The present study describes a new test of tonic pain in rats which can be used as an animal model of persistent pain. In the first experiment, the response to subcutaneous injection of various doses of bee venom into the hind paw of the rat was quantified. The second experiment investigated the effect of morphine and aspirin on the response to an intermediate dose of bee venom. Finally, the third experiment examined the response to concurrent injections of bee venom and formalin. Subcutaneous injection of bee venom produced local inflammation, marked edema, and tonic pain responses. Increasing doses of bee venom produced higher mean pain scores and increased durations of responding. Pain responses lasted up to approximately one hour and the inflammation and edema were virtually gone by 8 hours with the lower doses of bee venom tested and by 2 days with the two highest doses tested. Analgesia was produced by morphine and aspirin, indicating that the bee venom test can be used to test analgesic drugs. Concurrent administration of bee venom and formalin produced responses similar to formalin alone, with an increased duration of responding at higher intensities. The data suggest that the bee venom test is a valid animal model of experimental tonic pain.

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University of Macau / Institute of Chinese Medical Sciences

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Purpose Retinal pigmentosa (RP) is a heterogeneous group of retinal degeneration with a multitude of hereditary genetic defect. Photoreceptor degeneration usually starts with rods but invariably involves cones in later stages, leading to significant visual debilitation. Many animal models, in particular mouse models, have been used for the study of RP. The rd10 mouse, with mutation in the beta subunit of the rod phosphodiesterase gene, has been chosen as a model for an autosomal recessive form of human RP because of its later onset of retinal degeneration. The topographic and morphological patterns of cone photoreceptor degeneration following the loss of rods were studied. Methods The rd10 mice were sacrificed and enucleated at postnatal 14 days, 21 days, 1 month, 2 months and 3 months. The retina was processed with immuno-staining to differentiate different photoreceptor cells and mounted flat for microscopic examination. The topographic pattern of cone photoreceptor loss at different ages was identified. Confocal microscopy was used to examine the morphological changes of cone degeneration. The retina from an adult c35 mouse was chosen as a reference for comparison. Results Following the onset of rod degeneration, the orderly arrangement of cones became disrupted. Remodeling of cone cells was observed as the loss of outer segments, swelling of the somata, and redistribution of opsin. Subsequently the inner segment and part of their axon and pedicles were involved. Some cones then demonstrated neurite sprouting, restoring a new polarized morphology. However, with increasing age, extensive atrophy of cone cells ensued. The topographic pattern of cone degeneration advanced from central to the peripheral retina, with the cones in the superior part of the retina most resistant to degeneration. Conclusion Cone photoreceptors respond to the loss of rods by remodelling and maintain a relatively normal phenotype for a considerable period of time, especially those in the superior part of the retina. This may provide a therapeutic window for cone rescue for patients of RP. / published_or_final_version / Anatomy / Master / Master of Medical Sciences

