Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-06-14T19:34:21Z No. of bitstreams: 1 2015 - Veronica Cristina Lopes Menezes.pdf: 1388664 bytes, checksum: eb95b2be98ffd5f3a96140f14377f8f2 (MD5) / Made available in DSpace on 2017-06-14T19:34:21Z (GMT). No. of bitstreams: 1 2015 - Veronica Cristina Lopes Menezes.pdf: 1388664 bytes, checksum: eb95b2be98ffd5f3a96140f14377f8f2 (MD5) Previous issue date: 2015-07-30 / CAPES / The distribution of estrogen receptors in brain structures that are envolved in the hidroelectrolyte balance such as the lamina terminalis (LT), subfornical organ (SFO) and dorsal raphe nucleus (DRN) demonstrated that estradiol can modulate important responses in body fluids. In the literature experimental data support that estrogen can increase the triptofan-hydroxilase type 2 , the main enzyme for the serotonin synthesis. The primary question here is whether or not differences in the baseline or stimulated intake are a function of different levels of circulating gonadal hormones in female ovariectomized rats. Female Wistar intact rats (~230 g) were previously aclimated in metabolic cages during 5 days and ad libitum access to water and hypertonic saline (1.8%) bottles and food. Room temperature was maintained at 22?2 ?C with 12:12 h light-dark cycle (lights off at 19:00). Rats were anesthetized with intraperitoneal injections of a mixture of ketamine (75 mg/kg) and xylazine (5 mg/kg) and then bilaterally ovariohysterectomized. There were four experimental groups: OVX (replaced with corn oil), 2,5 ?g/kg (E2 2,5), 10,0 ?g/kg (E2 10,0) and 40,0 ?g/kg (E2 40,0), daily during seven days, s.c. After 24 h of the surgery the hormonal replacement initiated (estradiol cypionate, EC, Pfizer, Animal Health). We did three experimental protocols: baseline evaluations, sodium depletion and fluid replacement. The estrogen replacement exibitted a dose dependent effect in the following parameters under basal conditions: daily body weight, daily urinary volume and daily food intake. After sodium depletion there were no difference in the urinary volume after 2 and 24 hours of the experiment. But after fluid reposition we observed a dose dependent effect in the ingestive behaviour of water and hypertonic saline intake in sodium depleted and control animals. Our data support that estradiol can alter the natriorexigenic and dipsogenic responses especially after sodium depletion depending of the estrogenic status. / A distribui??o de receptores estrog?nicos em estruturas centrais envolvidas na regula??o da homeostase hidroeletrol?tica como o ?rg?o vasculoso da l?mina terminal, n?cleo subfornicial, n?cleo dorsal da rafe indica que o estradiol pode atuar nessas estruturas em resposta a altera??es nos fluidos corporais. Nosso objetivo foi verificar se a reposi??o hormonal pode influenciar de maneira concentra??o-dependente o status hidroeletrol?tico e neuroend?crino de ratas castradas com reposi??o hormonal em diferentes doses de forma comparativa. Ratas Wistar (~230 g) foram previamente adaptadas, por 5 dias, em gaiolas metab?licas, com acesso ad libitum aos bebedouros volum?tricos de ?gua e salina hipert?nica e ao alimento, sendo mantidas sob ciclo claro-escuro de 12 horas em sala com temperatura controlada em 22??2 ?C. Ao final da adapta??o, as ratas previamente anestesiadas com cetamina (75 mg/kg) e xilazina (5 mg/kg) foram submetidas ? cirurgia de ovariectomia bilateral. Os animais foram divididos em 4 grupos: OVX, reposi??o com ?leo de milho), repostos com ?leo de milho cipionato de estradiol (E2) 2,5 ?g/kg (E2 2,5), 10,0 ?g/kg (E2 10,0) e 40 ?g/kg (E2 40,0). O tratamento de reposi??o foi feito pela via subcut?nea, diariamente durante 7 dias tendo sido iniciado no dia seguinte ? cirurgia. Foram realizados tr?s protocolos experimentais: avalia??o sob condi??es basais, deple??o de ?ons s?dio e reapresenta??o de fluidos. Neste estudo o estradiol apresentou efeito dose dependente nos seguintes par?metros sob condi??es basais: peso corporal di?rio, volume urin?rio di?rio, ingest?o de alimento di?rio. Ap?s deple??o de s?dio n?o houve diferen?a em rela??o ao volume urin?rio de 2 e de 24 horas ap?s o experimento. No entanto ap?s a reapresenta??o dos fluidos houve efeito dose-dependente no comportamento ingestivo de ?gua e de salina hipert?nica tanto nos animais depletados de s?dio quanto nos animais controles.Os dados suportam que o estradiol modula o comportamento ingestivo dos animais sob condi??es basais e ap?s a deple??o de s?dio.

