No description available.

Chronic pain has been estimated to impact the economy of the United States by an annual cost of $635 billion per year and to affect approximately 100 million Americans (1). Pain is the primary reason patients seek medical attention yet physicians have few options for therapies and there remains a vast unmet medical need for effective and safe analgesics. Most of the drugs clinically available today either have limited efficacy or a variety of unwanted side effects. Discovery of novel therapeutics has been challenging with scientists struggling to find ways to better translate research from the bench-top to the bedside. One impediment in this process has been differences in preclinical and clinical assessment of pain. Preclinical models have historically relied heavily on evoked or reflexive endpoints in non-verbal animals while clinical measures of pain have the advantage of assessing changes in self-reported pain ratings. It is likely, and data from the studies reported in this dissertation show, that mechanisms that underlie threshold responses to evoked stimuli differ from those mediating affective (i.e., aversive) qualities of pain. A further confound is that many effective analgesics are narcotics that carry risk of addiction. Fear of addiction and possibly misuse for chronic treatment of pain may result in undertreatment in many patients. The most clinically relevant question in the management of pain is whether or not a treatment improves the patient's quality of life. Here, we demonstrate that the aversiveness of ongoing pain can be assessed using motivated behavior (conditioned place preference; CPP) and neurochemical output (in vivo NAc microdialysis). Additionally, we assessed the mechanistic effects of three clinically relevant analgesics. Our results show that: (1) pain relief is rewarding and activates reward circuitry that differs from circuits mediating addictive qualities of opiates, and (2) that drugs that mimic the consequences of engagement of descending inhibitory systems act by increasing spinal norepinephrine (NE) levels. These studies provide much needed information that helps build a platform from which more effective analgesics can be discovered and characterized in the preclinical setting and that may help in the introduction of new therapies for patients.

Pathogenesis of pain in pancreatitis is multifactorial, however little is known about the mechanisms by which inflammation in the pancreas causes pain. Here, we hypothesized that pancreatitis-induced pain is dependent upon sensitization of primary afferents by inflammatory mediators such as nerve growth factor (NGF) and interleukin-6(IL-6) and that such a pain is mediated through ascending pathways via the nucleus gracilis. Inflammation in the pancreas resulted in a significant increase in the levels of NGF in the pancreas. Pre-treatment with an anti-NGF peptibody delayed the development of pancreatitis-induced pain. Double injection of anti-NGF peptibody completely prevented the development of pancreatitis-induced pain. Post-treatment with anti-NGF peptibody significantly decreased the number of abdominal withdrawals, compared to the placebo group. Treatment with TRPV1 antagonists reversed the referred abdominal hypersensitivity. Intrathecal administration of p38 inhibitor blocked the pancreatitis-induced pain within minutes after administration. The levels of the TRPV1 in the pancreas, DRG and the spinal cord were not significantly different in the animals with pancreatitis compared to the controls. Pancreatic inflammation resulted in a significant increase in the levels of IL-6 in the pancreas. Treatment with an IL-6 receptor antagonist blocked the referred abdominal hypersensitivity in animals with pancreatitis after systemic and oral administration. Intrathecal administration of the antagonist did not reduce the number of abdominal withdrawals. The possibility that IL-6 might sensitize TRPV1 channels was tested. Cultured DRG neurons were stimulated with IL-6, IL-6 antagonist or a combination of IL-6 with an antagonist, followed by stimulation with capsaicin. Stimulation of DRG with IL-6 produced a marked increase in the levels of CGRP release; combination of IL-6 with an antagonist returned the levels of CGRP to the control levels. To determine the role of the nucleus gracilis in the pancreatitis-induced pain bilateral cannulation of the nucleus gracilis was performed. Microinjection of CNQX into the nucleus gracilis produced a reversal of pancreatitis-induced pain, suggesting an important role of ascending pathway to the n.gracilis in pancreatic pain transmission.

