My PhD research studies, described in this thesis, were designed to document the temporal development of mechanical allodynia, a hallmark symptom of painful diabetic neuropathy (PDN), as well as opioid hyposensitivity using two different rat models of diabetes mellitus (DM). Specifically, the studies were conducted using the streptozotocin (STZ)-diabetic rat model of chemically-induced Type 1 diabetes in two different rat strains, as well as the Zucker Diabetic Fatty (ZDF) rat genetic model of Type 2 diabetes. Additionally, a longitudinal investigation of the effect of basal insulin replacement therapy to restore euglycaemia from 7-days post-STZ administration, on the development of mechanical allodynia in the hindpaws of the STZ-diabetic Wistar rat model of PDN, was conducted. The studies herein also included a longitudinal study to document the temporal development of mechanical allodynia and opioid hyposensitivity in the ZDF rat, which also examined the influence of dietary composition on the time course for the development of mechanical allodynia in the hindpaws, together with opioid hyposensitivity in these animals. In the final section of this thesis, the experiments were designed to examine possible mechanisms that may contribute to the development of opioid hyposensitivity in ZDF diabetic rats. These experiments involved the quantification of opioid receptor messenger ribonucleic acid (mRNA) gene expression as well as μ-opioid receptor (MOP-r) functional responses in tissues collected from 29-wk old diabetic ZDF rats relative to 7-wk old pre-diabetic control ZDF animals. In Chapter One, diabetes mellitus and more specifically its longterm complication, PDN, the focus of this doctoral research program, has been reviewed. Specifically, possible pathogenic mechanisms underlying mechanical allodynia, the relevant diabetic rodent models of PDN, use of insulin replacement therapy in diabetic rodents and its impact on hallmark symptoms of PDN, role of opioid pharmacology, the comparative efficacy of opioids in the treatment of PDN, and possible mechanisms that may underpin the development of opioid hyposensitivity in PDN, including the impact of altered excitatory neurotransmitters, have been reviewed. In Chapter Two, a preliminary study was conducted to investigate the efficacy of 4-wks treatment with Linplants (subcutaneous (s.c.) sustained-release bovine insulin implants) alone and in combination with ActRapid® (s.c. human insulin; 0.05 U to 3.5 U/100 g/day) with respect to glycaemic control in STZ-diabetic Wistar rats, and on acute diabetes characteristics for a 5-wk post-STZ administration period. Briefly, STZ-diabetic rats were divided into three groups: (1) rats which received no insulin treatment, (2) rats which were implanted with one s.c. Linplant at Day 7 post-STZ administration, and (3) rats which received one s.c. Linplant plus a once-daily injection of ActRapid® once diabetes was confirmed at 7-days post-STZ administration. The findings were that following implantation of a single Linplant at Day 7 post-STZ administration, euglycaemia was achieved in 50% of STZ-diabetic rats, with glycaemic control maintained for up to 4-wks post-implantation. Furthermore, once-daily injection of ActRapid™ to animals whose blood glucose levels (BGLs) were not well-controlled through use of Linplants alone, failed to achieve euglycaemia. It is possible that the ActRapid™ doses administered were not sufficient to achieve euglycaemia, and that increasing the doses may provide more effective glycaemic control. However, doubling the mean ActRapid™ dose from 1.63 (+ 0.3) U administered at Day 28 to 2.56 (+ 0.6) U administered at Day 34 post-STZ administration effectively only reduced BGLs by 1.3 mM to 11.6 + 1.6 mM. This suggests that although administering additional large doses of ActRapid™ to STZ-diabetic rats may eventually achieve euglycaemia, this method would presumably not be a more efficient method in achieving euglycaemia compared with the use of dosage-adjustable s.c. Linplants. Group (1) STZ-diabetic rats which were not treated with insulin developed diabetic signs including polydipsia, hyperphagia, decreased rate of body weight gain, and mechanical allodynia. Group (2) rats in which insulin treatment from 7-days post-STZ administration restored euglycaemia and reversed polydipsia and hyperphagia, were protected against the development of mechanical allodynia and reduced weight gain for the 5-wk study duration, while rats from Group (3) with incomplete glycaemic control developed levels of polydipsia, hyperphagia, reduced weight gain and mechanical allodynia intermediate between rats in Groups (1) and (2). These findings collectively suggest a direct correlation between the level of glycaemic control and the extent to which mechanical allodynia, a defining symptom of PDN, develops. In Chapter Three, the findings from the preliminary 5-wk study in Chapter Two were used to design a 24-wk longitudinal study of the temporal development of mechanical allodynia and opioid hyposensitivity in STZ-diabetic Wistar rats for comparison with the findings of a similar study previously undertaken by our laboratory using STZ-diabetic Dark Agouti rats (Nielsen et al, 2007). Additionally, this study examined the effects of tight glycaemic control achieved through the use of insulin implants as a means of potentially preventing the development of mechanical allodynia and opioid hyposensitivity for up to 24 weeks in STZ-diabetic Wistar rats. Briefly, STZ-diabetic rats were divided into 3 groups: (1) non-insulin treated STZ-diabetic Dark Agouti rats to provide comparison data with our laboratory’s previously published data in this rat strain (Nielsen et al, 2007), (2) non-insulin treated STZ-diabetic Wistar rats to examine possible between-species differences, and (3) STZ-diabetic Wistar rats which were treated with adjustable-dose s.c. Linplants from Day 7 post-STZ administration to maintain euglycaemia for the remainder of the 24-wk study period. In this 24-wk longitudinal study in STZ-diabetic rats, body weight, 24-hr water intake, paw withdrawal