Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Faculty of Physical Education and Recreation, Center for Neuroscience. Title from pdf file main screen (viewed on September 17, 2009). Includes bibliographical references.

Submitted by Helmut Patrocinio (hell.kenn@gmail.com) on 2017-11-24T01:32:25Z No. of bitstreams: 1 Cl?udio_Queiroz_Disserta??o.pdf: 793741 bytes, checksum: f79a2a4bb52a8be9ba0b57baec9ed09c (MD5) / Approved for entry into archive by Ismael Pereira (ismael@neuro.ufrn.br) on 2017-11-27T16:10:35Z (GMT) No. of bitstreams: 1 Cl?udio_Queiroz_Disserta??o.pdf: 793741 bytes, checksum: f79a2a4bb52a8be9ba0b57baec9ed09c (MD5) / Made available in DSpace on 2017-11-27T16:11:41Z (GMT). No. of bitstreams: 1 Cl?udio_Queiroz_Disserta??o.pdf: 793741 bytes, checksum: f79a2a4bb52a8be9ba0b57baec9ed09c (MD5) Previous issue date: 1999 / Nos ?ltimos dois s?culos, o conhecimento sobre o sistema nervoso central expandiu-se consideravelmente, possibilitando atualmente o tratamento de muitas patologias do sistema nervoso central. Uma dessas patologias, entretanto, a discinesia tardia n?o apresenta nenhum tratamento terap?utico de efic?cia comprovada (Soares, 1997). A discinesia tardia ? uma s?ndrome caracterizada por movimentos involunt?rios repetitivos, normalmente envolvendo a l?ngua, boca e face, ocasionalmente atingindo tamb?m o pesco?o, membros superiores e quadris. Acredita-se ser a discinesia tardia um efeito colateral da exposi??o prolongada aos antipsic?ticos (neurol?pticos). Essa disfun??o motora pode persistir por meses ou anos ap?s a retirada do tratamento com neurol?ptico, podendo at? mesmo ser irrevers?vel (Karniol, 1979; Casey, 1985; Kane, 1995). Nesta tese de Mestrado, procuramos estudar os efeitos comportamentais da administra??o de buspirona sobre modelos animais de discinesia tardia. Os modelos animais utilizados foram: [1] a supersensibilidade dopamin?rgica induzida por um tratamento prolongado com haloperidol e quantificada pela atividade espont?nea de ratos em um campo aberto e [2] pelo comportamento estereotipado induzido pela apomorfina e [3] a quantifica??o dos movimentos orofaciais de ratos ap?s um tratamento repetido com reserpina. O tratamento prolongado com buspirona per se (3.0 mg/kg, i.p., duas vezes ao dia, por 30 dias) n?o resultou em uma supersensibilidade comportamental em nenhum dos dois modelos animais. O tratamento concomitante de buspirona foi capaz de diminuir os sintomas da supersensibilidade dopamin?rgica induzida pelo haloperidol (2.0 mg/kg, i.p., uma vez ao dia, por 30 dias) e quantificada pela atividade geral em campo aberto, mas n?o pelo comportamento estereotipado induzido pela apomorfina. Nos experimentos agudos, apesar de a buspirona per se diminuir tanto a atividade gera em campo aberto como o comportamento estereotipado induzido pela apomorfina, a co-administra??o de buspirona n?o foi capaz de modificar o efeitos agudos do haloperidol sobre esses dois modelos animais. No terceiro modelo, ratos foram tratados com salina ou buspirona (3.0 mg/kg, i.p., duas vezes ao dia) e ve?culo ou reserpina (0.1 mg/kg, s.c., dias intercalados) por 19 dias. No vig?simo dia, os animais foram observados para a quantifica??o de seus movimentos orofaciais: freq??ncia de protrus?o de l?ngua e movimentos mandibulares e dura??o do tremor facial. O tratamento com buspirona per se n?o foi capaz de induzir a movimentos orofaciais. Animais tratados com reserpina apresentaram maior freq??ncia de movimentos orofaciais em rela??o aos animais tratados com salina. A co-administra??o de buspirona foi capaz de atenuar o desenvolvimento da discinesia orofacial induzida pela reserpina. Verificou-se, tamb?m, que os animais tratados cronicamente com buspirona (3.0 mg/kg, i.p., duas vezes ao dia, 30 dias) desenvolvem maior resposta ao comportamento de bocejo induzido pela apomorfina. Assim, com este trabalho observamos que o tratamento prolongado com buspirona foi capaz de atenuar comportamentos dependentes da disponibilidade de dopamina end?gena (atividade geral em campo aberto e movimentos orofaciais induzidos pela reserpina) provavelmente por meio de uma supersensibilidade dos receptores pr?-sin?pticos (sugerida pelo aumento do comportamento de bocejo induzido por apomorfina). Os dados aqui apresentados, juntamente com a literatura cl?nica existente at? o momento, sugerem um poss?vel papel terap?utico da buspirona no tratamento da discinesia tardia. / In the last two centuries, the knowledgement about the central nervous systems increased enormously, making possible the treatment of patients who suffer of all sort of central nervous systems? diseases. One of this diseases is Tardive Dyskinesia, a syndrome characterized by repetitive involuntary movements, usually involving mouth, face and tongue and sometimes limb and trunk musculature. The syndrome is considered to be an adverse effect of prolonged administration of antipsychotic drugs (normally named neuroleptics). It persists for moths after neuroleptic has been discontinued and may be irreversible (Karniol, 1979; Casey, 1985; Kane, 1995). In a recent meta-analysis study, Soares (1997) concluded that there is no efficacious therapeutic interventions for tardive dyskinesia. In this thesis, we studied the behavior effects of buspirone administration on animal models of tardive dyskinesia. These models comprised the [1] dopaminergic supersensitivity induced by long-term haloperidol administration, which is quantified by the spontaneous activity (locomotion and rearing frequency) of rats observed in an open-field or [2] by the apomorphine-induced stereotyped behavior, and [3] the quantification of orofacial dyskinesia in rats repeatedly treated with reserpine. In the first an second models, buspirone per se (3.0 mg/kg, i.p., twice daily, for 30 days) did not produce dopaminergic supersensitivity. When buspirone was given in combination to haloperidol (2.0 mg/kg, i.p., once daily, for 30 days), it decreased the neuroleptic withdrawal symptoms as detected in open-field but not in apomorphine-induced stereotypy. Although single administration of buspirone per se decreased both open-field and apomorphine-induced stereotypy behavior, buspirone single administration did not modify the acute effects of haloperidol on these two behavioral models. In the third model, rats were co-treated with saline or buspirone (3.0 mg/kg, i.p., twice daily) and vehicle or reserpine (0.1 mg/kg, s.c., once every other day) for 19 days. On the day 20, the animals were observed for the quantification of the behavioral parameters of orofacial dyskinesia: tongue protrusion and vacuous chewing movements frequencies and duration of twitching of the facial musculature. Reserpine-treated rats exhibited a significant increase in the three behavioral parameters of orofacial dyskinesia relative to the saline-treated rats. The co-administration of buspirone in the reserpine-treated rats attenuated the development of orofacial dyskinesia, when compared to the reserpine-treated rats. We also verified that chronic (30 days) buspirone treatment was able to increase apomorphine-induced yawning behavior. The possibility is raised that buspirone attenuates haloperidol-induced increased locomotion and rearing and reserpine-induced orofacial dyskinesia through the development of dopamine autoreceptor supersensitivity. Taken together with previous clinical reports, the present data suggest that buspirone co-administration may lead to important clinical effects concerning different tardive dyskinesia treatment.