Estudos prévios demonstram projeções dos núcleos da rafe para áreas clássicas relacionadas ao controle do sistema cardiovascular, como locus coeruleus, núcleo do trato solitário, coluna intermédio lateral da medula espinal, núcleos motor dorsal do vago e ambíguo. Baseado nesta informação este estudo investigou a participação dos núcleos da rafe nas respostas adaptativas a estímulos de hipervolemia e hipovolemia. Foram utilizados ratos machos (Wistar), adultos, mantidos em biotério sob condições controladas, divididos em cinco grupos: hipovolemia (Hipo, submetidos à punção cardíaca com remoção sangüínea), controle da hipovolemia (C hipo, submetidos à punção cardíaca sem remoção sangüínea), hipervolemia (Hiper, submetidos à cirurgia para canulação da veia jugular externa e adição de solução salina no sexto dia pós-cirúrgico), controle da hipervolemia (C hiper, submetidos à cirurgia para canulação da veia jugular externa sem adição de solução salina) e basal (BS). Após 90 minutos do estímulo, os animais foram profundamente anestesiados e perfundidos transcardiacamente com salina seguida de fixador Paraformaldeído + Bórax 4%, 4ºC, pH 9,5. Os encéfalos foram removidos pós-fixados, crioprotegidos e seccionados em cortes coronais de 40 µm e processados com técnicas de imunohistoquímica contra Fos e 5-HT. Os estímulos de hipo e hipervolemia não causaram aumento significativo dos neurônios Fos-imunoreativos ou 5HT/Fos-imunoreativos. Nos núcleos Li, DR, MnR, RMg e ROb o grupo C hipo apresentou maior número de neurônios Fos-IR e 5HT/Fos-IR em relação ao grupo C hiper. Os resultados sugerem que os núcleos da rafe não participam nas repostas adaptativas aos estímulos aplicados. Entretanto, os mesmos parecem estar envolvidos em processos de nocicepção, seja através da 5-HT ou de outras substâncias neuroativas, presentes nestes núcleos. / Previous studies presented projections from the raphe nuclei to areas related to the cardiovascular control, like the locus coeruleus, nucleus of the solitary tract, spinal cord intermedium lateral column, dorsal motor vagus and ambiguus nuclei. Based in this information, the present work analyzed the participation of the raphe nuclei in the adaptative responses to hypovolaemia and hypervolaemia. Were used wistar rats, kept in controlled conditions, divided in 5 groups: hypovolaemia (Hypo, cardiac puncture and blood extraction), hypovolaemia control (C Hypo, cardiac puncture without blood extraction), hypervolaemia (Hyper, external jugular vein canulation surgery and saline addition), hypervolaemia control (C Hyper, external jugular vein canulation surgery and no saline addition) and basal condition (BS). Ninety minutes after the stimulus, the animals were anesthetized and perfused with saline followed by 4% paraformaldehyde plus borax, pH 9.5 at 4ºC. The brains were removed, pos-fixated, cryoprotected, sectioned in 40µm thickness coronal slices and proceeded with double-labeling immunohistochemistry techniques against Fos protein and serotonin. The Hypo and Hyper stimuli presented no significant increase of Fos-IR and double-labeled neurons. The C Hypo group presented higher number of labeled neurons in the Li, DR, MnR, RMg and ROb nuclei in comparison to the group C Hyper. These results suggest that the raphe nuclei have no participation in the adaptative responses to the volemy stimuli. However, this nuclei seems to be related to the nociception no modulated just by the serotonin but by others neurotransmitters too.