thresholds (PWTs) and BGLs were monitored at fortnightly intervals in all animals in order to document possible temporal changes in the development of diabetic signs and mechanical allodynia in the hindpaws respectively. STZ-diabetic rats underwent 6-wkly opioid antinociceptive testing, using single bolus doses of each of morphine and oxycodone with a 2-3 day washout period between individual opioids in order to assess the potential influence of both diabetes and glycaemic control on opioid potency in these animals. The findings demonstrate that non-insulin treated STZ-diabetic rats of both strains exhibited a decreased rate of body weight gain and polydipsia, as well as progressive development of mechanical allodynia in the hindpaws and loss of morphine potency. Importantly, STZ-diabetic Wistar rats which were treated with insulin to maintain euglycaemia from Day 7 post-STZ administration failed to develop these diabetic symptoms for the duration of the 24-wk study period, highlighting the importance of chronic hyperglycaemia in the development of mechanical allodynia and morphine hyposensitivity in the STZ-diabetic rodent model of PDN. The research described in Chapter Four involved a 22-wk longitudinal study of the development of diabetes and its longterm sensory nerve complications, viz mechanical allodynia and opioid hyposensitivity, in the ZDF rodent model of Type 2 diabetes commencing at 7-wks of age. This study also examined the influence of four different diets fed to separate groups of ZDF rats from 7-wks age, on the time course for the development of diabetes, mechanical allodynia in the hindpaws and opioid hyposensitivity in these animals. Briefly, ZDF rats were sub-divided into four dietary groups, each of which was fed one of the four following diets for 22-wks commencing at 7-wks of age, viz: (a) Purina 5008™, (b) a domestically-produced rat chow of similar composition to Purina 5008 (termed Purina Composition diet), (c) a Diabetogenic diet, or (d) Standard Rat Chow. All rats underwent once-fortnightly measurement of BGLs, body weight, 24-hr water intake, and measurement of PWTs in the hindpaws. Additionally, ZDF rats underwent opioid antinociceptive testing, similar to that previously described for STZ-diabetic rats (Chapter Three), to investigate the influence of diabetes and dietary composition on the antinociceptive potency of single bolus doses of morphine and oxycodone administered at 6-weekly intervals over a 22-wk study period. The afore-mentioned data were compared with the respective data obtained from the pre-diabetic control group of ZDF rats that were euthanised at 7-wks of age prior to the development of hyperglycaemia. The results demonstrate that the ZDF rat develops mechanical allodynia in the hindpaws and opioid hyposensitivity in a temporal fashion, in a manner similar to that previously documented for the STZ-diabetic Wistar rat model of Type 1 diabetes (Chapter Three). For the four diets assessed, there did not appear to be significant differences between dietary groups with respect to the time course and extent of development of hyperglycaemia, mechanical allodynia or opioid hyposensitivity in the ZDF rat model of PDN. The study described in Chapter Five investigated the effect of both diabetes and dietary composition on opioid receptor mRNA expression in tissue samples collected from the five groups of ZDF rats used in the behavioural studies described in Chapter Four and outlined above. Briefly, mRNA expression for each of the - (MOP), - (DOP), and - (KOP) receptors were quantified in mid-brain and spinal cord tissues prepared from 29-wk old diabetic ZDF rats maintained on one of four diets from 7-wks age, and compared with the respective expression levels in samples prepared from pre-diabetic ZDF rats euthanised at 7-wks of age. Overall, the findings suggest that diabetes does not alter opioid receptor mRNA expression in the mid-brain or spinal cord of diabetic ZDF rats at 29-wks of age relative to the corresponding levels of mRNA expression in the mid-brain and spinal cord of pre-diabetic ZDF rats at 7-wks of age. Hence, the marked reduction in the anti-allodynic potency of morphine and oxycodone observed in diabetic ZDF rats at 29-wks of age relative to that observed in pre-diabetic ZDF rats at 7-wks of age (Chapter Four) does not appear to be associated with a decrease in opioid receptor mRNA expression. In Chapter Six, the effect of both advanced diabetes and dietary composition on opioid-agonist stimulated [35S]GTPγS binding was examined in spinal cord tissue membranes from the ZDF rat. Specifically, [35S]GTPγS binding assays were used to assess the ability of a -opioid ligand (DAMGO) to stimulate -opioid receptor coupling to inhibitory G proteins in homogenates prepared from spinal cord samples of 29-wk old ZDF rats maintained on one of four different diets from 7-wks age (Chapter Four), relative to [35S]GTPγS binding in homogenates prepared from spinal cord samples of pre-diabetic 7-wk old ZDF rats. As specific MOP agonist-stimulated [35S]GTPγS binding was significantly decreased in spinal cord homogenates from diabetic ZDF rats at 29-wks of age relative to that for pre-diabetic ZDF rats (7-wks), this may contribute, at least in part, to the morphine hyposensitivity observed in diabetic ZDF rats at 29-wks of age relative to the pre-diabetic ZDF group. However, closer examination of these data revealed that specific MOP agonist-stimulated [35S]GTPγS binding above basal did not differ significantly between the pre-diabetic group and the longterm diabetic group of ZDF rats. Instead, there was significantly lower basal [35S]GTPγS binding in the spinal cord of ZDF rats at 29-wks c.f. 7-wks of age. Together, the findings suggest that impaired basal G-protein function rather than impaired coupling of MOP-r to its inhibitory G-protein may, at least in part, underpin -opioid agonist hyposensitivity in 29-wk ZDF rats. Finally, Chapter 7 contains a brief description of the main conclusions and discussion of the relevance of this doctoral research project, including potential future research directions.