Glial cell line derived neurotrophic factor (GDNF) and artemin maintain the structural and functional integrity of the adult nervous system and regulate the plasticity of the injured or diseased adult nervous system apparently by interacting with GFRalpha1/RET and GFRalpha3/RET systems.The clinical management of neuropathic pain is particularly challenging. Current therapies for neuropathic pain modulate nerve impulse propagation or synaptic transmission; these therapies provide limited efficacy due in part to dose-limiting and undesirable side effects. Here we show that chronic infusion of GDNF normalizes nerve injury-induced neurochemical changes and prevents the expression of neuropathic pain. Systemic artemin produces partial to complete normalization of multiple morphological and neurochemical features of the injury state.Damaged axons in the dorsal root of adult mammals rarely regrow into the spinal cord, leading to the permanent loss of sensory function. This continues to be a major unmet clinical challenge relevant to a host of disease and trauma-induced injuries to peripheral nerves. Here we show that systemic artemin restores sensory function, apparently permanently, in an experimental dorsal root injury model in rats, including responses to noxious heat, mechanical and chemical stimuli and sensory input-required proprioceptive responses of placement stabilization, targeting and grasping. These effects are likely to result from successful support of multiple classes of sensory afferents which cross the dorsal root entry zone into the spinal cord and make functional connections with spinal neurons. Delayed artemin treatment defines the "therapeutic window" for artemin application following injury to the nerve roots, indicating that this strategy may ultimately be of clinical benefit.Our results indicate that the behavioral symptoms of neuropathic pain states can be treated successfully, and that partial to complete reversal of associated morphological and neurochemical changes can be achieved with artemin. The damaged axons can re-grow perhaps into their original region of occupation, and make functional connections with spinal neurons, resulting in apparently permanent restoration of the lost sensory function following dorsal root injury. Our present studies provide experimental evidence that the neurotrophic factors GDNF and artemin may serve as clinically viable drugs in treating peripheral nerve injury or other neurodegenerative diseases.

Intrathecal injection of dynorphin or des-Tyr-dynorphin fragments, which do not bind to opioid receptors, produce tactile and thermal hypersensitivity in rodents. The maintenance, but not initiation, of experimental neuropathic pain depends upon pronociceptive effects of elevated levels of spinal dynorphin. Recent findings implicated a direct excitatory action of dynorphin A at bradykinin receptors in vitro. Here, the possibility that the pronociceptive actions of pharmacological dynorphin or of pathological levels of endogenous spinal dynorphin are mediated by interaction with bradykinin receptors was explored.While spinal administration of a wide range of bradykinin did not produce hyperalgesia in rats, intrathecal injection of non-opioid des-tyrosyl-dynorphin A(2-13) produced reversible tactile and thermal hypersensitivities that were reversed by bradykinin receptor antagonists. Dynorphin-induced behavioral hyperesthesias were observed in bradykinin B2 receptor wild-type but not in B2 receptor knockout mice. Spinal administration or infusion of B1, and especially B2, receptor antagonists reversed experimental neuropathic pain behaviors in rats with peripheral nerve injury but only when the antagonists were given at times at which dynorphin was upregulated. After nerve injury, both B1 and B2 receptor mRNA were increased in the dorsal root ganglion, but not in the spinal cord. While a marked increase in mRNA expression for prodynorphin in the lumbar spinal cord was found following nerve injury, expression of mRNA for kininogen was below detection levels. The possible interaction of spinal dynorphin with bradykinin receptors as a basis of the pronociceptive action of this peptide was further tested in the CFA-induced inflammatory pain and DBTC-induced pancreatitis pain. Intrathecal administration of bradykinin receptor antagonists or dynorphin antiserum reversed DBTC-induced abdominal hypersensitivity and CFA-induced hyperalgesia only when spinal dynorphin or prodynorphin is upregulated. The antihyperalgesic effect of the bradykinin receptor antagonists was not due to de novo production of bradykinin.Taken together, our results unravel a novel, non-opioid molecular target of dynorphin, and indicate that dynorphin acts at bradykinin receptors to produce persistent psin in the pathological pain states. This novel pronociceptive mechanism offers new approaches to the development of therapy for pathological pain states.