Vários estudos mostram que o núcleo mediano da rafe (NMR) e o hipocampo dorsal (HD) estão envolvidos no medo condicionado Pavloviano. Além disso, mecanismos serotoninérgicos do NMR parecem participar da expressão da resposta de medo condicionado contextual. Entretanto, ainda não existe uma abordagem experimental que integre os mecanismos do circuito NMR-HD. Neste trabalho, o paradigma do medo condicionado foi utilizado para testar a influência dos mecanismos serotoninérgicos do circuito NMR-HD no medo condicionado contextual. As respostas de sobressalto e congelamento foram avaliadas após a administração de drogas serotoninérgicas intra-NMR e no HD, 6 h depois a sessões treino, nas quais os ratos eram condicionados com choques nas patas. A redução da transmissão serotoninérgica no NMR é devido a microinjeção do 8-hidroxi-2(di-n-propilamino)-tetralin (8-OH-DPAT), um agonista de receptores 5-HT1A, no NMR promoveu redução das respostas de congelamento, mas não alterou a resposta de sobressalto. Estes resultados são consistentes com a ideia de que mecanismos serotoninérgicos no NMR regulam as respostas de congelamento a um contexto aversivo. A diminuição pós-sináptica da serotonina nas áreas de projeção do NMR ocorre devido a ativação de autoreceptores 5-HT1A nesta estrutura. Com relação ao hipocampo, a microinjeção de cetanserina, um antagonista de receptores 5-HT2, não promoveu alteração nas respostas de congelamento e sobressalto potencializado pelo medo, porém a ativação de receptores 5-HT1A pela injeção de 8-OH-DPAT 6 h após o treino inibiu essas respostas. De acordo com esses resultados, um mecanismo inibitório deva se interpor entre os processos associados à chegada de informação aversiva e os associados à saída delas no HD. As projeções HD-amígdala e córtex pré-frontal medial podem constituir a porta de saída dos processos neurais subjacentes a expressão do medo condicionado contextual, conforme foi observado no experimento em que estudou a imunorreatividade destas estruturas á proteína Fos em ratos submetidos ao mesmo procedimento experimental de medo condicionado contextual / Several studies have shown that the median raphe nucleus (MRN) and dorsal hippocampus (DH) are involved in Pavlovian conditioned fear. Moreover, previous findings have also implicated serotonergic mechanisms of the MRN in the retrieval of contextual conditioned fear. However, studies that examine the integrated involvement of serotonergic mechanisms of the MRN-DH are lacking. This study, a fear conditioning paradigm was used to test whether the serotonergic projections from the MRN to DH can influence contextual fear conditioning. Startle and freezing responses were avaliated after administration of serotoninergics drugs into the MRN or DH, 6 h previously rats received footshocks in the training session. A reduction of 5-HT transmission in the MRN by local infusions of the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) decreased freezing in response to the CS but did not reduce fear-potentiated startle. This pattern of results is consistent with the hypothesis that MRN serotonergic mechanisms selectively modulate the freezing response to the aversive context. As for the DH, a decrease in postsynaptic 5-HT receptor activity at projection areas has been proposed to be the main consequence of 5-HT1A receptor activation in the MRN. Infusions of the 5-HT2 receptor antagonist ketanserin into the DH had no effect, but activation of 5-HT1A receptors through intra-DH injections of 8-OH-DPAT inhibited both the freezing and fear-potentiated startle response to the CS. To reconcile these findings, an inhibitory mechanism may exist between the incoming DH 5-HT pathway from the MRN and the presynaptic 5-HT neurons that are part of the DH output to other structures. The DH-amygdala and medial prefrontal cortex projections could well be this output circuit modulating the expression of contextual fear conditioning as revealed by measurements of Fos immunoreactivity in these areas.

A perimenopausa representa a transição da vida reprodutiva para não reprodutiva. É geralmente caracterizada por alterações neuroendócrinas, metabólicas e comportamentais, um possível resultado da depleção folicular ovariana e consequente redução do número de folículos ovarianos. É o período em que as mulheres podem apresentar maior susceptibilidade a manifestar transtornos afetivos e de ansiedade. A exposição de roedores ao resíduo químico 4-diepóxido de vinilciclohexeno (VCD) é um modelo bem estabelecido para estudos sobre perimenopausa, pois o VCD acelera o processo natural de atresia folicular. Embora as concentrações plasmáticas de estradiol estejam normais ou elevadas durante a perimenopausa, a terapia com estradiol pode ser benéfica para mulheres sintomáticas na perimenopausa. Portanto, o objetivo do presente trabalho foi investigar se a depleção folicular gradativa acelerada pelo VCD resulta em alterações na neuroquímica de ratas fêmeas em núcleos cerebrais que controlam o humor, além de avaliar se o estradiol seria capaz de reverter as possíveis alterações. Ratas da linhagem Wistar (28 dias pós-natal) receberam diariamente, durante 15 dias consecutivos, injeções subcutâneas de VCD (160 mg / kg) ou óleo de milho (O). Aproximadamente 55 dias após a primeira injeção, cápsulas de silastic contendo 17?