Provoked Vestibulodynia (PVD) is a common form of chronic genital pain, affecting approximately 12% of premenopausal women. Even though knowledge of vulvodynia has been present in the medical field for many years, it was previously thought to be of psychogenic origin and has never been thoroughly investigated for the purpose of pain classification. When investigating any pain condition, one of the most important distinctions to make is whether or not the pain is neuropathic. Even though this possibility has never been investigated in women with PVD, some have claimed that PVD pain contains elements of neuropathy, even treating this pain with medication created for neuropathic pain conditions. The purpose of this study was to use standardized measures and determine whether PVD may have a neuropathic component. Women with PVD completed an online survey assessing various pain and psychosocial variables. Their responses were compared with those of pain-free controls and women experiencing an established neuropathic pain condition, post-herpetic neuralgia (PHN). Women with PVD scored above established cut-offs on measures of neuropathic pain (NP). Further, for some NP measures there was no difference in scores between PVD and PHN women. Women with PVD also had similar psychosocial profiles as those with PHN, although women with PHN reported poorer health-related quality of life. Interestingly, the number of NP symptoms did not predict pain/psychosocial disturbance, or vary as a function of pain duration or intensity. Overall, these results lend support to the argument that PVD is a chronic pain condition. Further, these results indicate that women with PVD likely experience some form of NP. These results add to the understanding and classification of PVD, justifying further investigation, for example, via psychophysical testing and functional magnetic resonance imaging. / Thesis (Master, Psychology) -- Queen's University, 2011-09-21 16:25:34.216

In this thesis, the detection of global proteomics alteration and changes in neuropeptide distribution caused by neuropathic pain in rat spinal cord tissue was the main focus. Neuropathic pain (NP) is a major clinical syndrome caused by disease or dysfunction of the nervous system and often mediated by neuronal networks in the spinal cord. The estimated prevalence of NP is 6-8% in general population. Only in the United States, the indirect cost associated with chronic pain has been estimated to 100 billion dollars each year and NP substantially contributes to this cost. So far, the underlying mechanisms of NP are not well understood. Proteomics techniques are commonly used in biology system studies, due to its high throughput, capability of unbiased analysis and sensitivity. It builds up a bridge to link genes, peptides, proteins, and the disease. Two proteomic/peptidomic approaches were developed, evaluated and discussed in this thesis. Both of them were further applied in the studies of neuropathic pain. First approach is a quantitative proteomic approach using liquid chromatography combined with Fourier transform mass spectrometry (LC-FTMS), which is developed for quantitative analysis of proteins originated from small central nervous system (CNS) samples. This approach was successfully applied in the study of the rat spinal cord tissue proteome in a neuropathic pain model. Another approach is using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) for the visualization of the distribution of neuropeptides in rat spinal cord, which in the future will be applied in investigating the ongoing signal transmission under neuropathic pain conditions. Results provided by these two methods are of high importance for the general understanding of the underlying pathophysiological mechanisms and potential identification of new targets for novel treatment of neuropathic pain.

Background: Pain is frequently reported by people with multiple sclerosis (MS). It has been associated with decreased quality of life, psychiatric morbidity, interference with day to day activities, and frequent healthcare attendance. It has been reported by people with multiple sclerosis to be one of their most important symptoms, and available treatments are limited in their effectiveness. Despite this, our understanding of the epidemiology and mechanisms of pain in people with MS are limited. Our understanding of the interactions of central nervous system mechanisms and pain states overall is growing. However, the application of this knowledge to MS is incomplete. Previous studies have shown that the descending pain modulatory system (DPMS) is an endogenous network of cortical and subcortical brain structures which act to limit, or accentuate, an individual’s perception of pain, via descending brainstem pathways. Associated clinical measures include depression, anxiety, and cognitive flexibility. Our understanding of the function or dysfunction of this system in MS is limited. We do not know if the MS disease process may adversely affect the structure or function of the DPMS. Hypothesis: In people with neuropathic limb pain in relapsing remitting MS (RRMS), compared to people with RRMS who do not have pain, there will be disruption of the endogenous descending pain modulatory system. This will manifest as impaired descending inhibition of pain. Aims and Methods Establishing the background using systematic reviews: The first aim of this thesis was to establish the prevalence, natural history and associations of pain (and pain syndromes) occurring in people with MS. The second aim was to explore existing knowledge of how the MS disease process may contribute to pain states, using a systematic review of neuroimaging studies. Prospective clinical study: A case-control study of 47 