Osteoarthritis (OA) has been one of the most prevalent joint disorders which cause pain, impair mobility of patients and bring heavy burden to social economics. Unfortunately up to now we still know little about OA. We are not sure what exactly cause OA. The pathogenesis of OA is not clearly understood. Besides, we don’t have effective treatment for OA. When joint degeneration goes to the end-stage, Total Joint Replacement surgery seems to be the only way to relieve pain and restore joint function. Since cartilage degeneration is quite difficult to be stopped or reversed, people are trying to looking into the upstream events which occurred at early stage of Osteoarthritis. Nowadays, early OA changes have drawn attention. It is considered to play an important role in OA initiation and progression and may be the key for OA treatment. However, the exact change at each component of the joint including cartilage, bone and osteochondral junction at early stage of OA is still poorly elucidated. Because it’s quite difficult to obtain information from early staged OA patients, and it’s also difficult to conduct longitudinal observation on human OA patients, we need animal models to obtain knowledge about OA. In this study, we adopted the Dunkin-Hartley (DH) strain guinea pig, which has been widely accepted as aging-related spontaneous OA model. Meanwhile, the pigmented guinea pig was used as OA-resistant control. The aim of this study was to: 1) observe and compare the difference of OA progression between DH and pigmented guinea pigs; 2) characterize the early changes of subchondral bone, cartilage and osteochondral junction in DH strain comparing with pigmented guinea pig; 3) preliminarily investigate the disease-modifying effect of Transforming Growth Factor beta 1 (TGFβ1) receptor inhibitor on OA progression. Significant cartilage degeneration was found in DH strain with cartilage surface disruption, cleft, proteoglycan loss and diffused hypertrophic chondrocytes clustering at age of 12 months. In comparison, cartilage of pigmented guinea pigs remains intact and smooth. At early stage with age from 1 to 3 months, in DH strain, subchondral bone was found to be sclerotic, denser with ultrastructure change, and chondrocytes was found with elevated level of proliferation and apoptosis concurrently, while osteochondral junction was found to be thicker, denser and less porous comparing with pigmented strain. Additionally, TGFβ1 receptor inhibitor was found effective to suppress formation of Bone Marrow Lesion, which is the early sign of OA detected by Magnetic Resonance Imaging. It also preserved the volume of cartilage and subchondral plate against OA degeneration. In conclusion, OA-prone Dunkin-Hartley guinea pigs showed significant cartilage degeneration at age of 12 months. In comparison, pigmented guinea pigs are OA-resistant. At early stage of OA, DH guinea pigs were found with subchondral bone ultrastructure change and chondrocytes hyperproliferation as well as apoptosis. Disease-modifying drugs such as TGFβ1 receptor inhibitor is potentially an option for OA treatment. The finding of this study may contribute to new insight and understanding of early OA pathogenesis and potential development of specific therapeutics strategies. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Schizophrenia is a chronic, common and debilitating illness which causes serious psychosocial impairments. Despite the adverse impact of schizophrenia on public health, progress in understanding its pathophysiology is frustratingly slow, which hinders discovery of new therapeutic mechanisms. The major factors that have impeded this exploration are the complex neurobiology of higher brain function and the ethical and practical difficulties of investigating the living brain. Thus, animal models are useful to investigate the pathophysiology and therapeutics of schizophrenia and related conditions. A useful animal model is an important tool to illuminate pathophysiology and signpost a target for treatment development. But animal models also have limitations and not all the phenotypic traits thought relevant to schizophrenia are expressed in all models. However, in-bred mouse strains have proved useful in the field of research into neurodevelopmental disorders. Therefore, in the first part of this thesis, behavioural and protein expression of C57BL/6N and 129 X1/SvJ mice were compared. The 129X1/SvJ strain, like the C57BL/6N strain, is a widely used background strain for behavioral research. Both 129X1/SvJ and C57BL/6N are routinely used in gene-targeting research. The results suggested that C57BL/6N mice mimic aspects of schizophrenia, at least in comparison with 129X1/SvJ mice. Therefore C57BL/6N mouse may have application in pre-translational screening of new treatments for schizophrenia. Oxytocin has been proposed as a possible treatment for neurodevelopmental conditions such as schizophrenia. However, there are gaps in the understanding of its therapeutic potential, in particular the extent to which it may have effects on non-social as well as social behaviors in both sexes. In the second part of the thesis, female and male C57BL/6N mice were used to study the effects of oxytocin on social and non-social behaviours relevant to schizophrenia. Oxytocin generally ‘improved’ behaviours analogous to those reported to be impaired in neurodevelopmental disorders, but effects were observed at different doses in each sex. The work here suggests oxytocin has potential for treatment of both social and non-social features of schizophrenia. Further research into the clinical application of this peptide hormone, which may in turn significantly extend treatment options across a spectrum of neurodevelopmental conditions, should be encouraged. / published_or_final_version / Psychiatry / Doctoral / Doctor of Philosophy

The etiology and pathophysiology of schizophrenia are poorly understood, although increasing evidence suggests an important role for altered GABA neurotransmission. Animal models of schizophrenic symptoms include administration of noncompetitive N-methyl-D-aspartate (NMDA) glutamate receptor antagonists, such as dizocilpine (MK-801), and post-weaning social isolation. The present study tested the hypothesis that a “double-hit” model, in which subchronic MK-801 administration and post-weaning social isolation are combined, produces greater behavioural and neurochemical effects than either insult alone. As a secondary objective, the present study also assessed whether the timing of the subchronic MK-801 injections (early adolescence vs. early adulthood) influences these measures. Male Sprague-Dawley rats (N = 74) were obtained at weaning (P21) and were either socially isolated (n = 42) or group housed (n = 32) for the duration of the experiment. Subgroups received subchronic treatment with MK-801 (0.5 mg/kg ip) or saline injections (1.0 ml/kg ip) twice daily for seven days either during early adolescence (P25-P32) or early adulthood (P56-63). At P70, all groups were tested for locomotor activity and subsequently sacrificed to assess the function of the GABA membrane transport protein, GAT-1, and GABAA receptor expression in the frontal cortex and hippocampus. For animals treated in early adulthood, post-weaning social isolation, in comparison to group housing, resulted in an increase in (1) locomotor activity (2) GAT-1 activity in frontal cortex and hippocampus and (3) GABAA receptor expression in the frontal cortex. MK-801 treatment in early adulthood increased GABAA receptor expression in the hippocampus, whereas post-weaning social isolation had no effect on GABAA receptor expression in the hippocampus. Previous studies have demonstrated that increased GAT-1 activity is associated with suppression of GABA-mediated inhibitory synaptic transmission. Furthermore, increased GABAA receptor expression may be a compensatory response to decreased availability of GABA. These data indicated that combined post-weaning social isolation and subchronic MK-801 treatment do not produce additive or synergistic effects on locomotor behaviour or GABA signalling, but rather induced differential effects on GABAA receptor binding. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2010-09-01 15:21:58.474