Submitted by Socorro Pontes (socorrop@ufersa.edu.br) on 2017-05-23T13:58:52Z No. of bitstreams: 1 MaisaOF_DISSERT.pdf: 767871 bytes, checksum: d17c478d78b4175dd0a3f62dd37f0234 (MD5) / Made available in DSpace on 2017-05-23T13:58:52Z (GMT). No. of bitstreams: 1 MaisaOF_DISSERT.pdf: 767871 bytes, checksum: d17c478d78b4175dd0a3f62dd37f0234 (MD5) Previous issue date: 2013-08-29 / This study aimed to evaluate the regenerative and analgesic effects of the aqueous and hydroalcoholic extract of Caesalpinia ferrea pods – daily dosage of 300 mg/kg – on experimental sciatic neuropathy and non-neuropathic peripheral pain. An amount of 82 male Wistar rats was used in the experiment. There were 40 neuropathic rats divided in four groups of ten animals. They were treated with aqueous extract, hydroalcoholic extract, saline solution, and one them was pseudo-operated. The other 42 rats were distributed in six groups of seven animals. Three groups were led to writhing test induced by acetic acid administration, and the other three groups to termal test induced by carrageenan. The groups had different treatments with aqueous and hydroalcoholic extract and saline solution. Spontaneous (motor and pain) and induced (hyperalgesia and flare) behavioural studies were conducted. There were not significant difference between the groups treated or not with the extracts. However, in the third week, the induced behaviors of interdigital and lateral pinch presented significant difference between the groups treated with extracts and the ones treated with saline solution, which suggests analgesic effect for this behaviour. The dosage of 300 mg/kg of aqueous and hydroalcoholic extracts of Caesalpinia ferrea pods in the experimental traumatic sciatic neuropathy may not be used as analgesic. This dosage suggests analgesic effects for the induced behaviour of pinch. It shows they may be tested in studies evaluating axonal regeneration. Histological results suggest a higher axonal integrity in animals treated with the extracts, similar to the sham group / Este estudo avaliou os efeitos regenerativo e analgésico do extrato aquoso (EACF) e hidroalcoólico (EHCF) da vagem do jucá na neuropatia ciática experimental e na dor periférica não neuropática em ratos, em uma dose diária de 300 mg/kg. Foram utilizados 82 ratos machos da linhagem Wistar, distribuídos da seguinte forma: 40 animais neuropáticos divididos em quatro grupos de dez animais, tratados com o EAFC, EHCF, solução salina e um pseudo-operado. Quarenta e dois animais foram distribuídos em seis grupos de sete animais cada, sendo três para realização de testes de contorção abdominal induzida por ácido acético e três para realização de teste térmico induzido por carragenina. Os grupos receberam tratamentos com EAFC, EHCF e solução salina. Estudos comportamentais espontâneos (motricidade e dor) e induzidos (hiperalgesia e reflexos) foram avaliados, não havendo diferença significativa entre os grupos tratados e não tratados, entretanto nos comportamentos induzidos de pinçar interdigital e lateral da região plantar, houve uma diferença significativa entre os grupos tratados com os extratos e tratados com solução salina, , sugerindo um efeito analgésico. A dose de 300mg/kg de EACF e EHCF na neuropatia traumática experimental não pode ser utilizada como analgésico, porém sugerem a existência de efeitos analgésicos para os comportamentos induzidos de pinçar. Os resultados histológicos sugerem uma maior integridade axonal dos animais tratados com os extractos, semelhante ao grupo Sham / 2017-05-23

Submitted by ALESSANDRA MANCHINI CARDOSO null (lemanchini@hotmail.com) on 2017-01-30T15:58:06Z No. of bitstreams: 1 ficha+dissertação.pdf: 3142311 bytes, checksum: fd89634d15b20bb2b50e11cbbb2d7552 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-02-03T13:34:35Z (GMT) No. of bitstreams: 1 cardoso_am_me_sjc.pdf: 3142311 bytes, checksum: fd89634d15b20bb2b50e11cbbb2d7552 (MD5) / Made available in DSpace on 2017-02-03T13:34:35Z (GMT). No. of bitstreams: 1 cardoso_am_me_sjc.pdf: 3142311 bytes, checksum: fd89634d15b20bb2b50e11cbbb2d7552 (MD5) Previous issue date: 2017-01-16 / O objetivo deste ensaio clínico foi avaliar a influência de dois comprimentos de trabalho foraminal diferentes na dor pós-operatória e alodinia mecânica após o tratamento endodôntico concluído em sessão única ou em duas sessões. Quarenta e oito pacientes adultos, indicados para tratamento endodôntico primário de dente com periodontite apical assintomática, foram randomizados em 4 grupos (n = 12): SV0 - tratamento em sessão única e instrumentação do canal radicular até o forame apical; SV+1 - tratamento em sessão única e instrumentação do canal radicular 1 mm além do forame apical; TV0 - tratamento em duas sessões e instrumentação do canal radicular até o forame apical; TV+1 - tratamento em duas sessões e instrumentação do canal radicular 1 mm além do forame apical. Todos os participantes receberam um questionário baseado em uma escala visual analógica para registrar sua avaliação da dor em 3 horas, 6 horas, 12 horas, 24 horas, 48 horas, 72 horas e 7 dias após o término do tratamento endodôntico. Para a avaliação mecânica da alodinia, a medição da força da mordida foi realizada utilizando um gnatodinanômetro digital imediatamente antes do tratamento e 7 dias após a sua conclusão. Não houve diferença estatisticamente significante entre os 4 grupos em relação a dor pós-operatória