people with RRMS was then carried out. 31 of these had neuropathic pain in the limbs, and 16 did not have pain. Using targeted assessments, function of the descending pain modulatory system was assessed in the following ways: First: Detailed clinical, behavioural and neuropsychological assessment, focussing on cognitive, behavioural and affective features known to be closely related to the DPMS. Second: MRI imaging of brain structure, focussing on the volume and location of MS lesions, as well as the volume of key grey-matter structures involved in the DPMS. Third: Resting state functional MRI imaging of the brain, focussing on functional connectivity between the rostral anterior cingulate cortex and two other key DPMS structures (dorsolateral prefrontal cortex, and periaqueductal gray). Results: Systematic reviews: Meta-analysis of existing prospective studies confirmed that pain is very common in MS, affecting about 63% of people with MS on average (95%CI between 55 and 70%). Many different types of pain contribute to this overall estimate. No significant associations with disease course or stage emerged. Several neuroimaging studies have assessed people with MS-associated pain using MRI. These studies were often small, and with associated methodological issues. It is likely that location of MS lesions is implicated in aetiology of pain syndromes in some cases, though our overall knowledge is limited. Prospective study: In a prospective study, people with and without pain were matched for age and gender. Furthermore, groups were balanced for a range of other variables. The pain group more frequently received gabapentinoid medications. The presence of pain was significantly associated with increased scores for depression, fatigue and catastrophising, as well as with specific impairments at neuropsychological assessment, including cognitive flexibility. Many of these impairments are directly relevant to existing models of the DPMS. Overall volume of MS lesions was not different in people with pain, though lesions were more likely to occur in the brainstem. Some alterations of grey-matter volumes in people with pain which mirrored studies of pain disorders outside MS were found, but these did not involve structures key to the DPMS. Affected structures included trigeminothalamic nucleus (relative volume increase in pain group), posterior cingulate cortex and parahippocampal gyrus (volume decrease in pain group). Functional connectivity of the rostral anterior cingulate cortex to the periaqueductal grey matter, a key structure in the descending modulation of pain, was stronger in the group without pain. Conversely, functional connectivity to the dorsolateral prefrontal cortex, repeatedly implicated in the DPMS and thought to be involved in cognitive evaluation and flexibility, was stronger in the pain group. MS lesion volume appeared to account for some of this difference in a multivariate analysis. Limitations: Key limitations of this work include cross-sectional design, small sample size, and number of statistical comparisons carried out. Conclusions: Systematic reviews examined the prevalence, natural history and associations of pain in MS, as well as examining existing neuroimaging studies which investigated how the MS disease process could contribute to pain states. A prospective study found evidence of both emotional/affective and cognitive dysfunctions relevant to the hypothesis of dysfunction in the DPMS. Higher likelihood of MS lesions in the brainstem could be relevant to DPMS function. Separately, there were structural grey-matter volume alterations reflecting those found in many pain studies outside MS. Importantly, however, these did not affect key DPMS structures. Resting state functional MRI however demonstrated altered connectivity of core DPMS structures, which may be partly mediated by MS lesion volume. Functional connectivity findings could be consistent with the hypothesis of impaired descending pain inhibition, in people with relapsing remitting MS affected by neuropathic limb pain.

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Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / O diabetes é uma doença de alta prevalência que, frequentemente, induz o comprometimento do sistema nervoso periférico. Na neuropatia diabética periférica, os sintomas mais encontrados são os sensitivos, no qual a dor neuropática, condição crônica caracterizada por alodinia e hiperalgesia, é a mais debilitante. Esta, prejudica a qualidade de vida do paciente, sendo muitas vezes não responsiva aos métodos farmacológicos convencionais de tratamento. Diante desse panorama, o desenvolvimento de novas abordagens terapêuticas que possuam ação efetiva neste tipo de dor é de grande relevância. O uso da terapia celular no tratamento de lesões do sistema nervoso tem demonstrado resultados promissores e o potencial terapêutico de células-tronco na neuropatia experimental tem sido proposto. Neste estudo, avaliou-se o efeito de células-tronco mesenquimais derivadas da medula óssea (CMsMO) na neuropatia diabética periférica estabelecida em modelo experimental de diabetes induzido por estreptozotocina (ETZ). Quatro semanas após a indução do modelo por ETZ (80 mg/kg; ip; 3 dias consecutivos), os animais receberam uma administração endovenosa de CMsMO (1 x 106) ou veículo. O tratamento com gabapentina (30 mg/kg; v.o. a cada 12 horas durante seis dias consecutivos) foi usado como padrão ouro. Os limiares nociceptivos térmico e mecânico foram avaliados durante todo o período experimental (90 dias), pelos métodos de hargreaves e von Frey. A avaliação da função motora foi realizada pelo teste de rota-rod. Em diferentes tempos e para todos os grupos experimentais, foram realizadas coletas de segmentos