Changes in cerebral venous blood volume (DCBVv) is a critical component of the BOLD fMRI signal (instead of total DCBV), but its role has remained relatively unexplored, predominantly because measuring CBVv non-invasively is challenging. Motivated by this challenge, this thesis focuses on the development and use of the venous refocusing for volume estimation (VERVE) technique to non-invasively measure DCBVv. Driven by the substantial signal-to-noise (SNR) gain at high field, VERVE was re-designed for 3 T. This technique is strongly field-dependent through its reliance on venous blood transverse relaxation (T2) variations as a function of the Carr-Purcell Meiboom-Gill (CPMG) refocusing interval and blood oxygenation. To characterize this dependence, human whole blood T2 relaxometry was performed at 3 T. The results reveal significantly enhanced blood T2 dependence relative to 1.5 T, one best modelled as a diffusion process. In addition, human grey and white matter T2 relaxometry results support venous blood T2 variation being the predominant source of VERVE contrast at 3 T. The subsequent design of VERVE was based on the blood relaxometry results. Also, to minimize signal biases due to gradient-echo BOLD effects, greatly amplified at 3 T, a turbo spin-echo approach was adopted, further boosting SNR. VERVE was then used with arterial spin labeling (ASL) to assess the steady-state venous flow-volume relationship in humans under visual and sensorimotor stimulation. The results demonstrated a spatially-invariant flow-volume relationship characterized by a power-law coefficient (a) of 0.23, significantly lower than Grubb’s value of 0.38 (derived using total DCBV). The assumption of the latter in calibrated BOLD introduced a significant underestimation in cerebral oxygen metabolism changes (DCMRO2). Finally, the interactions giving rise to the controversial BOLD post-stimulus undershoot were examined with respect to the prevalent biomechanical, metabolic and neuron / Les changements du volume sanguin cérébral veineux (DCBVv) est un élément essentiel du signal BOLD (par opposition à l’ensemble de DCBV). Pourtant, jusqu'ici le rôle du CBVv est resté relativement inexploré, et ce du aux difficultés liées aux mesures non-invasives du CBVv. Motivée par ce défi, cette thèse se rapporte sur le développement et l'utilisation de la méthode VERVE (refocalisation veineuse pour l’estimation du volume), qui permet l’estimation non-invasive de DCBVv. D’abord, l’augmentation importante du rapport signal-sur-bruit (SNR) aux champs magnétiques élevés a mené à réviser VERVE pour 3 T. Le contraste VERVE est basé sur les variations du temps de relaxation transversale (T2) sanguin veineux en fonction de l’intervalle de refocalisation Carr-Purcell Meiboom-Gill (CPMG) et de l’oxygénation sanguine. Pour caractériser cette dépendance, qui dépend fortement du champ magnétique, une étude relaxométrique du sang humain a été réalisé à 3 T. Les résultats indiquent que la dépendance du T2 sanguin est amplifiée de façon importante entre 1.5 T et 3 T. Un modèle de diffusion décrit le mieux cette dépendance. D’autre part, une étude relaxométrique de la matière grise et blanche a été réalisée, confirmant la dominance de l’effet T2 sanguin dans le contraste VERVE. La composition de VERVE se rapporte aux résultats relaxométriques sanguins. En plus, afin de minimiser l’effet de l’écho de gradient, amplifié à 3 T, une approche turbo spin-écho a été adoptée. VERVE est ensuite utilisée avec le marquage des spins artériels (ASL) pour mesurer les changements du CBVv et du débit sanguin cérébral (CBF) chez les sujets sains lors de stimulations visuelles et sensorimotrices. Les résultats démontrent une relation débit-volume invariante à travers le cortex, caractérisée par a = 0.23, inférieure à la valeur de Grubb (0.38, calculée a partir du CBV total). En employant cett

No description available.

Sindbis virus (SINV) is a member of the Alphavirus genus and belongs to the family Togaviridae. The virus has a positive sense RNA genome of 11700 bases which encodes for both structural and non structural proteins. Infections are frequently diagnosed based on clinical, epidemiological and laboratory criteria. Laboratory confirmation is essential as SINV infections must be distinguished from various conditions that share similar clinical manifestations. The most frequently used methods for identification are haemagglutination inhibition, enzyme-linked immunosorbent assay, plaque reduction neutralization tests as well as conventional in-vitro neutralization assays. Serological assays for the detection of SINV are not readily available commercially and due to the non-specific symptoms caused by SINV infection the number of infections per annum may be under diagnosed. The purpose of this study was to develop serological assays such as ELISA and a novel neutralization assay that could be used in serological surveys for the detection of IgG antibodies against SINV. Furthermore to develop assays that could be used to determine the level of viral replication in mammalian cells for characterizing infection in mammalian cells as well as investigate the influence of interferon on viral replication and look for evidence of apoptosis caused by SINV infection. An in house ELISA was developed and used to screen 146 sera for IgG antibodies against SINV. The in-vitro neutralization assay is the gold standard for serology and 43 samples in total were tested in both the ELISA and the in-vitro neutralization assay. Analysis and comparison of the results obtained using the in-house ELISA and the neutralization assay indicated that the sensitivity of the ELISA was 68.9% and the specificity of the in house ELISA was 78.57 - 85.71% depending on the use of the percentage positive or optical density values to differentiate positive and negative samples. A forward and reverse primer for the amplification of a conserved 181bp region of the nsp2 gene encoding the nsp2 protein of SINV were designed along with a TaqMan hydrolysis probe to be used in a real time quantitative TaqMan PCR. The infection of mammalian cells, human macrophages and HeLa cells, was determined by measuring viral loads with a real time quantitative TaqMan RT-PCR. Two strains of SINV were used in attempts to infect macrophages, a strain from Egypt and a strain from South Africa. Small increases in viral load suggested possible low levels of viral replication but were considered insufficient to warrant further investigation and insufficient to investigate occurrence of antibody dependent enhancement of disease in macrophages. The mechanism possibly interfering with replication of virus in the human macrophages was investigated. Supernatant fluid samples from macrophage infections were tested for the release of interferon gamma which could inhibit viral replication. There were nine to fifteen fold differences in the concentration of interferon gamma detected in the supernatant fluid at baseline and 24h after infection. HeLa cells were treated with similar concentrations of human interferon gamma at different time intervals. Pretreatment and concurrent treatment with infection showed reduced levels of viral load compared with no treatment or delay in treatment. Hence the suggestion that interferon could have played a role in inhibiting viral replication in the human macrophages. DNA was extracted from HeLa cells infected with SINV and the DNA fragments separated through agarose gel electrophoreses. There were multiple bands visible in the infected samples whereas the negative control did not show multiple bands, only one large band of genomic DNA. The presence of multiple DNA fragments in infected cells and absence of those fragments from uninfected cells were suggestive of virus induced apoptosis.