em todos os momentos avaliados (α = 5%, teste de Kruskal-Wallis). Os valores de força de mordida foram significativamente maiores 7 dias após o tratamento endodôntico, indicando que houve uma redução significativa da dor mecânica em todos os grupos, sem diferença significativa entre eles (α = 5%, ANOVA e teste de Tukey). Todos os grupos apresentaram a mesma taxa de dor pós-operatória nos momentos avaliados e efetivamente aumentaram os limiares mecânicos de dor. / The objective of this clinical trial was to evaluate the influence of two different foraminal working lengths on postoperative pain and mechanical allodynia after endodontic treatment completed in single-visit or two-visit. Forty-eight adult patients indicated for primary endodontic treatment of tooth with asymptomatic apical periodontitis were randomly assigned to 4 groups (n = 12): SV0 – single-visit root canal treatment and instrumentation up to the apical foramen; SV+1 – single-visit root canal treatment and instrumentation 1 mm beyond the apical foramen; TV0 – two-visit root canal treatment and instrumentation up to the apical foramen; TV+1 – two-visit root canal treatment and instrumentation 1 mm beyond the apical foramen. All participants received a questionnaire based on a visual analog scale to record their assessment of pain at 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours and 7 days after the endodontic treatment concluded. For mechanical allodynia evaluation, bite force measurement was performed using a digital gnatodynanometer just before treatment and 7 days after its conclusion. No statistically significant difference was found among the 4 groups in relation to postoperative pain at all time points assessed (α= 5%, Kruskal-Wallis test). Bite force values were significantly higher 7 days after endodontic treatment, indicating that there was a significant reduction of mechanical pain in all groups, with no significant difference among them (α= 5%, ANOVA and Tukey’s test). All groups exhibited the same rate of postoperative pain at the time points assessed and effectively increased the mechanical pain thresholds.

A dor é um importante sintoma que sinaliza danos teciduais ou agentes potencialmente prejudiciais ao organismo, evocando respostas sensoriais e motoras de proteção. A dor orofacial apresenta alta prevalência na sociedade atual, sendo esta condição associada a tecidos duros e moles da cabeça, face, pescoço e a estruturas intraorais. Considerando as dores orofaciais de origem músculo-esquelética, destacam-se àquelas causadas pela Disfunção Temporomandibular (DTM). A DTM apresenta etiologia multifatorial, caracterizada por quadros crônicos envolvendo a região cervical, a musculatura mastigatória e a articulação temporomandibular (ATM). Desde que a inflamação das ATMs é considerada a principal causa da dor em pacientes portadores de DTM, a busca por novas opções terapêuticas para esta disfunção envolve estudos desta articulação, abrangendo aspectos fisiológicos, morfológicos e moleculares. Considerando o processo inflamatório e os aspectos moleculares envolvidos no desenvolvimento desta condição, é possível que as enzimas proteolíticas extracelulares, destacando-se as Metaloproteinases da Matriz (MMPs), as quais estão envolvidas na reabsorção de colágeno e de outras macromoléculas, tenham participação ativa neste processo. Em particular, estudos demonstraram que as MMPs estão envolvidas na modulação da dor neuropática, bem como estão presentes no líquido sinovial de portadores de inflamação da ATM. Sendo assim, o objetivo deste trabalho foi avaliar a influência da administração do Adjuvant de Freund (CFA) intraarticular, bilateralmente nas ATMs de ratos, na sensibilidade mecânica e nociceptiva, bem como avaliar a expressão das MMPs, em particular da MMP-2 e MMP-9, no gânglio trigeminal, nas diferentes fases de desenvolvimento da inflamação. Os resultados mostraram que a inflamação das ATMs promoveu alodinia mecânica e hiperalgesia orofacial. Em adição, a administração de doxiciclina (inibidor inespecífico das MMPs) reduziu as alterações na sensibilidade mecânica e nociceptiva. A quantificação das MMPs no gânglio trigeminal demonstrou que o início da inflamação promove aumento da MMP-9 (1 e 3 dias), enquanto que nas fases tardias do processo inflamatório acompanha-se o aumento da expressão da MMP-2 (3, 7 e 10 dias). / Pain is an important symptom that signals tissue damage or potentially harmful agents to the body and evokes sensory and motor protection. The orofacial pain is a type of symptoms that appears in high prevalence in modern society. This painful condition is associated with hard and soft tissues of the head, face, neck and intraoral structures. Considering the pain of musculoskeletal origin, we can highlight those caused by temporomandibular dysfunction (TMD). The TMD has a multifactorial etiology, characterized primarily by chronic conditions involving the neck, the chewing muscles and temporomandibular joint (TMJ). Inflammation of the TMJ is considered the main cause of pain in patients with TMD. Thus, the search for new therapeutic options for this disorder involves studies in the TMJ region encompassing physiological, morphological and molecular aspects. Considering the inflammatory process as the main cause of pain present in TMD, it is extremely important to understand the molecular aspects involved in developing this condition. In this