da medula espinal (L4-L5) para dosagem de citocinas por ELISA e segmentos do nervo isquiático foram também coletados para avaliação de alterações morfológicas por microscopia óptica e eletrônica de transmissão. Os dados comportamentais demonstraram que o tratamento com CMsMO reduziu a mecanoalodinia e a hipoalgesia térmica, levando os limiares nociceptivos de animais neuropáticos a níveis similares aos de animais não neuropáticos. Do mesmo modo, a administração de CMsMO normalizou a função motora dos animais neuropáticos. Dados de microscopia mostraram que animais neuropáticos apresentaram atrofia axonal, redução do número de fibras mielínicas e aparente redução do numero de fibras amielínicas no nervo isquiático. Animais neuropáticos tratados com CMsMO tiveram menor ocorrência de atrofia axonal e não apresentaram redução do numero de fibras mielínicas ou amielínicas, em relação aos neuropáticos tratados com salina. Além disso, animais neuropáticos tratados com CMsMO apresentaram menores níveis espinais de IL-1β e TNF-α, e maiores de IL-10 e TGF-β, em relação aos animais neuropáticos não tratados. Esse conjunto de resultados indica que CMsMO produzem efeito antinociceptivo duradouro na neuropatia diabética, seguido de modificações no padrão fisiopatológico da doença, o que aponta a terapia celular como uma interessante alternativa para o controle da neuropatia diabética periférica dolorosa. / Diabetes is a highly prevalent disease which frequently compromises the peripheral nervous system. In peripheral diabetic neuropathy, the most frequent symptoms are sensitive, in which the neuropathic pain, chronic condition characterized by allodynia and hyperalgesia, is the most debilitating. Neuropathic pain affects the quality of patients’ lives, and is often not responsive to pharmacological conventional treatment methods. Against this background, the development of new therapeutic approaches that have an effective action in this type of pain is of great importance. The use of cell therapy in the treatment of lesions in the nervous system has shown promising results and the therapeutic potential of stem cells in experimental neuropathy has been proposed. In this study, we evaluated the effect of mesenchymal stem cells derived from bone marrow (CMsMO) in peripheral diabetic neuropathy established in experimental model of streptozotocin (STZ) induced diabetes in mice. Four weeks after the induction of the model by administration of STZ (80 mg/kg, ip; 3 days) the animals received an CMsMO by intravenous administration (1x106) or vehicle. The treatment with gabapentin (30 mg/kg, orally every 12 hours for six days) was used as the gold standard. The thermal and mechanical nociceptive thresholds were assessed throughout the entire experimental period (90 days), using Hargreaves and von Frey methods, respectively. Motor function evaluation of was conducted using the rotarod test. At different times, were analyzes conducted in spinal cord segments (L4-L5) to determine cytokines profile by ELISA. Sciatic nerve segments were also collected for evaluation of morphological changes by optical and electron transmission microscopy. According to the behavioral data, the CMsMO treatment reduced the mecanoalodinia and the thermal hypoalgesia, leading nociceptive thresholds of neuropathic animals to levels similar to those of non-neuropathic animals. Similarly, CMsMO administration normalized motor function of neuropathic animals. Microscopy data demonstrated that neuropathic animals had axonal atrophy and an apparent decrease of the number of myelinated fibers as well a reduction in the number of unmyelinated fibers in the sciatic nerve, but neuropathic animals treated with CMsMO had a lower incidence of axonal atrophy, showed no decrease in the number of myelinated fibers and no apparent decrease in the amount of unmyelinated fibers in relation to neuropathics treated with saline. Furthermore, neuropathic animals treated with CMsMO presented lower levels of spinal IL-1β and higher levels of TNF-α, and IL-10 and TGF-β compared to neuropathic animals that received saline. These data indicate that CMsMO produces a lasting analgesic effect in diabetic neuropathy, followed by changes in the pathophysiological disease pattern, which indicates cell therapy as an interesting alternative for the control of painful peripheral diabetic neuropathy.

Dor neuropática (DN) é definida como “dor iniciada ou causada por lesão primária ou disfunção em sistema nervoso”, porém sua prevalência depende do tipo de trauma e da disfunção relacionada. Apesar desta condição dolorosa ser considerada altamente prevalente e debilitante, os tratamentos disponíveis são relacionados a efeitos adversos dificultando a adesão. Devido a isso, buscam-se alternativas não farmacológicas para o tratamento deste tipo de dor, entre elas, as técnicas de neuromodulação periférica, como acupuntura (AC) e eletroacupuntura (EA). Estas técnicas podem ser combinadas com intervenções farmacológicas e não farmacológicas e têm apresentado resultados promissores no tratamento da dor neuropática. No entanto, seus mecanismos de ação não estão totalmente elucidados, desta forma a utilização de modelos animais é de grande valia para o estudo destes mecanismos no tratamento da dor neuropática e da patofisiologia deste tipo de dor crônica. É importante salientar que a aplicação de AC e EA em animais acordados é complexa, visto que gera desconforto e pode alterar a analgesia induzida pelo tratamento. Em muitos estudos a anestesia com isoflurano é utilizada durante a aplicação dos tratamentos, porém sua utilização pode gerar um viés no estudo, considerando a possível interferência do fármaco nos resultados comportamentais e neuroquímicos. Outro importante foco de