Chapter 5 reported the study to assess whether the urinary metabolic alterations linked to early pathophysiological changes in the TgCRND8 mouse model of AD. An unbiased metabolomics approach using high resolution Orbitrap mass spectrometry coupled with hydrophilic interaction liquid chromatography was conducted to uncover the metabolic alterations as a relevant readout of biochemical activity that implicated in the pathogenesis and progression of AD in the TgCRND8 mice. A total of 73 differential metabolites of urine sample sets was identified in 12-week and 18-week transgenic mice compared to wild-type littermates, covering perturbations of aromatic amino acids metabolism, TCA cycle and one-carbon metabolism. Of particular interest, divergent tryptophan metabolism, such as up-regulation of serotonin pathway while down-regulation of kynurenine pathway, was observed. The accumulation of both N-acetylvanilalanine and 3-methoxytyrosine indicated the aromatic L-amino acid decarboxylase deficiency. The microbial metabolites derived from tryptophan metabolism and drug-like phase II metabolic response via the glycine conjugation reactions were also highlighted, indicating that genetic modification in mouse brain not only alters genotype but also disturbs gut microbiome. Together, our study demonstrated that the integrative approach employing mass spectrometry-based metabolomics and a transgenic mouse model for AD might provide new insights into the metabolic phenotypes of AD with a noninvasive approach.;For cancer metabolism research, much effort has been focused on development of ultrahigh performance liquid chromatography triple quadrupole mass spectrometry (UPLC-MS/MS)-based targeted metabolomics method and its emerging applications in exploiting oncogene-induced metabolic alterations. To achieve our goal, more than one hundred intermediate and/or metabolite were selected and broadly categorized into cationic species and anionic species. Tandem mass spectrometric conditions were extensively optimized for each analyte by using energy-resolved collision-induced dissociation. Two crucial operating parameters of tandem mass spectrometry, namely, cone voltage and collision energy were finely tuned to get the highest signal response of the parent ion and fragment ions. Multiple reaction monitoring (MRM) transitions were created for each targeted compound, providing foundation for MRM-based assays. Meanwhile, to enhance the retention and separation of the water-soluble metabolites on reversed-phase C18 column, hydrophobic ion-pairing interactions separation (HIPS) strategies were proposed and established via complementary use of two ion-pairing reagents, heptafluorobutyric acid and tributylamine, for the cationic species and anionic species, respectively. The HIPS strategies led to efficient retention and resolution of polar intermediates/metabolites, covering the majority of components involved in central carbon metabolism and amino acid metabolism. Even isomeric pairs, like citrate-isocitrate and leucine-isoleucine, were almost baseline resolved. The performance evaluation of the developed UPLC MRM-based assays showed that nanomolar levels of limit of quantification were achieved. The developed methods enabled quantitative analysis of central carbon metabolism in mammalian cells. The altered metabolism induced by the overexpression of the oncogene EIF5A2 in human normal liver cell line LO2 was studied. We found that the altered aerobic glycolysis and pentose phosphate pathway dysregulated the tricarboxylic acid (TCA) cycle and amino acid imbalances presented as distinct metabolic features in EIF5A2 overexpressed LO2 cells.;In chapter 3, we performed quantitative analysis of central carbon metabolism and amino acid metabolism via the established UPLC-MRM-based metabolomics, which was combined with pharmacological inhibition of the catalytic enzymes, O-linked N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and β-N-acetylglucosaminidase (O-GlcNAcase, OGA) in order to uncover the contribution of protein (including the glycolytic enzymes) O-GlcNAc modification to metabolic alterations in cancer cells. We found that OGA inhibition led to decreased levels of intermediates in both glycolysis and TCA cycle, but increased level of pentose phosphate pathway. Interestingly, the opposite phenotypes were obtained in OGT inhibition, i.e., the increased levels of glycolysis and TCA cycle were observed. Our data suggested that O-GlcNAc modification could direct switches of glucose metabolism through coordinated glycolysis and TCA cycle pathways in HCC cell line.;In Chapter 4, an improved UPLC-MS/MS method for accurate and rapid assessment of the content and redox state of coenzyme Q10 (CoQ10) and the crucial component of electron transport chain (ETC) was described. Non-aqueous reversed phase liquid chromatography on a C18 column was hyphenated with tandem mass spectrometry working in the electrospray ionization positive MRM mode, with methanol serving dual roles as sample preparation solvent and mobile phase. This rapid extractive and analytical method could avoid artificial auto-oxidation of reduced form of CoQ10, enabling the native redox state assessment. To demonstrate the utility of the developed method, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposed mice liver tissue were analyzed, revealing the down-regulated mitochondrial ETC in TCDD exposed mice group.;This thesis research concentrates on the development and applications of mass spectrometry-based metabolomics to elucidate biochemical alterations involved in basic research models for two common human diseases: mammalian cell culture model of hepatocellular carcinoma (HCC) and transgenic mouse model of Alzheimer's disease (AD). Two major approaches were developed: (1) targeted quantitative metabolomics for elucidation of altered cancer metabolism in human liver cell lines caused by the overexpression of the oncogene eukaryotic translation initiation factor 5A2 (EIF5A2) and O-Linked β-N-acetylglucosamine (O-GlcNAc) modification; (2) non-targeted metabolite profiling for early discovery of potential non-invasive urinary metabolite markers in the transgenic mouse model TgCRND8 of AD.

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