A protocol of establishing radiochromic film based reference dosimetry for high dose rate Ir-192 brachytherapy source was assessed and described. A comparison between calibration curves created in water and Solid Water are provided. Solid Water was shown to be a viable alternative to water in establishing calibration curve for Ir-192 radiation beam. A Monte Carlo correction factor was calculated to convert the dose to water into dose to Solid Water and the experimental methods that we performed agreed with the Monte Carlo results where the ratio (DSW/DW)Ir-192 was found to be 0.9808 ± 0.14% (1σ). EBT-2 GAFCHROMIC film model was also investigated for absorption properties and found to be a less sensitive than its predecessor (EBT-1) in terms of net change of absorbance, but that did not affect the dosimetric value that this film possesses. A dose error assessment method has been described for EBT-2 film model (and is applicable to other types as well) that can establish the time error constraints on the post-irradiation scanning time that will still provide an acceptable dose error for clinical applications if the protocol employing the shorter post-irradiation scanning time is implemented in the clinic. We show that for two post-irradiation scanning times of 30 minutes and 24 hours the 1% dose error can be granted if the scanning time window is less than ± 5 minutes and ± 2 hours, respectively. Performance of EBT-2 model was also evaluated in water and it was concluded that a suggested correction protocol is necessary for immersion times that exceed 2 hours. This correction was tested with the calibration curve created from water setup and found to be effective when compared to the dose-corrected calibration curve in Solid Water. / Un protocole d'établir film radiochromique dosimétrie de référence en fonction de débit de dose élevé source Ir-192 curiethérapie été évalués et décrits. Une comparaison entre les courbes d'étalonnage créé dans l'eau et Solid WaterTM sont fournis. Solid WaterTM s'est révélée être une alternative viable à l'eau dans l'établissement de la courbe d'étalonnage pour les Ir-192 faisceau de rayonnement. Un facteur de correction de Monte Carlo a été calculé pour convertir la dose à l'eau en dose à Solid WaterTM et les méthodes expérimentales que nous avons réalisé d'accord avec les résultats de Monte Carlo où le ratio (DSW/DW)Ir-192 a été trouvé à 0.9808 ± 0.14% (1σ). EBT-2 modèle GAFCHROMICTM film a également été étudiée pour les propriétés d'absorption et jugé être un moins sensible que son prédécesseur (EBT-1) en termes de variation nette de l'absorbance, mais cela n'a pas d'incidence sur la valeur dosimétrique que ce film possède. Une méthode d'évaluation des doses d'erreur a été décrit pour le modèle EBT-2 film (et est applicable à d'autres types ainsi) qui permet d'établir les contraintes de temps d'erreur sur le post-irradiation temps de balayage, qui va encore donner une erreur de dose acceptable pour des applications cliniques, si le protocole emploie le plus court post-irradiation de numérisation temps est mis en œuvre dans la clinique. Nous montrons que pour deux post-irradiation de numérisation fois de 30 minutes et 24 heures, la dose d'erreur de 1% peut être accordée si la fenêtre de temps de balayage est inférieure à ± 5 minutes et de ± 2 heures, respectivement. Performance de la EBT-2 modèle a également été évaluée dans l'eau et il a été conclu un protocole de correction proposé est nécessaire pour que les temps d'immersion supérieure à 2 heures. Cette correction a été testé avec la courbe de calibration créée à partir d'installation de l'eau et ont été jugés effic