estudo consiste em comparar as duas técnicas, AC e EA, buscando determinar qual destas é a mais eficaz no tratamento da dor neuropática. Considerando o exposto acima, os objetivos desta tese foram: 1) avaliar os parâmetros comportamentais e neuroquímicos dos efeitos da utilização de anestesia na aplicação de AC e EA em ratos submetidos ao modelo de DN; 2) comparar os efeitos da AC e EA em modelo animal de DN por meio de parâmentros comportamentais, neuroquímicos e histológicos. Considerando os resultados obtidos nesta tese, concluímos que o isoflurano aumenta a analgesia promovida por AC e EA, provavelmente diminuindo o efeito do estresse gerado pela aplicação dos tratamentos em animais acordados, resultado que é corroborado pela diminuição do nível de S100β periférico (marcador de morte neuronal central); Por outro lado, o isoflurano diminuiu os níveis de fator de crescimento neuronal (NGF) no nervo periférico lesado, indicando diminuição do processo de regeneração neural, enquanto a EA aumentou. Ao mesmo tempo, o isoflurano alterou os efeitos dos tratamentos nos comportamentos exploratórios e nos níveis de N-metil D-aspartato em tronco encefáfio e medula espinhal. A AC apresentou-se mais eficaz no tratamento da DN em comparação à EA, porém nenhum dos tratamentos foi capaz de alterar os danos causados pela indução da DN no músculo gastrocnemio esquerdo dos animais demonstrado na histologia. Todavia, este resultado não alterou a analgesia gerada pelos tratamentos. / Neuropathic pain (NP) is defined as "pain initiated or caused by primary injury or dysfunction in the nervous system," but its prevalence depends on the type of trauma and related dysfunction. Although this painful condition is considered to be highly prevalent and debilitating, the available treatments are related to adverse effects, making adherence difficult. Because of this, non-pharmacological alternatives for the treatment of this type of pain are sought, among them, the techniques of peripheral neuromodulation, such as acupuncture (AC) and electroacupuncture (EA). These techniques can be combined with pharmacological and non-pharmacological interventions and have shown promising results in the treatment of neuropathic pain. However, its mechanisms of action are not fully elucidated, so the use of animal models is of great value for the study of these mechanisms in the treatment of neuropathic pain and the pathophysiology of this type of chronic pain. It is important to emphasize that the application of AC and EA in awake animals is complex, since it generates discomfort and can alter the analgesia induced by the treatment. In many studies, anesthesia with isoflurane is used during the application of the treatments, but its use may generate a bias in the study, considering the possible interference of the drug in the behavioral and neurochemical results. Another important focus of the study is to compare the two techniques, AC and EA, seeking to determine which is the most effective in the treatment of neuropathic pain. Considering the above, the objectives of this thesis were: 1) to evaluate the behavioral and neurochemical parameters of the effects of the use of anesthesia in the application of AC and EA in rats submitted to the DN model; 2) to compare the effects of AC and EA on animal model of DN by means of behavioral, neurochemical and morphological parameters. Considering the results obtained in this thesis, we conclude that isoflurane increases the analgesia promoted by AC and EA, probably decreasing the effect of the stress generated by the application of the treatments in agreed animals, a result that is corroborated by the decrease in the level of peripheral S100β (biomarker of central neuronal injury); On the other hand, isoflurane decreased the levels of neural grown factor (NGF) in the injured peripheral nerve, indicating a decrease in the neural regeneration process, while the EA increased. At the same time, isoflurane altered the effects of treatments on exploratory behaviors and N-metil-D-aspartato (NMDA) levels in the brainstem and spinal cord. AC was more effective in the treatment of DN compared to EA, but none of the treatments was able to alter the damage caused by DN induction in the left gastrocnemius muscle of the animals showed in histology. However, this result did not alter the analgesia generated by the treatments.

Introdução: Um fator que pode contribuir para o desenvolvimento da dor neuropática é a modulação negativa da via descendente da dor pelo aumento da degradação do triptofano pela ativação da enzima indoleamina 2,3-dioxigenase 1 (IDO1) ou a ativação da via descendente facilitatória da dor por um agonista glutamatérgicos produzido pela enzima quinurenina 3 monoxigenase (KMO). Objetivo: Foi avaliar a participação das enzimas IDO1 e a KMO presente na substância cinzenta periaquedutal (PAG) e no bulbo rostral ventromedial (RVM) no desenvolvimento da dor neuropática em camundongos induzida pelo modelo SNI. Metodologia: A indução da neuropatia experimental foi realizada de acordo com (Bourquin et al., 2006). A expressão da IDO1 e KMO foi realizada pela técnica de Western blotting. A administração de drogas foi realizada por via oral, intraperitoneal, intratecal e intracerebroventricular (i.c.v.). Resultados: Foi observado o aumento da expressão da enzima IDO1 no RVM (7 dias) e PAG (3, 7, 14 e 21 dias) após SNI. A microinjeção de Norharmane no espaço i.c.v. reduziu a hipersensibilidade mecânica no 7, 14 e 21 dias após SNI. Corroborando com esses achados, animais deficientes para a enzima IDO1 submetidos a SNI não desenvolvem a hipersensibilidade mecânica. Além disso, a expressão da enzima KMO aumenta significativamente no 7 e 14 dias no RVM e 7 dias na PAG após SNI. Por conseguinte, a