context, extracellular proteolytic enzymes, highlighting the metaloproteniases matrix metalloproteinases (MMPs) play major role in the resorption of collagen and other macromolecules. The proteolytic activity of these MMPs is controlled by tissue inhibitors of metalloproteinases (TIMPs), which contribute to the maintenance of metabolic balance and structure of the extracellular matrix. Therefore, the objective of this study was to assess whether the type MMP gelatinases (MMP-2 and MMP-9) of the trigeminal ganglion participate in the development of mechanical allodynia and hyperalgesia in rats orofacial chronic inflammation bilateral TMJ. Our results demonstrated the presence of orofacial hyperalgesia, as well as mechanical allodynia in animals with temporomandibular inflammation induced by CFA and an increase in the expression and activity of gelatinases in the trigeminal ganglion of these animals. Still, there was a decrease in nociceptive orofacial hipersensitivity in animals that received a non-specific inhibitor for MMPs (doxycycline, 30mg/kg/day) for 10 days.

The endogenous cannabinoids (endocannabinoids) anandamide (AEA) and 2-arachidonylglycerol (2-AG) exert their effects predominantly through cannabinoid CB1 and CB2 receptors, but these actions are short-lived because of rapid hydrolysis by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. Selective inhibition of either enzyme elevates CNS levels of the appropriate endocannabinoid and produces analgesic effects with fewer psychomimetic side effects than Δ9-tetrahydrocannabinol (THC), the primary active constituent of marijuana. While cannabinoid receptor agonists and FAAH inhibitors reliably produce anti-inflammatory and anti-hyperalgesic effects in the carrageenan test and other inflammatory pain models, much less is known about the consequences of inhibiting MAGL in these assays. Here, we tested whether the selective MAGL inhibitor JZL184 would reduce nociceptive behavior in the carrageenan test. JZL184 significantly attenuated carrageenan-induced paw edema and mechanical allodynia, whether administered before or after carrageenan. Complementary genetic and pharmacological approaches revealed that JZL184’s anti-allodynic effects required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-edematous actions. Importantly, the anti-edematous and anti-allodynic effects of JZL184 underwent tolerance following repeated injections of high dose JZL184 (16 or 40 mg/kg), but repeated administration of low dose JZL184 (4 mg/kg) retained efficacy. Interestingly, the anti-allodynic effects of the combination of low dose of JZL184 (4mg/kg) and high dose of the selective and long-acting FAAH inhibitor PF-3845 (10 mg/kg) was augmented compared with each drug alone. On the contrary, the combination treatment did not reduce edema more than either JZL184 or PR-3845 given alone. These results suggest that low doses of MAGL inhibitors alone or in combination with FAAH inhibitors, reduce inflammatory nociception through the activation of both CB1 and CB2 receptors with no evidence of tolerance following repeated administration.

La gestion de la douleur neuropathique reste un challenge en médecine, malgré le nombre de traitements actuellement disponible. L'expérimentation animale a généré beaucoup d'informations concernant la douleur, mais ces connaissances demeurent insuffisantes pour développer de nouveaux analgésiques plus efficaces tout en restant sécuritaires. La douleur est un symptôme clinique complexe avec de multiples origines, et les mécanismes de douleur centraux et périphériques dépendent de l'évolution de la pathologie. Il est donc essentiel d'investiguer plus profondément les mécanismes moléculaires responsables de l'initiation et du maintien de la douleur, afin de cibler de nouvelles voies de transmission de la nociception plus prometteuses pour soulager la neuropathie et développer de meilleures stratégies thérapeutiques. Ce projet s'est donc intéressé plus particulièrement à la famille des tachykinines issues du gène TAC1 (substance P, ses précurseurs et métabolites, et neurokinine A sont les peptides ciblés pour ce projet de recherche), une famille de neuropeptides qui joue un rôle critique dans la transmission nociceptive. Pour réaliser cette étude, nous avons d'abord développé une stratégie de quantification afin de quantifier les expressions des différents neuropeptides bioactifs cibles, par HPLCMS/ MS. Puisqu'il existe différentes stratégies de quantification des peptides par HPLCMS/ MS, une méthode analytique fiable et robuste était nécessaire pour répondre aux objectifs de recherche. Nous avons développé une méthode utilisant la quantification relative avec un étalon interne stable marqué isotopiquement. En effet, pour quantifier les neuropeptides d'intérêt de l'étude, c'est la stratégie qui s'est avérée la plus reproductible et précise. Suite à la mise au point de la stratégie de quantification, nous avons utilisé des modèles animaux, souvent nécessaires pour faire progresser la recherche scientifique sur la compréhension de la douleur / The management of neuropathic pain remains a challenge in medicine, despite the availability of numerous drugs. Animal experimentation has generated a tremendous amount of information about pain, but this knowledge is still insufficient for new more efficient and safe analgesics. Pain is a complex clinical symptom with multiple origins, and peripheral and