-estradiol (E) ou O foram inseridas subcutaneamente (Grupos O+O; VCD+O; VCD+E). Cerca de 21 dias após o implante das cápsulas, as ratas dos grupos O+O e VCD+O foram decapitadas na manhã do diestro, enquanto que as do grupo VCD+E foram decapitadas exatamente 21 dias após o implante das cápsulas contendo estradiol, entre 0900 h e 1100 h. O sangue foi colhido para avaliação das concentrações plasmáticas de estradiol e progesterona por radioimunoensaio (RIE). Os cérebros foram removidos para microdissecção do hipocampo, amígdala, Locus coeruleus (LC) e Núcleo Dorsal da Rafe (NDR), para posterior análise dos níveis de RNAm para os receptores de progesterona (PR) e estradiol do tipo beta (ER?) por meio de RT/PCR. Este experimento foi replicado para remoção do hipocampo e amígdala para dosagem dos conteúdos de noradrenalina (NA) e serotonina (5-HT) por meio de cromatografia líquida de alta performance, seguida de detecção eletroquímica (HPLC/ED). Outro conjunto de ratas submetidas às mesmas condições10 experimentais foi perfundido para imunohistoquímica para TPH no NDR e TH no LC. Como esperado, na periestropausa (grupo VCD+O) as concentrações plasmáticas de estradiol não foram diferentes daquelas das ratas controles (O+O). As concentrações plasmáticas de progesterona na periestropausa foram menores que as do grupo controle, o que foi revertido pelo estradiol. No LC, a expressão de PR na periestropausa foi igual à das ratas controles, enquanto a expressão do ER? foi menor; a terapia com estradiol não modificou a expressão de nenhum destes receptores. A densidade de neurônios noradrenérgicos (TH+) no LC não foi alterada nem pela depleção folicular nem pela terapia estrogênica. Na periestropausa, o conteúdo de NA foi menor na amígdala, mas não no hipocampo, e o estradiol não alterou este conteúdo em nenhuma das áreas. No NDR, a expressão de PR e de ER? nas ratas na periestropausa foi menor que nas ratas controles; o estradiol preveniu o declínio da expressão de ER?, mas não de PR. O NDR foi analisado separadamente por toda a extensão rostro-caudal em 3 níveis anatômicos: rostral, médio e caudal, cada um dividido em 3 sub-regiões: lateral, dorsal e ventral. O número de neurônios serotonérgicos (TPH+) no NDR foi menor na periestropausa, e o estradiol foi capaz de reverter esse efeito, atuando principalmente na região caudal. A expressão gênica de PR não foi alterada nem pela depleção folicular nem pela terapia estrogênica tanto na amígdala como no hipocampo. A expressão de ER? também não foi diferente na periestropausa, quando comparada ao grupo controle, mas o estradiol aumentou esta expressão no hipocampo. Tanto na amígdala como no hipocampo houve redução no conteúdo de 5-HT na periestropausa e estradiol foi capaz de reestabelecer os níveis deste neurotransmissor aos valores controles apenas no hipocampo. Estes dados elucidam, pelo menos em parte os mecanismos do efeito positivo da terapia estrogênica nos sintomas de mulheres normoestrogênicas na perimenopausa. Estes efeitos parecem não envolver de forma importante o sistema noradrenérgico central, mas resultar do aumento da biossíntese de progesterona periférica em associação com a regulação positiva de ER? no NDR e hipocampo, que parece potencializar a via serotonérgica NDR/HPC. Portanto, o desenvolvimento de novas terapias que ativem os ER? pode ser uma alternativa para obter os efeitos positivos da ação do estradiol, eliminando os efeitos colaterais das terapias de estradiol que normalmente resultam da ativação do ER?. / Perimenopause represents the transition from reproductive to non-reproductive life. It is usually characterized by neuroendocrine, metabolic and behavioural changes, which result from a follicular depletion and reduced number of ovarian follicles. During this period, women are more likely to express mood disorders and anxiety. The exposure of animals to diepoxide 4-vinylcyclohexene (VCD) is a well-established experimental model for perimenopause studies, as VCD induces loss of ovarian small follicles (primary and primordial) in mice and rats by accelerating the natural process of atresia. Although estrogens levels are normal or even high during perimenopause, estrogen therapy can be beneficial for symptomatic perimenopausal women. The aim of this study was to investigate whether gradual follicular depletion induced by VCD results in changes in the neurochemistry of female rats in brain nuclei that control mood and the role of estradiol on these changes. Female rats (28 days) were daily injected with VCD or corn oil (O) for 15 days. Around 55 days after the first injection, pellets of 17?-estradiol (E) or O were inserted s.c (Groups O+O; VCD+O; VCD+E). Around 21 days after, rats O+O and VCD+O were decapitated between 0900 h and 1100 of diestrus while rats VCD+E were decapitated exactly 21 days after the onset of E therapy. Another set of rats followed the same experimental design and were perfused for TH and TPH immunohistochemistry in Locus coeruleus (LC) and Dorsal Raphe Nuclei (DRN), respectively. Blood was collected for estradiol and progesterone measurement by radioimmunoassay (RIA). The brains were removed from decapitated rats to punch out LC, DRN, hippocampus and amygdala to analyse the expression of mRNA for ER? and PR by RT/PCR. This experiment was replicated to punch out the hippocampus and amygdala for the determination of noradrenaline (NE) and serotonin (5-HT) contents by High Performance Liquid Chromatography, followed by Electrochemical Detection (HPLC/ED). As expected, plasma concentrations of estradiol were not different from those of control rats (O + O). Plasma concentrations of progesterone in the periestropause were lower than those in the control group, which was reversed by estradiol. In the LC, the PR expression in the periestropause was similar to that of the control rats, whereas