administração oral de JM6, pró-droga de liberação lenta do Ro61-8048, ou Ro61-8048 (inibidor da KMO) no espaço i.c.v. reduziu significativamente a hipersensibilidade mecânica nos dias 7, 14 ou 21 após SNI. Sabendo que a expressão da enzima IDO1 é modulada pela citocina IFN-, verificamos que os animais deficientes para a citocina IFN- apresentam hipersensibilidade mecânica reduzida. Ainda, os animais IFN- KO possuem expressão reduzida da IDO1 no RVM 7 dias e na PAG 14 dias após a SNI. Em adição, a microinjeção de doses crescentes de IFN- no espaço i.c.v. induz uma hipernocicepção mecânica em camundongos naives. Constatamos também que animais CD4+ KO, mas não os animais CD8+ KO apresentam reduzida expressão da enzima IDO1 no RVM e na PAG e consequentemente menor hipersensibilidade mecânica após SNI. A microinjeção dos metabolitos da via das quinureninas, no espaço i.c.v. de camundongos causou hipersensibilidade mecânica, sendo o QUIN o mais potente. Sugerimos que a ativação da via das quinureninas seja dependente da ativação do receptor NMDA, visto que o pré-tratamento local com o MK801 (antagonista seletivos dos receptores NMDA) reverte os efeitos nociceptivos induzidos pelos metabólitos. Além disso, o efeito nociceptivo induzido por QUIN depende ativação da via descendente facilitatória. Constatamos que os animais neuropáticos exibem um comportamento do tipo depressivo e esse comportamento não é observado em animais IFN- KO e CD4KO. Por último, avaliamos a participação da via das quinureninas no desenvolvimento do comportamento depressivo associado à SNI e constatamos que esse comportamento depende da ativação das enzimas IDO1 e KMO. Conclusão: Os resultados sugerem que as enzimas IDO1 e KMO, localizadas em regiões supraespinais desempenham um importante papel no desenvolvimento da dor neuropática, assim como da comorbidade depressão. Além disso, a expressão da IDO1 é dependente da sinalização via citocina IFN- e células CD4+. O mecanismo responsável pelo desenvolvimento da hipersensibilidade neuropática deve-se tanto a redução dos níveis de triptofano/5-HT, diminuição da eficiência da via descendente inibitória, quanto ao aumento dos níveis de QUIN, que ativa a via descendente facilitatória da dor. / Introduction: One factor that may contribute to the development of neuropathic pain is the negative modulation of the descending pain pathway by increased degradation of the activation of tryptophan by enzyme indoleamine 2,3-dioxygenase1 (IDO1) or activation of the descending facilitatory pain pathway for a glutamate agonist produced by the enzyme kynurenine 3 monooxygenase (KMO). Aim: We evaluate the role of IDO1 and KMO in the periaqueductal gray (PAG) and the rostral ventromedial medulla (RVM) in the development of neuropathic pain in mice induced by SNI model. Methods: Induction of experimental neuropathy was performed according to (Bourquin et al. 2006). The expression of IDO1 and KMO was carried out by Western blotting technique. The drug administration was performed orally, intraperitoneally and intracerebroventricularly (i.c.v) Results. We observed increased IDO1 expression in the RVM (7 days) and PAG (3, 7, 14 and 21 days) after SNI. The microinjection Norharmane in i.c.v. space reduced mechanical hypersensitivity in the 7, 14 and 21 days after SNI. Corroborating these findings, mice deficient for the enzyme IDO1 undergoing SNI did not develop mechanical hypersensitivity. Furthermore, the KMO expression was significantly increased in the 7 and 14 days in the RVM and 7 days in PAG after SNI. Therefore, oral administration of JM6, prodrug slow release from Ro61-8048 or Ro61-8048 (KMO inhibitors) within i.c.v. significantly reduced the mechanical hypersensitivity at day 7, 14 or 21 after SNI. Knowing that the expression of IDO1 enzyme is modulated by IFN- cytokine, it was found that animals deficient for IFN- cytokine have reduced mechanical hypersensitivity. Moreover, IFN- ko animals have reduced expression of IDO1 RVM 7 days and 14 days after SNI in the PAG. In addition, microinjection of increasing doses of IFN- in i.c.v. induced mechanical hyperalgesia. We also found that CD4 + KO animals, but not CD8 + KO animals showed reduced expression of the enzyme IDO1 RVM and PAG and consequently lower mechanical hypersensitivity after SNI. The microinjection of the main metabolites of kynurenine pathway into the i.c.v. spaces induced mechanical hypersensitivity, QUIN being the most potent. We suggest that the activation of the kynurenine pathway was dependent of NMDA receptor activation, whereas the spot pre-treatment with MK801 (selective NMDA receptor antagonist) reverses the effects induced by noxious metabolites. After that, the microinjection into i.c.v. spaces of MK801 reduced mechanical hypersensitivity after SNI. Furthermore, nociceptive effect induced by QUIN depends activation of the descending facilitatory. We found that the neuropathic animals exhibit depressive-like behavior and this behavior is not observed in IFN- KO and CD4KO mice. Finally, we evaluate the participation of kynurenine pathway in the development of depressive-like behavior associated with SNI and found that this behavior depends on the activation of IDO1 and KMO Conclusion: These results suggest that IDO1 and KMO enzyme, located in supraspinal regions play a role in the development of neuropathic pain as well as comorbidity depression. Furthermore, the expression of IDO1 are dependent on signaling via cytokine IFN- and CD4+ cells. The mechanism responsible for the development of neuropathic hypersensitivity is due to both reduced levels of tryptophan/5-HT decrease the descending inhibitory pain pathway efficiency, as the increased levels of QUIN, which activates the descending facilitatory pain pathway.