central pain mechanisms depend on the pathology evolution. Thus, it is essential to further investigate the mechanisms responsible for the initiation and maintenance of pain in order to develop better effective therapies. This project is particularly focused on the tachykinin family encoded by TAC1 gene (substance P, its precursors and metabolites, neurokinin A), a family of neuropeptides that plays a critical role in nociceptive transmission. We initially developed a quantification strategy in order to study the targeted bioactive neuropeptide expression modulation by HPLC-MS and HPLC-MS/MS. And it is critical to develop reliable and robust analytical methods to reach the objectives. So, we developed a method using relative quantification with stable isotopic labeled internal standards. In fact, in order to quantify target neuropeptides, this strategy was the most reproducible and accurate. Following the development of the relative quantification strategy, we used validated animal models, fundamental to better knowledges of painful molecular mechanisms

There is an unmet medical need for the efficient treatment of neuropathic pain, a condition that affects approximately 10% of the population worldwide. Current therapies need to be improved due to the associated side effects and lack of response in many patients. Moreover, neuropathic pain causes great suffering to patients and puts an economical burden on society. The work presented in this thesis addresses SP1-7, (Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), a major metabolite of the pronociceptive neuropeptide Substance P (SP). SP is released in the spinal cord following a noxious stimulus and binds to the NK1 receptor. In contrast to SP, the degradation fragment SP1-7 is antinociceptive through binding to specific binding sites distinct from the NK1 receptor. The aim of this thesis was to investigate the impact of SP1-7 on neuropathic pain. To understand how SP1-7 exerts its effect, a series of N-truncated forms of the heptapeptide were biologically evaluated. A set of small high-affinity ligands was evaluated in animal models of neuropathic pain. To confirm a clinical relevance the levels of SP1-7 in human neuropathic pain were assessed incerebrospinal fluid (CSF) collected from neuropathic pain patients. The results showed that SP1-7 could alleviate thermal as well as mechanical hypersensitivity in three different animal models of neuropathic pain. C-terminal amidation was connected with increased efficacy. N-terminal truncation of SP1-7 indicated a necessity of five amino acids in order to retain biological effect. One small high-affinity ligand showed a significant anti-allodynic effect. CSF levels of SP1-7 in neuropathic pain patients were lower compared to controls. Taken together, these findings demonstrate that the formation of SP1-7 may be attenuated in neuropathic pain. C-terminal amidation and a majority of its amino acids are necessary for stability and permeability. Clearly, SP1-7 and SP1-7 mimetics with high affinity to the SP1-7 binding site ameliorate neuropathic pain-like behaviors in animal models of neuropathic pain. Overall, the findings presented in this thesis contribute to new knowledge regarding the role of SP1-7 and related analogues and fragments in neuropathic pain. In a future perspective, this could be essential for the development of efficient strategies for managing patients with neuropathic pain.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Recent studies have indicated that nearly half of all surgical patients present a picture of moderate to severe pain what become important to understand the mechanisms involved postoperative pain to be better treat it. Previous studies have shown that incisions can cause mast cell degranulation. Thus, the aim of this study was to investigate the involvement of mast cells and its inflammatory mediators, histamine, serotonin and tryptase in a model of postoperative pain in mice. The depletion of mast cell mediators produced by repeated pre-treatment with compound 48/80 (1, 3, 10 and 10 μg/paw), that promote mast cell degranulation, prevented postoperative nociception (98 ± 23% of inhibition) and reduced histamine and serotonin levels (88 ± 4% and 68 ± 10%, respectively) and tryptase activity (82 ± 14% of reduction) in paw tissue. Furthermore, plantar surgery produced immense mast cell degranulation, as assessed by histology and confirmed by the increased levels of serotonin (three fold higher) and histamine (fifteen fold higher) and by increased activity of tryptase (two fold higher) in the perfused tissue after surgery. Accordingly, pre-treatment with the mast cell membrane stabilizer cromoglycate (200 μg/paw, i.pl.) prevented mechanical allodynia (inhibition of 96 ± 21%) and an increase in histamine (44 ± 10% of inhibition) and serotonin (73 ± 5% of inhibition) levels and prevented the tryptase release (100% of inhibition) induced by plantar surgery. Finally, local treatment with H1 (promethazine, 100 μg/paw, i.pl.), 5-HT3 (ondansetron, 10 μg/paw, i.pl.), 5-HT2A (ketanserin, 5 μg/paw, i.pl.) or PAR-2 (ENMD-1068, 10-100 nmol/paw) receptor antagonists or with the tryptase inhibitor (gabexate, 0.01-1 nmol/paw) partially decreased postoperative nociception in mice. Thus, mast cell activation mechanisms as well as release of mast cells inflammatory mediators and activation of its respective receptors are interesting