the ER? expression was lower; estradiol therapy did not modify the expression of any of these receptors. The12 density of noradrenergic (TH +) neurons in LC was not altered by either follicular depletion or estrogen therapy. In periestropause, NA content was lower in the amygdala, but not in the hippocampus, and estradiol did not alter this content in any of the areas. In NDR, the expression of PR and ER? in periestropausal rats was lower than in controls; estradiol prevented the decrease of ER? expression, but not PR. The NDR was analyzed separately for the entire rostrocaudal axis in three anatomical levels: rostral, middle and caudal, each divided into three sub-regions: lateral, dorsal and ventral. The number of serotonergic neurons (TPH +) in NDR was lower in the periestropause, and estradiol was able to reverse this effect, acting mainly in the caudal region. PR gene expression was not altered by either follicular depletion or estrogen therapy in either the amygdala or the hippocampus. ER? expression was also no different in periestropause compared to the control group, but estradiol increased this expression in the hippocampus. Both in the amygdala and in the hippocampus there was a reduction in 5-HT content in the periestropause, and estradiol was able to reestablish the levels of this neurotransmitter at the control values only in the hippocampus. These data elucidate, at least in part, the mechanisms of the positive effect of estrogen therapy on the symptoms of normoestrogenic women in perimenopause. These effects do not appear to significantly involve the central noradrenergic system but result from increased peripheral progesterone biosynthesis in association with positive regulation of ER? in the NDR and hippocampus, which appears to potentiate the serotonergic NDR/HPC pathway. Therefore, the development of new therapies that activate ER? may be an alternative to obtain the positive effects of the estradiol action, eliminating the side effects of the estradiol therapies that normally result from the activation of ER?.

M.A. / Irritable bowel syndrome [IBS] has been described as a chronic relapsing condition, characterised by a change in bowel habit and abdominal pain, that cannot be explained by an organic disease. Some research indicates that IBS may be psychogenic in origin, however, the aetiology of this complex syndrome is still unclear. Some researchers have postulated that IBS is primarily a motility disorder of the gut, while others have indicated that the symptoms of IBS are mediated by the central nervous system. Thus it is not surprising that the care of patients with IBS poses a particular challenge to physicians, especially because of its biologic and symptomatic heterogeneity and, particularly for patients with refractory symptoms, its association with psychological disturbances. The literature study indicates that there that there may be a possible connection between the ingestive disorders, the menstrual cycle fluctuations associated with premenstrual dysphoric disorder and IBS. All three of these disorders also appear to be mediated, to some extent, by the neurotransmitter serotonin. In terms of these suggested correlations one of the aims of this study was to determine whether blood-serotonin levels significantly influence the symptomatology of IBS. Extensive literature exists documenting the potential role that serotonin plays in gastrointestinal functioning. However, none of the existing studies refer specifically to blood-serotonin levels. Thus the present study attempted to address this problem. A second aim of the present study was to determine the possible serotonergic connection in the ingestive disorders and premenstrual dysphoric disorder in women with IBS. All the subjects were required to go for a blood test in order to determine whether their serotonin levels were low, normal, or high. In addition, three self-report questionnaires were used in this investigation. The Irritable Bowel Syndrome Client Questionnaire; The Eating Disorder Inventory -2, of which four subscales out of 11 subscales were included, namely the Drive for Thinness, Bulimia, Body Dissatisfaction and Introceptive Awareness subscales; and the Premenstrual Assessment Form, of which six subscales out of 18 were included, namely Endogenous Depressive Features, Atypical Depressive Features, Signs of water Retention, General Physical Discomfort, Autonomic Physical Changes and Miscellaneous Physical Changes. In order to address the above mentioned aims, research was conducted at the Research and Counselling Centre for Psychogastroenterology at the Rand Afrikaans University. The Research and Counselling Centre for Psychogastroenterology is a facility developed to investigate the psychological constituents of IBS. Researchers at the centre are aiming to explore the multidimensional components of IBS with the purpose of gaining some understanding into the development and maintenance of this syndrome. A variety of topics are being investigated at the Research and Counselling Centre for Psychogastroenterology, including the role that stress, depression and coping styles play in IBS. Initially a sample group of (N = 60) women with IBS were selected for this research from a population of South Africans who were referred from gastroenterologists and general practitioners to the Centre for Gastroenterology at the Rand Afrikaans University. A number of women (N = 40) without IBS, from the north eastern suburbs of Johannesburg, were also asked to participate in this study in order to compile the comparison group.