Photoneutrons generated in the linac head are a byproduct of the radiotherapy process and can be potentially harmful to clinical personnel. The lack of accuracy associated with analytical photoneutron shielding methods has generated much interest in the Monte-Carlo (MC) method as a more flexible and precise tool for radiotherapy bunker design. The purpose of this work was to use MC simulations to characterize photoneutron fluence, dose, and spectrum throughout various radiotherapy bunker configurations and for various room design features, such as the presence of a maze, a bulkhead, and the addition of borated polyethylene on the maze walls. Three existing rooms at the MUHC and two hypothetical doorless rooms were modelled with the MCNP5 code and using the Visual Editor GUI. The analytical spectrum of an 18 MV linac served as the point source of photoneutrons and was surrounded with a 10 cm radius tungsten sphere placed 100 cm above the isocenter. The next-event estimator variance reduction technique was used and simulations were performed with 20 million particle histories yielding un- certainties under 1%. Physical measurements were also attempted with bubble detectors and a He-3 neutron spectrometer. The latter was unsuccessful because of pulse pile-up caused by the Linac's pulsed mode of operation, whereas the former gave us qualitative information on neutron equivalent dose distribution in the maze and around the linac. Simulation results showed a marked decrease in neutron equivalent dose near the bunker entrance when maze walls are lined with BPE and when a bulk-head is added in the inner maze passage. It was found that the high thermal neutron cross-section of BPE was key in reducing the portion of thermal photoneutrons in the spectrum along the maze. The bulkhead was also useful in reducing photoneutron fluence entering the maze and hence reducing overall photoneutron dose near the entrance of the bunker. Future work will focus on validating simulations with accurate physical measurements and refining the MC code to make it more user friendly and flexible in reproducing bunker geometry. / Les photoneutrons générés par le linac sont un produit secondaire de la radiothérapie et peuvent être nuisibles au personnel médical. Le manque de précision des équations analytiques pour le blindage contre les photoneutrons a accéléré le développement des méthodes Monte-Carlo (MC), qui sont considérées plus flexibles et précises pour le design des salles de radiothérapie. L'objectif de cette étude est d'utiliser les simulations MC afin de caractériser le flux, la dose, et le spectre des photoneutrons pour différentes configurations de salles de radiothérapie, telles que la présence d'un corridor, d'un bloc d'atténuation, et l'addition de borate de polyéthyléne sur les murs du corridor. Trois chambres du MUHC et deux chambres hypothétiques ont été modélisées avec le code MCNP5 et le logiciel Visual Editor. Le spectre d'énergie analytique d'un linac opérant à 18 MV a été utilisée comme source ponctuelle de photoneutrons. Ce point est entouré d'une sphére de Tungsténe de 10 cm de rayon positionnée 100 cm au dessus de l'isocentre. L'estimateur du prochain événement est la technique de réduction de variance qui a été utilisée et les simulations ont été effectuées avec 20 millions de particules résultant en des incertitudes inférieures à 1%. Des mesures physiques ont aussi été tentées à l'aide de compteurs à bulles et un spectrométre de neutrons à He-3. Ce dernier n'a pas eu de succés à cause de l'effet d'accumulation du signal pulsé. Les tests avec compteurs de bulles ont permis d'avoir une idée qualitative sur la distribution de la dose équivalente dans le corridor et autour du linac. Les résultats des simulations ont montré une diminution de la dose équivalente de neutron prés de l'entrée de la chambre quand les murs du corridor sont couverts de borate de polyéthyléne et quand un bloc d'atténuation est présent dans le passage de la chambre centrale vers le corridor. Il a été confirmé que la haute probabilité d'interaction des neutrons de basses énergies avec le borate de polyéthyléne est essentiel à la réduction de la portion de photoneutrons à basses énergies à travers le corridor. Le bloc atténuateur contribue aussi à la réduction du flux de photoneutrons entrant dans le corridor et réduit ainsi la dose totale à l'entrée de la chambre. La suite des travaux vise à mettre l'emphase sur la validation des simulations à l'aide de mesures expérimentales et sur le perfectionnement du code MC pour donner plus de flexibilité à l'utilisateur dans la reproduction des salles de radiothérapie.