Dor neuropática (DN) é definida como “dor iniciada ou causada por lesão primária ou disfunção em sistema nervoso”, porém sua prevalência depende do tipo de trauma e da disfunção relacionada. Apesar desta condição dolorosa ser considerada altamente prevalente e debilitante, os tratamentos disponíveis são relacionados a efeitos adversos dificultando a adesão. Devido a isso, buscam-se alternativas não farmacológicas para o tratamento deste tipo de dor, entre elas, as técnicas de neuromodulação periférica, como acupuntura (AC) e eletroacupuntura (EA). Estas técnicas podem ser combinadas com intervenções farmacológicas e não farmacológicas e têm apresentado resultados promissores no tratamento da dor neuropática. No entanto, seus mecanismos de ação não estão totalmente elucidados, desta forma a utilização de modelos animais é de grande valia para o estudo destes mecanismos no tratamento da dor neuropática e da patofisiologia deste tipo de dor crônica. É importante salientar que a aplicação de AC e EA em animais acordados é complexa, visto que gera desconforto e pode alterar a analgesia induzida pelo tratamento. Em muitos estudos a anestesia com isoflurano é utilizada durante a aplicação dos tratamentos, porém sua utilização pode gerar um viés no estudo, considerando a possível interferência do fármaco nos resultados comportamentais e neuroquímicos. Outro importante foco de estudo consiste em comparar as duas técnicas, AC e EA, buscando determinar qual destas é a mais eficaz no tratamento da dor neuropática. Considerando o exposto acima, os objetivos desta tese foram: 1) avaliar os parâmetros comportamentais e neuroquímicos dos efeitos da utilização de anestesia na aplicação de AC e EA em ratos submetidos ao modelo de DN; 2) comparar os efeitos da AC e EA em modelo animal de DN por meio de parâmentros comportamentais, neuroquímicos e histológicos. Considerando os resultados obtidos nesta tese, concluímos que o isoflurano aumenta a analgesia promovida por AC e EA, provavelmente diminuindo o efeito do estresse gerado pela aplicação dos tratamentos em animais acordados, resultado que é corroborado pela diminuição do nível de S100β periférico (marcador de morte neuronal central); Por outro lado, o isoflurano diminuiu os níveis de fator de crescimento neuronal (NGF) no nervo periférico lesado, indicando diminuição do processo de regeneração neural, enquanto a EA aumentou. Ao mesmo tempo, o isoflurano alterou os efeitos dos tratamentos nos comportamentos exploratórios e nos níveis de N-metil D-aspartato em tronco encefáfio e medula espinhal. A AC apresentou-se mais eficaz no tratamento da DN em comparação à EA, porém nenhum dos tratamentos foi capaz de alterar os danos causados pela indução da DN no músculo gastrocnemio esquerdo dos animais demonstrado na histologia. Todavia, este resultado não alterou a analgesia gerada pelos tratamentos. / Neuropathic pain (NP) is defined as "pain initiated or caused by primary injury or dysfunction in the nervous system," but its prevalence depends on the type of trauma and related dysfunction. Although this painful condition is considered to be highly prevalent and debilitating, the available treatments are related to adverse effects, making adherence difficult. Because of this, non-pharmacological alternatives for the treatment of this type of pain are sought, among them, the techniques of peripheral neuromodulation, such as acupuncture (AC) and electroacupuncture (EA). These techniques can be combined with pharmacological and non-pharmacological interventions and have shown promising results in the treatment of neuropathic pain. However, its mechanisms of action are not fully elucidated, so the use of animal models is of great value for the study of these mechanisms in the treatment of neuropathic pain and the pathophysiology of this type of chronic pain. It is important to emphasize that the application of AC and EA in awake animals is complex, since it generates discomfort and can alter the analgesia induced by the treatment. In many studies, anesthesia with isoflurane is used during the application of the treatments, but its use may generate a bias in the study, considering the possible interference of the drug in the behavioral and neurochemical results. Another important focus of the study is to compare the two techniques, AC and EA, seeking to determine which is the most effective in the treatment of neuropathic pain. Considering the above, the objectives of this thesis were: 1) to evaluate the behavioral and neurochemical parameters of the effects of the use of anesthesia in the application of AC and EA in rats submitted to the DN model; 2) to compare the effects of AC and EA on animal model of DN by means of behavioral, neurochemical and morphological parameters. Considering the results obtained in this thesis, we conclude that isoflurane increases the analgesia promoted by AC and EA, probably decreasing the effect of the stress generated by the application of the treatments in agreed animals, a result that is corroborated by the decrease in the level of peripheral S100β (biomarker of central neuronal injury); On the other hand, isoflurane decreased the levels of neural grown factor (NGF) in the injured peripheral nerve, indicating a decrease in the neural regeneration process, while the EA increased. At the same time, isoflurane altered the effects of treatments on exploratory behaviors and N-metil-D-aspartato (NMDA) levels in the brainstem and spinal cord. AC was more effective in the treatment of DN compared to EA, but none of the treatments was able to alter the damage caused by DN induction in the left gastrocnemius muscle of the animals showed in histology. However, this result did not alter the analgesia generated by the treatments.

Alexithymia represents a deficit in identifying and expressing emotions, paucity of fantasies, and an externally oriented cognitive style. Currently, numerous studies document that alexithymia and several mental and somatic disorders are significantly related. Several findings also indicate that this association might be caused by alexithymia related dysregulation of neuroendocrine and immune functions. Together these findings indicate that stressors related to alexithymia could underlie the process of neuroendocrine and immune dysregulation that likely may present a significant risk, sustaining and mediating pathogenesis of several disorders and particulary psychosomatic illnesses. In this context, it is also known that several proinflammatory cytokines may play a role in pain generation and that alexithymia is significantly associated with pain symptoms in several pain disorders. Following these findings this study includes several new data developing current state of the art and showing some alexithymia specific changes in patients with neurological disorders. Main finding of this study shows that alexithymia and anxiety in their specific interactions are linked to increased levels of interleukine-8 (IL-8) in cerebrospinal fluid (CSF) in the group of patients with non-inflammatory neurological...