L'hypertension artérielle pulmonaire (HTAP) est caractérisée par un intense remodelage de la microcirculation pulmonaire affectant principalement les artérioles pulmonaires musculaires. Lorsqu'elle survient en l'absence de condition associée, l'HTAP est considérée comme idiopathique (HTAPi). L'HTAP représente une pan-vasculopathie au cours de laquelle chaque type cellulaire (cellules endothéliale, musculaire lisse, fibroblaste) constituant la paroi vasculaire joue un rôle spécifique dans la réponse à l'agression. Les buts de ce travail étaient d'explorer l'implication de différentes voies de signalisation dans l'initiation ou la progression de la maladie. Les différentes études ont été réalisées à partir de cultures de cellules musculaires lisses (CML) d'artère pulmonaire et de cellules endothéliales (CE) pulmonaires obtenues à partir de prélèvements pulmonaires humains obtenus lors de transplantation chez des patients souffrant d'HTAP réfractaire.Des études antérieures avaient souligné le rôle majeur de la sérotonine au cours de l'HTAP idiopathique. Dans une première étude, nous avons étudié le rôle respectif de la sérotonine (5-HT), de son transporteur (5-HTT) ou de ses récepteurs (5-HT1B, 5-HT2A et 5-HT2B) dans le remodelage vasculaire pulmonaire mis en évidence dans l'HTP associée à diverses conditions. Les résultats de cette première étude montraient qu'une surexpression du 5-HTT dans les CML d'artère pulmonaire est une voie physiopathologique commune impliquée dans le remodelage vasculaire pulmonaire observé dans l'HTAP idiopathique, la maladie veino-occlusive et l'HTAP associée à différentes pathologies.Des mécanismes inflammatoires jouent probablement un rôle important dans la physiopathologie du remodelage microvasculaire pulmonaire. En effet, des infiltrats composés de cellules inflammatoires mononucléées (macrophages, lymphocytes T et B et cellules dendritiques) sont fréquemment mis en évidence autour des lésions vasculaires pulmonaires de patients présentant une HTAP idiopathique. Les mécanismes impliqués dans le recrutement de ces cellules mononucléées demeurent mal compris et nous avons étudié le rôle d'une chimiokine, CC chemokine ligand 2 (CCL2). Les résultats de cette seconde étude montraient que CCL2 était surexprimée au cours de l'HTAP idiopathique. La source de cette surexpression semblait provenir des cellules endothéliales pulmonaires. CCL2 agissait non seulement sur le recrutement des monocytes mais également sur les cellules musculaires lisses vasculaires pulmonaires en stimulant leur prolifération et leur migration.Des mutations germinales de gènes codant pour des membres de la famille des récepteurs du TGF tels que BMPR2 (Bone Morphogenic Protein Receptor type 2) sont retrouvées dans près de 70% des cas d'HTAP familiale mais également chez 10 à 30 % des cas d'HTAPi apparemment non familiales. Ces patients sont regroupés sous le terme d'HTAP « héritable » (HTAPh). Nous avons, dans une troisième étude, évalué si la dysfonction des voies de signalisation secondaires aux mutations de BMPR2 pouvait avoir des conséquences sur la voie de l'endothéline 1 (ET-1) qui représente l'une des cibles thérapeutiques de choix au cours de l'HTAP. Les résultats de cette troisième étude montraient que l'ET-1 était surexprimée au cours de l'HTAP avec ou sans mutation de BMPR2. En revanche, une surexpression des récepteurs ET-A dans les CML était mise en évidence au cours de l'HTAPh et était associée à une augmentation de l'effet pro-proliférant de l'ET-1 sur les CML.Ces résultats révèlent que des facteurs vaso-actifs (ET-1, 5-HT) et inflammatoires jouent un rôle déterminant dans la physiopathologie de l'HTAP et pourraient représenter de nouvelles cibles thérapeutiques. / Pulmonary arterial hypertension (PAH) is characterized by intense pulmonary vascular remodelling affecting mainly the muscular pulmonary arteries and leading to increased pulmonary vascular resistance. When it occurs in the absence of associated conditions, PAH is regarded as idiopathic (iPAH). PAH represents a panvasculopathy in which each cell type constituting the vascular wall (endothelial cells, smooth muscle cells, fibroblast) plays a specific role. The aims of this work were to explore the implication of various pathways in the initiation or the progression of the disease. The various studies were carried out using pulmonary artery smooth muscle cells (PASMC) and pulmonary endothelial cells (PEC) obtained during lung transplantation from patients with refractory PAH.Former studies have emphasized the major role of serotonin (5-HT) in the process of pulmonary vascular remodelling in iPAH. In a first study, we studied the respective role of 5-HT, the 5-HT transporter (5-HTT) and several 5-HT receptors (5-HT1B, 5-HT2A and 5-HT2B) on PASMC proliferation in cells from patients with PH associated with various conditions. The results of this first study showed that 5-HTT overexpression in PASMC is a common pathogenic mechanism in various forms of PH.Inflammatory cytokines may affect pulmonary vascular remodelling in iPAH. Indeed, iPAH