targets for the development of novel therapies to treat postoperative pain. / Estudos recentes indicam que praticamente a metade de todos os pacientes submetidos à procedimentos cirúrgicos apresentam um quadro de dor moderada à severa, o que torna importante entender os mecanismos envolvidos na dor pós-operatória para melhor tratá-la. Dados da literatura demonstram que incisões podem causar a degranulação de mastócitos. Assim, o objetivo deste estudo foi investigar o envolvimento dos mastócitos e seus mediadores inflamatórios, serotonina, histamina e triptase, em um modelo de dor pós-operatória em camundongos. A depleção dos mediadores dos mastócitos produzida pelo pré-tratamento repetido com o composto 48/80 (1, 3, 10 e 10 μg/pata), que promove degranulação de mastócitos, preveniu a nocicepção pós-operatória (98 ± 23% de inibição) e reduziu os níveis de histamina e serotonina (88 ± 4 % e 68 ± 10%, de redução, respectivamente) e a atividade da triptase (82 ± 14% de redução) no tecido da pata. Além disso, a cirurgia plantar produziu grande degranulação dos mastócitos, como avaliado por histologia e confirmado pelo aumento dos níveis de serotonina (três vezes maior) e histamina (quinze vezes maior) e pelo aumento na atividade da triptase (duas vezes maior) no perfusato tecidual após a cirurgia. O pré-tratamento com o estabilizador da membrana celular dos mastócitos, cromoglicato (200 μg/pata, i.pl.), preveniu a nocicepção mecânica (inibição de 96 ± 21%) e o aumento dos níveis de histamina (44 ± 10% de inibição) e serotonina (73 ± 5% de inibição), bem como preveniu a liberação de triptase (100% de inibição) induzida pela cirurgia plantar. Finalmente, o tratamento local com os antagonistas dos receptores H1 (prometazina, 100 μg/pata, i.pl.), 5-HT3 (ondansetrona, 10 μg/pata, i.pl.), 5-HT2A (cetanserina, 5 μg/pata, i.pl.) ou PAR-2 (ENMD-1068, 10-100 nmol/pata) ou com o inibidor da triptase (gabexato, 0,01-1 nmol/pata) reduziu parcialmente a dor pós-operatória em camundongos. Assim, os mecanismos de ativação de mastócitos, bem como a liberação dos seus mediadores inflamatórios e conseqüente ativação dos seus respectivos receptores são alvos interessantes para o desenvolvimento de novas terapias para tratar a dor pós-operatória.

Les canaux sodiques dépendants du voltage, ou canaux Nav, jouent un rôle capital dans l'excitabilité neuronale, dans la genèse et dans la propagation des potentiels d'action. Le canal Nav1.9 se distingue par une expression restreinte aux nocicepteurs et par des propriétés électrophysiologiques uniques qui, si elles excluent sa contribution à la phase dépolarisante du potentiel d'action, lui confèreraient un rôle dans la modulation de l'excitabilité des nocicepteurs. Ce travail de thèse vise à caractériser son implication dans la physiopathologie de la douleur par une approche comportementale, moléculaire et fonctionnelle. La première partie de ce travail consiste à étudier la contribution du canal Nav1.9 à la douleur inflammatoire. Nous avons donc réalisé différents tests comportementaux chez des souris knock-out (KO) et des rats traités par antisens (knock-down) modèles de douleur inflammatoire (aigu, subaigu, chronique). L'expression du canal ainsi que ses propriétés électrophysiologiques sont ensuite analysées chez ces mêmes modèles animaux. Notre premier constat est que le canal Nav1.9 n'est pas impliqué dans la réponse à une stimulation mécanique ou thermique chaude nociceptive chez des animaux sains. En revanche, l'hypersensibilité douloureuse thermique et mécanique induite par une inflammation subaiguë (carragénine intraplantaire) ou chronique (monoarthrite) est significativement réduite chez la souris KO Nav1.9. Un résultat similaire est obtenu par traitement antisens chez le rat, sur le modèle d'inflammation subaiguë. Chez la souris, suite à l'induction d'une inflammation subaiguë, une légère diminution suivie d'une forte augmentation de l'expression protéique du canal Nav1.9 est observée dans les ganglions rachidiens innervant la patte enflammée. Une augmentation de la quantité de canaux est également observée au niveau des troncs nerveux cutanés innervant cette même zone. Les canaux néosynthétisés ne contribuent pas au courant sodique enregistré en patch clamp dans les corps cellulaires des neurones des ganglions rachidiens, mais nos données suggèrent qu'ils sont exportés en direction des terminaisons nerveuses, où ils pourraient devenir fonctionnels et augmenter l'excitabilité cellulaire. La deuxième partie de ce travail de thèse consiste à caractériser l'implication de canal Nav1.9 dans la perception du froid et dans l'hypersensibilité au froid induite par l'oxaliplatine. Nous avons en effet observé de manière inattendue que les souris KO Nav1.9 présentent des seuils de douleur au froid (<10°C) plus élevés que les souris sauvages. Ce phénomène est confirmé par plusieurs tests comportementaux chez les souris KO et chez des rats traités par antisens anti-Nav1.9. L'oxaliplatine, prescrit dans le traitement des cancers colorectaux, est connu pour induire une hypersensibilité au froid invalidante chez la majorité des patients. Nous avons donc décidé d'étudier la contribution du canal Nav1.9 à ce symptôme. Suite à une injection unique d'oxaliplatine, une forte hypersensibilité au froid apparait chez les souris dès 20°C. Nous montrons que le KO Nav1.9 permet de supprimer l'hypersensibilité au froid aux températures normalement