractile function was similarly >2-fold augmented in SHR vs. WKY and dependent on COX-1 and TP receptor and ROCK effectors, ACh-stimulated CCA EDCF signaling activity was only minimally affected by in-bath ROS manipulating compounds. Additional biochemical and molecular analysis revealed that ACh stimulation was associated with PG over-production from an over-expressed COX-1 in SHR CCA, and with CCA plasma membrane localization and activation of RhoA. Study III demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from Aging SD rats, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to Adult SD rats. Specific examination of ACh-stimulated contractile function revealed that EDCF activity did exist in Adult CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in Aging CCA could not fully suppress its >3-fold augmented EDCF activity vs. Adult CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the Adult-Aging SD rat CCA model revealed that EDCF signaling activity was completely dependent on COX-1, but while exogenous ROS was able to elicit a COX-dependent CCA contractile response, in-bath ROS manipulating compounds were found to be without effect on ACh-stimulated CCA EDCF signaling activity. Furthermore, biochemical analysis revealed that aging was not associated with a change in tissue (liver and vascular) GSH content or ROS accumulation. Chronic in vivo BSO treatment was effective in depleting tissue GSH content and increasing ROS accumulation, to a similar extent, in both Adult and Aging SD rats. However, regardless of age, neither ACh-stimulated NO-mediated EDRF signaling activity nor COX-mediated EDCF signaling activity were affected by these BSO-induced perturbations. Conclusions and Perspective: In the CCA of animals at the early pathological stages of either essential hypertension (young adult SHR) or normotensive aging (Aging SD rats), endothelial vasomotor dysfunction can be caused solely by over-active EDCF signaling, apparently disconnected from changes in NO bioavailability or oxidative stress. While NO and ROS may act, respectively, as negative and positive modulators of the established COX-PG-TP receptor-RhoA-ROCK cell-signaling axis mediating endothelium-dependent contractile activity, these factors do not appear to be essential to the mechanism(s) underlying the development of over-active EDCF signaling. Further elucidation of the cellular-molecular causes of over-active EDCF signaling, and its patho-biological consequences, in the SHR-WKY and Adult-Aging SD rat CCA models of EDCF activity established and hemodynamically characterized in this thesis, may help to identify new or more effective targets to be used in prevention or treatment strategies to combat the pathogenesis of CVD.

Long-term potentiation (LTP) and depression (LTD) are important forms of activity-dependent synaptic plasticity believed to play a role in memory at the cellular level. It has previously been described that synthesis of new proteins is needed to maintain LTP longer than a few hours. Other reports argue that sufficient proteins for stable LTP are already available. The present study aims to examine the role of protein synthesis in LTD, the presumed mirror mechanism of LTP. Experiments were carried out in hippocampal slices from young (12-45 days) and old (12-18 weeks) Sprague-Dawley rats. Extracellular techniques were used to study synaptic responses in the Schaffer-collateral-commissural pathway. Plasticity was induced electrically by low frequency stimulation (2-3 trains at 1 Hz for 15 min) or chemically by brief exposure to certain glutamate receptor agonists (NMDA at 20 µM for 3 min or DHPG at 100 µM for 10 min). Whole slice protein synthesis was quantified by assessing 3H-leucine incorporation. Stable LTD (&gt; 8 h) was be obtained by either electrical or chemical activation. Protein synthesis inhibitors anisomycin (40 uM) and cycloheximide (100 uM) both failed to influence the magnitude of LTD. Moreover, no age difference was found, in terms of stable LTD in both young and old rats under inhibition of protein synthesis. The potency of the inhibitors was found to be high, depressing synthesis down to a few percent. It is concluded that sufficient proteins for generating stable LTD are normally present in the brain, implying a large safety-margin for cellular memory.

Cerebral ischemia is the principal cause of morbidity and mortality worldwide. In addition to neuronal loss associated with hypoxic-ischemic damage, cerebral ischemia is characterized by a neuromicrovascular injury. Nitrative stress and lipid peroxidation increase in hypoxic-ischemic damages and play an essential role in neuromicrovascular injury leading to cerebral ischemia. We hypothesized that newly described lipid peroxidation products, termed trans-arachidonic acids (TAA), could be implicated in the pathogenesis of hypoxia-ischemia by affecting the cerebral vasomotricity and microvascular integrity. / The effects of TAA on neuromicrovascular tone were tested ex vivo by monitoring the changes in vascular diameter of rat cerebral pial microvessels. Four isomers of TAA, namely 5 E-AA, 8E-AA, IIE-AA and 14 E-AA induced an endothelium-dependent vasorelaxation. Possible mechanisms involved in TAA-induced vasorelaxation were thoroughly investigated. Collectively, data enclosed revealed that TAA induce cerebral vasorelaxation through the interactive activation of BKCa channels with heme oxygenase-2. This interaction leads to generation of carbon monoxide which in turn activates soluble guanylate cyclase and triggers vasorelaxation. / Chronic effects of TAA on microvascular integrity were examined by generating a unilateral hypoxic-ischemic (HI) model of cerebral ischemia on newborn rat pups. Our HI model showed microvascular degeneration as early as 24h post-HI, preceded by an increase in cerebral TAA levels. HI-induced microvascular lesions were dependent on nitric oxide synthase activation and ensued TAA formation. Although the molecular mechanisms leading to TAA-induced microvascular degeneration were, in part uncovered for the retina, the primary site of action of TAA remains unknown. We demonstrated that TAA binds and activates GPR40 receptor, a newly described free fatty acid receptor. Importantly, GPR40 receptor knock-out prevents TAA-induced reduction in cerebral microvascular density and limits HI-induced brain infarct.