frequently reveals inflammatory infiltrates corresponding to macrophages, lymphocytes and dendritic cells in the range of plexiform lesions as well as in other vascular lesions. The mechanisms underlying pulmonary vessel infiltration by monocytes / macrophages are unclear and the role for inflammatory cells in pulmonary vascular remodeling remains to be elucidated. This second study showed that iPAH is associated with an overexpression of CCL2. PEC are a major source of CCL2, which behaves as chemoattractant for circulating inflammatory cells and as growth factor for PASMC.Germline mutations of bone morphogenetic protein (BMP) receptor type 2 (BMPR-2), a member of the transforming growth factor (TGF)-β receptor family, have been reported in nearly 70% of patients with the heritable form of the disease (hPAH), and in 10–30% of patients with sporadic iPAH. In a third study, we evaluated the functional consequences of BMPR-2 mutations on the endothelin 1 (ET-1) pathway which represents one of the therapeutic targets on PAH. The results of this third study showed that iPAH and hPAH were associated with a similar overexpression of ET-1. In contrast, ETA receptor mRNA levels which were increased in PASMC from patients with iPAH and hPAH compared to controls were much higher in hPAH than in iPAH cells. Consequently, the growth promoting effect of ET1 on PASMC was higher in PASMC from patients with iPAH, and was markedly elevated in PASMC from patients with hPAH. No changes in ETB receptor mRNA levels could be detected in PASMC from patients with iPAH or hPAH in comparison with controls.These results reveal that vasoactive factors (ET-1, 5-HT) and inflammatory factors play a determining role in the pathophysiology of PAH and could represent new therapeutic targets.

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.

Ectothermic animals are susceptible to temperature changes such as cold shock with seasons. To survive through a cold shock, ectotherms have developed unique strategies. My interest is focusing on the physiological function of during cold shock and prolonged cold exposure in the fruit fly (Drosophila melanogaster) and crayfish (Procambarus clarkii). I used Drosophila melanogaster as a model system to investigate cardiac function in response to modulators (serotonin, acetylcholine, octopamine, dopamine and a cocktail of modulators) in acute cold shock and chronic cold shock conditions as possible mechanism to regulate heart rate in the cold. To examine if the dampened heart rate in the cold could still be enhanced by modulators or calcium loading, modulators and light-sensitive channelrhodopsin proteins were utilized to stimulate the heart. This light induced cardiac activation increased heart rate in all conditions, and potentially can be used for cardiac therapy in mammals. Also, the acute and chronic cold conditioned heart showed responsiveness to the above mention modulators. In examining how synaptic transmission is influenced by acute and chronic cold, the crayfish neuromuscular junction was used as a model. This is a good model as there are high and low output synapses to be investigated. The low output neuromuscular junction was enhanced in response to acute cold. The high output nmj increased in synaptic response to acute cold. In addressing chronic cold conditions, the nmj were physiologically assayed in their response to acute warm changes as well as influence of serotonin and octopamine. In chronic cold condition, the synaptic output was varied in enhanced and dampened responses to an acute warm environment. These junctions were enhanced in their synaptic output by serotonin and octopamine (100nM). In assessing, by HPLC assay, octopamine concentration increased in chronic cold crayfish. This suggests compensation in synaptic transmission in cold acclimation possibility via endocrine responses.

Abstract Primary intracerebral hemorrhage (pICH) is a severe, suddenly occurring disease involving high mortality and poor functional outcome. In the absence of curative treatment patient management is mainly supportive with the emphasis on preventing hematoma enlargement and complications. Better understanding of the factors predicting outcome are needed to define effective treatments. An unselected population-based registry study of 982 pICH patients admitted to Oulu University Hospital during the years 1993 to 2008 was conducted The study revealed that concomitant use of warfarin and serotonin-modulating antidepressants at the time of pICH increases the case fatality rate compared to patients with warfarin alone. An elevated C-reactive protein value on admission was an independent predictor of unfavorable outcome after pICH. This association was not explained by pre-existing heart disease, diabetes, severity of the bleeding, or infections. Patients undergoing surgical hematoma evacuation were observed to have improved 3-month survival compared to conservatively treated patients. Improved survival was noticed especially in patients with ≤70 years of age with ≥30ml supratentorial