non douloureuses (20 et 15°C, allodynie), et de réduire l'hypersensibilité aux températures douloureuses (10 et 5°C, hyperalgie). Le même effet est observé chez le rat après traitement antisens. En conclusion, ce travail permet de mettre en évidence l'intérêt du canal Nav1.9 en tant que cible pharmacologique potentielle pour le traitement de douleurs inflammatoires et de l'hypersensibilité au froid induite par l'oxaliplatine. Il est de plus intéressant de constater que les seuils de réponse à des stimuli nociceptifs ne sont pas perturbés chez les souris KO Nav1.9 saines, à l'exception de la douleur provoquée par des températures froides extrêmes. Le blocage du canal Nav1.9 aurait donc des propriétés anti-hyperalgiques plutôt qu'antalgique, ce qui est conceptuellement intéressant. / Voltage-gated sodium channels, or Nav channels, play a key role in neuronal excitability and in the emission and propagation of action potentials. Among the different Nav isoforms, Nav1.9 is only expressed in nociceptors and shows atypical electrophysiological properties which, if they exclude a possible contribution to the depolarizing phase of the action potential, could be important for the modulation of nociceptors' excitability. This study aims to characterize the Nav1.9 implication in the pathophysiology of pain using behavioral, molecular and functional approaches. The first part of this work is to assess the Nav1.9 contribution to inflammatory pain. Therefore we have performed several behavioral tests in different inflammatory pain models (acute, subacute, chronic), using knock-out (KO) mice and rats treated with antisense oligodeoxynucleotides. Nav1.9 expression and electrophysiological properties are then analyzed within the same animal models. First, we observe that Nav1.9 channels do not contribute to pain perception in response to noxious heat or pressure in healthy animals. However, thermal and mechanical pain hypersensitivity induced by subacute (intraplantar carrageenan) or chronic (monoarthritis) inflammation is significantly lowered in Nav1.9 knock-out mice. Similar results are obtained on the subacute inflammation model using a knock-down strategy in rats. A weak reduction followed by a strong increase in Nav1.9 protein expression is observed in mice dorsal root ganglions innervating the inflamed paw during subacute inflammation. We also observe an increase in Nav1.9 immunolabeling in cutaneous nerve trunks innervating this zone. Whereas the newly produced channels do not contribute to the sodium current recorded in dorsal root ganglion cell bodies, as assessed by patch clamp, our data suggest that they are transported to nerve terminals where they could become functional and increase neuronal excitability. In the second part of this study, we aim to characterize the implication of Nav1.9 channels in cold perception and in oxaliplatin-induced cold hypersensitivity. Indeed, we surprisingly observed that Nav1.9 KO mice showed higher pain thresholds to intense cold (<10°C) than wild-type mice. This observation is confirmed by several behavioral tests in KO mice and in antisense-treated rats. As oxaliplatine (a platinum salt used to treat colorectal cancer) is known to induce cold pain hypersensitivity in most of the patients, we decided to study the Nav1.9 contribution to this symptom. Following acute oxaliplatin injection, a strong cold hypersensitivity is observed in wild-type mice at 20°C and below. We show that Nav1.9 KO results in a suppression of cold hypersensitivity to non-noxious temperatures (20 and 15°C, allodynia), and a reduction of hypersensitivity to noxious cold (10 and 5°C, hyperalgesia). A similar observation is made using Nav1.9 knock-down in rats. To conclude, our data shows that Nav1.9 could be potentially a good target to treat acute to chronic inflammatory pain, as well as oxaliplatin-induced cold hypersensitivity. Furthermore, as Nav1.9 is not involved in defining pain thresholds of healthy animals (except for noxious cold), its blockade would have anti-hyperalgesic rather than analgesic effects, which is conceptually interesting.

Neuropathic pain is one of the most debilitating disorders. Currently available treatments for neuropathic pain are still unsatisfactory as they have only limited treatment effect and patients may suffer from unwanted side effects. Mechanism-based approaches to neuropathic pain treatment are considered to be more effective. Therefore multiple studies are dedicated to study the pathophysiological mechanisms of neuropathic pain. One of the possible underlying mechanism that causes neuropathic pain is neuroinflammation. Recent studies suggested that angiotensin II ( main effector molecule of the renin-angiotensin system) via its receptors in the central nervous system may be involved in the neuroinflammatory processes. The aim of this study was to investigate the role of angiotensin receptor type 1 in the developement and maintenance of neuropathic pain induced in animal model. Spinal nerve ligation (L5) was used as a model of peripheral neuropathy. Our results showed that treatment with AT1R blocker losartan markedly reduced thermal hyperalgesia and reduced increased sensitivity to mechanical stimuli in the SNL-operated rats.This indicates a possibly significant role of AT1 receptors in the development of neuropathic pain, probably due to reduction of neuroinflammation in the nervous system. These findings...