A Síndrome de Ardência Bucal (SAB) caracteriza - se por sensação de ardor, com ausência de sinais clínicos ou laboratoriais associados. A etiopatogenia é desconhecida, inexistindo protocolo terapêutico satisfatório. O objetivo deste trabalho foi avaliar a eficácia da aplicação tópica de capsaicina, como alternativa terapêutica, em um grupo de pacientes portadores da SAB, além de investigar, nessa população, indicativos de ansiedade e depressão, correlacionando estes últimos aspectos com a resposta à terapêutica aplicada. Constituiu-se um grupo de vinte indivíduos portadores da síndrome, todos de acordo com os termos do consentimento esclarecido. Quinze sujeitos constituíram o grupo teste (GT) e foram tratados com capsaicina, em aplicações diárias, durante três semanas, repetindo-se o ciclo por quatro semanas após uma semana de intervalo. O grupo controle (GC) foi tratado com o creme base utilizado como veículo da capsaicina, durante o mesmo período. Ambos foram controlados após 30 dias do término da medicação. A evolução dos sintomas foi controlada por escala visual de sintomatologia (EVS) e questionário acerca do efeito global percebido (EGP). A intensidade média do sintoma de ardência antes do início dos ciclos de tratamento, mensurado pela EVS, foi de 5,1 (GT) e 4,4 (GC). Ao final da quarta semana o GT mostrou redução dos sintomas (EVS=3,6), enquanto o GC declarou aumento da sintomatologia (EVS=4,8). No GT, entre a quarta e a oitava semana houve redução dos sintomas da ordem de 8,3%, e entre a oitava e a décima segunda semana observou-se aumento de 13,5% da sintomatologia. No GC houve 22.8% de piora (EVS=5,75) entre o início e a décima segunda semana. Ao EGP houve pelo menos algum alívio do sintoma em seis pacientes (40%) do GT e em um paciente do GC (20%). Quatro pacientes (26,6%) reportaram remissão total do sintoma após tratamento com capsaicina e um paciente (20%) do controle. Para três pacientes do GT e dois do GC não houve modificações do sintoma. Houve relato de piora em dois pacientes (13,3%) do GT e um (20%) do GC. Oito pacientes do GT apresentaram alto nível de ansiedade e sete níveis médios. No GC um paciente apresentou nível baixo, três mostraram valores médios e um classificou-se como alto. Ao CES-D valores indicativos de depressão foram registrados por dez pacientes (66,6%) do GT e 40% (02) do GC. Concluímos que a capsaicina apresentou efetividade no controle da sintomatologia da SAB, parecendo haver correlação com a intensidade inicial de sintomas e manutenção do uso do medicamento. Além disso, houve correlação entre alto nível de ansiedade e indicativos de depressão, embora não se tenha percebido influência destes aspectos sobre a resposta terapêutica. / Burning mouth syndrome (BMS) is characterized by an oral burning sensation, with no corresponding clinical signs or laboratory abnormalities. The etiology is unknown, and there was no satisfactory treatment available. The objective of this study was to evaluate the effectiveness of topical use of capsaicin, as an alternative therapy in a group of BMS patients, as well as to correlate anxiety and depression levels to response to the therapy applied. Twenty BMS individuals in accordance to the terms to informed consent comprised the study group. Fifteen subjects were allocated to the test group (TG) and were treated with capsaicin, in daily applications for three weeks, one-week interval and an additional treatment cycle of four weeks. The control group (CG) was treated with the cream base used as a vehicle of capsaicin preparation, during the same period. All patients were examined 30 days after discontinuation of the medication. Results were assessed through a visual analogue scale (VAS) and a questionnaire on the global perceived effect (GPE). The average symptoms intensity before treatment, on EVS, was 5.1 (TG) and 4.4 (CG). At the fourth week control, TG presented reduction on the level of symptoms (EVS = 3.6), while CG presented an increase of symptoms intensity (VAS = 4.8). In the TG, between fourth and eighth week of follow-up, symptoms decreased around 8.3%, and between the eighth and twelfth week there was an increase of 13.5% on symptoms intensity. In the CG it was registered 22.8% of worsening (EVS = 5.75) between the beginning of the study and the twelfth week of control. On GPE assessment, six patients (40%) of TG and one patient of CG (20%), presented some relief of symptoms; four patients TG (26.6%) reported total remission of symptoms after treatment with capsaicin and one patient (20%) of control; three patients of TG and two of the CG remained unaltered. There were reports of worsening in two patients (13.3%) of TG and one (20%) of the CG. Eight patients of TG showed a high level of anxiety and seven moderate levels. In CG one patient presented low level, three showed a moderate level and one was ranked as having a high level of anxiety. CES-D suggested traits of depression in ten patients (66.6%) of TG and 40% (2) of the CG. We concluded that capsaicin is effective in controlling the burning symptom of BMS, suggesting some correlation with initial symptoms intensity and the maintenance of drug use. Moreover, there was some correspondence between high levels of anxiety and traits of depression, but it was not perceived influence of these aspects to the therapeutic response.