ICHs. Hematoma evacuation did not improve functional outcome. Earlier ischemic stroke was found to be an independent predictor of recurrent pICH. Diabetes seemed to increase and treated hypertension decrease the risk for fatal recurrence. Aspirin or serotonin-modulating antidepressants did not seem to increase the risk of recurrence. / Tiivistelmä Primääri aivoverenvuoto (pICH) on vakava, yhtäkkisesti alkava sairaus, johon liittyy korkea kuolleisuus ja vaikea vammautuminen. Parantavan hoidon puuttuessa on hoito lähinnä elintoimintoja tukevaa vuodon laajenemisen ja komplikaatioiden estämistä. Ennusteeseen vaikuttavien tekijöiden parempi tunteminen on ehto tehokkaiden hoitojen löytämiseksi. Väitöskirjatutkimustani varten kerättiin Oulun yliopistollisen sairaalan alueelta vuosien 1993-2008 aikana 982 aivoverenvuotoon sairastuneen potilaan väestöpohjainen aineisto. Tutkimus osoitti, että varfariinin ja selektiivisen serotoniinin takaisinoton estäjän (SSRI) yhteiskäyttö aivoverenvuodon aikana lisäsi kuolevuutta pelkkään varfariiniin nähden. Alkuvaiheen koholla oleva C-reaktiivinen proteiini oli itsenäinen aivoverenvuodon jälkeistä vammautuneisuutta ennustava tekijä. Yhteys ei selittynyt olemassa olevalla sydänsairaudella, diabeteksella, aivoverenvuodon vaikeudella tai infektioilla. Kirurginen aivoverenvuodon poistoleikkaus paransi kolmen kuukauden ennustetta verrattuna potilaisiin ilman leikkausta. Erityisesti leikkaus auttoi alle 70-vuotiaita potilaita, joilla oli yli 30 millilitran kokoinen pinnallisempi vuoto. Leikkaus ei parantanut fyysistä kuntoutumista. Aiempi sairastettu aivoinfarkti oli itsenäinen aivoverenvuodon uusiutumista ennustava tekijä. Diabetes saattaa lisätä ja hoidossa oleva verenpainetauti laskea riskiä tappavaan uusintavuotoon. Aspiriinin tai SSRI:n käyttö eivät lisänneet uusintavuodon riskiä.

Les traitements actuels de la dépression présentent une efficacité partielle et nécessitent une administration pendant plusieurs semaines avant d’obtenir un effet thérapeutique. Il est donc urgent de trouver de nouvelles stratégies antidépressives. La vortioxetine (Lu AA21004) est un nouvel antidépresseur en cours de développement. À la différence des inhibiteurs sélectifs de recapture de la sérotonine (ISRS), il est multi-cibles. Il bloque non seulement le transporteur de la sérotonine (SERT) mais aussi les récepteurs 5-HT3. Afin de caractériser les effets de ce composé et d’évaluer l'implication du blocage des récepteurs 5-HT3 dans son mécanisme d’action, plusieurs marqueurs précliniques de la réponse antidépressive ont été évalués. Nous avons utilisé des approches électrophysiologiques, immunohistochimiques, comportementales et de microdialyse chez le rat. La vortioxetine augmente la prolifération cellulaire hippocampique et induit une désensibilisation des autorécepteurs 5-HT1A dès 3 jours contre 2 à 3 semaines pour les antidépresseurs classiques. Elle induit également une importante libération de sérotonine malgré une occupation partielle du SERT. Ces effets sont liés, au moins en partie, au blocage des récepteurs 5-HT3. Nous avons ensuite montré qu’un antagoniste des récepteurs 5-HT3, l’ondansetron, à très faible dose, potentialisait l’effet d’un ISRS, la paroxetine. L’ensemble de nos données in vivo et ex vivo prouvent que le blocage des récepteurs 5-HT3 participe à l’efficacité pseudo-antidépressive de la vortioxetine. Les récepteurs 5-HT3 sont donc une cible intéressante pour améliorer l’efficacité des antidépresseurs et raccourcir leur délai d’action / Therapeutic effects of current antidepressant drugs only appear after several weeks of treatment and a significant number of patients do not respond to any treatment. Thus, more effective treatments for major depression are still needed. Vortioxetine (Lu AA21004), a novel antidepressant in development, displays effective properties in human. To the difference of selective serotonin reuptake inhibitors (SSRIs), it is a multimodal serotoninergic agent. Not only does it block the 5-HT transporter but it is also a potent 5-HT3 receptor antagonist. This current study was undertaken to characterize the effects of this compound and the role of 5-HT3 blockade. Using electrophysiological, immunohistochemical, autoradiography and behavioral approaches in rats, several pre-clinical markers of antidepressant-like response were assessed. Vortioxetine increased hippocampal cell proliferation and desensitized 5-HT1A autoreceptors from 1-3 days versus 2-3 weeks for classical antidepressants. In contrast to SSRIs, it also increased 5-HT hippocampal release with an incomplete SERT occupancy. Later effects are at least partly due to 5-HT3 receptors blockade. In parallel, we also showed that the 5-HT3 receptor antagonist ondansetron potentiated the effect of the SSRI paroxetine. Taken together, our in and ex vivo findings highlight the crucial role of 5-HT3 receptor blockade in the antidepressant-like efficacy of vortioxetine. Thus, we propose that the 5-HT3 receptors are an interesting target to improve antidepressant efficacy and reduce the therapeutic delay