O tramadol é um analgésico de ação central eficaz na atenuação de dores agudas e crônicas, entre elas a dor neuropática em pacientes diabéticos. Encontra-se disponível na clínica como mistura de (+)-tramadol e (-)-tramadol. O tramadol é metabolizado pelo CYP2D6 em O-desmetiltramadol (M1) e pelo CYP3A4 e CYP2B6 em N-desmetiltramadol (M2). Ambos enantiômeros do tramadol e o (+)-M1 contribuem para a atividade analgésica: o (+)-tramadol e o (+)-M1 agem como agonistas do receptor -opióide; o (+)-tramadol inibe a recaptação de serotonina; e o (-)-tramadol inibe a recaptação de noradrenalina. O estudo investiga a influência do diabetes mellitus (DM) tipo 1 e tipo 2 descompensados na disposição cinética, metabolismo e farmacocinética-farmacodinâmica dos enantiômeros tramadol em pacientes com dor neuropática. Os pacientes não diabéticos (Grupo Controle, n=12), os pacientes com DM tipo 1 (n=9) e os pacientes com DM tipo 2 (n=9), todos portadores de dor neuropática e fenotipados como metabolizadores extensivos do CYP2D6, receberam dose única oral de 100 mg de tramadol racêmico. Amostras seriadas de sangue foram coletadas até 24 h após a administração do tramadol para o estudo farmacocinético e para a avaliação das concentrações de noradrenalina. A dor dos pacientes foi avaliada através da escala analógica visual de dor nos mesmos tempos de coleta de sangue. Os pacientes foram avaliados quanto à atividade in vivo do CYP3A utilizando midazolam como fármaco marcador e genotipados para o CYP2B6. As concentrações plasmáticas total e livre dos enantiômeros do tramadol, M1 e M2 foram analisadas por LC-MS/MS usando a coluna Chiralpak® AD. A disposição cinética do tramadol é enantiosseletiva nos pacientes dos Grupos Controle e DM tipo 1, com acúmulo plasmático do (+)-tramadol. O DM tipo 1, mas não o DM tipo 2, reduz a AUC do metabólito ativo (+)-M1 e simultaneamente aumenta sua fração livre. Portanto, a concentração plasmática livre do eutômero (+)-M1 permanece inalterada nos pacientes portadores de DM tipo 1 e DM tipo 2. Não foram observadas diferenças entre os Grupos Controle, DM tipo 1 e DM tipo 2 quanto às razões metabólicas plasmáticas e urinárias do metoprolol/-hidroximetoprolol e quanto ao clearance do midazolam. Correlações significativas entre as razões metabólicas de AUC (+)-tramadol/(+)-M1 ou (-)-tramadol/(-)-M1 e a atividade in vivo do CYP2D6 avaliada em plasma ou urina empregando o metoprolol como fármaco marcador sugerem a aplicação do tramadol como fármaco marcador do CYP2D6. Os dados também mostram uma tendência de aumento do clearance do (+)-tramadol e do (-)-tramadol em virtude da presença do alelo mutante T no polimorfismo 516G>T do CYP2B6. O modelo sigmóide de efeito máximo fracional foi empregado para descrever a relação farmacocinética-farmacodinâmica do tramadol em pacientes com dor neuropática, relacionando as concentrações plasmáticas livre do (+)-M1 com o efeito analgésico do tramadol. O presente estudo mostra a importância da análise da concentração livre dos enantiômeros individuais do tramadol e seus metabólitos nos estudos de farmacocinética-farmacodinâmica. / Tramadol is a centrally acting analgesic that effectively relieves acute and chronic pain, including neuropathic pain in diabetic patients. The drug is available in clinical practice as a mixture of the (+)-tramadol and (-)-tramadol enantiomers. Tramadol is metabolized by CYP2D6 to O-desmethyltramadol (M1) and by CYP3A4 and CYP2B6 to N-desmethyltramadol (M2). Both tramadol enantiomers and (+)-M1 contribute to the analgesic activity of the drug: (+)-tramadol and the (+)-M1 metabolite act as -opioid receptor agonists; (+)-tramadol inhibits serotonin reuptake; and (-)-tramadol inhibits the reuptake of norepinephrine. This study investigated the influence of uncontrolled type 1 and type 2 diabetes mellitus (DM) on the kinetic disposition, metabolism and pharmacokinetics-pharmacodynamics of tramadol enantiomers in patients with neuropathic pain. Nondiabetic patients (control group, n = 12), patients with type 1 DM (n = 9), and patients with type 2 DM (n = 9), all with neuropathic pain and phenotyped as extensive metabolizers of CYP2D6, received a single oral dose of 100 mg racemic tramadol. Serial blood samples were collected up to 24 h after administration of the drug for pharmacokinetic study and for the analysis of noradrenaline in plasma. Pain was rated on a visual analog pain scale at the same time as blood sampling. The patients were evaluated for in vivo CYP3A activity using midazolam as a probe drug and genotyped for CYP2B6. Total and unbound plasma concentrations of the tramadol, M1 and M2 enantiomers were analyzed by LC-MS/MS using a Chiralpak® AD column. The kinetic disposition of tramadol was enantioselective in the control and type 1 DM groups, with the accumulation of (+)-tramadol. Type 1, but not type 2, DM reduced the AUC of the active (+)-M1 metabolite and simultaneously increased its unbound fraction. Therefore, unbound plasma concentrations of the (+)-M1 eutomer remain unchanged in patients with type 1 and type 2 DM. No differences in the plasma and urinary metabolic ratios of metoprolol/-hydroxymetoprolol or in midazolam clearance were observed between the control, type 1 and type 2 DM groups. The significant correlations seen between (+)-tramadol/(+)-M1 or (-)-tramadol/(-)-M1 AUC metabolic ratios and in vivo CYP2D6 activity evaluated in plasma or urine using metoprolol as a probe drug suggest the application of tramadol as a marker for CYP2D6. The data also showed a trend towards increased clearance of (+)-tramadol and (-)-tramadol as a result of the presence of mutant allele T in the 516G>T polymorphism of the CYP2B6 gene. The fractional sigmoid maximum drug effect model was used to describe the pharmacokinetic-pharmacodynamic relationship of tramadol in patients with neuropathic pain, associating the unbound plasma concentrations of (+)-M1 with the analgesic effect of tramadol. The present study highlights the importance of analyzing unbound concentrations of the individual tramadol enantiomers and its metabolites in pharmacokinetic-pharmacodynamic studies.