Il n'existe actuellement aucun traitement efficace pour les patients présentant une blessure au niveau de la moelle épinière. Ce triste constat est, en partie, dû à la présente d'une cicatrice empêchant la repousse des tissus. Dans ce contexte, des biomatériaux (composés non-toxiques) pourraient être implantés afin de réduire la cicatrice en formation et de fournir un support de repousse aux fibres nerveuses. Parmi ces biomatériaux, certains semblent induire de nombreuses améliorations chez le rat. Nous avons renforcé ces résultats, à l'aide de techniques électrophysiologiques. De plus, nous avons développé un nouveau matériau dégradable afin de limiter toutes réactions délétères à long terme. Après avoir synthétisé notre matériau, combinant les qualités de dégradabilité de l'acide poly(lactique) et les propriétés mécaniques du poly(méthacrylate d'hydroxyéthyle), nous avons évalué ses différentes caractéristiques et ses effets thérapeutiques. Les résultats obtenus sont encourageants. Il serait maintenant intéressant de coupler notre biomatériau à des molécules bioactives ou à des cellules. / Currently, there is no treatment for patients with spinal cord injury. This pessimistic statement is, in part, due to the presence of a scar that prevents tissue regrowth. In this context, biomaterials (non-toxic compounds) could be implanted in order both to reduce the scar formation and to provide a growth support for nervous fibers. Among those biomaterials, many seem to induce numerous benefic effects in the rat model. We confirmed these data by the use of electrophysiological techniques. In addition, we developed a new degradable material so as to limit any long term deleterious reactions. After having synthesized our material, combining the degradable quality of the poly(lactic acid) and the mechanical properties of the poly(hydroxyethyl methacrylate), we analyzed its different characteristics and its therapeutic effects. The obtained results are encouraging. Now, it would be interesting to couple bioactive molecules or cells with our biomaterial scaffold.

Introdução: A microcefalia é um sinal clínico associado à heterogeneidade etiológica. As principais causas de microcefalia são as infecções congênitas e as anomalias congênitas. Em 2015, após o surto do zika vírus (ZIKV) e o aumento de casos de microcefalia, o Ministério da Saúde (MS) instituiu notificação compulsória para os recém-nascidos (RN) com microcefalia e/ou alterações do Sistema Nervoso Central (SNC). No Brasil, a distribuição geográfica do ZIKV ocorreu de maneira diferenciada nas regiões norte e sul, sendo que no extremo sul do país houve um menor número de infecções por esse vírus. Assim, o estado do Rio Grande do Sul (RS) tornou-se um local importante para a avaliação sistemática das causas de microcefalia neste país, independente da presença de transmissão continuada do ZIKV. Objetivos: Avaliar e descrever as causas de microcefalia dos RN notificados por microcefalia e/ou alterações do SNC no RS. Métodos: Estudo descritivo dos 162 RN com microcefalia notificada no período de dezembro de 2015 a dezembro de 2016. Destes, 99 casos foram avaliados de forma retrospectiva com base em revisão de banco de dados, e 63 casos foram avaliados de forma prospectiva em ambulatório específico do HCPA-Brasil. As etapas propostas para a avaliação (ou informações coletadas dos bancos de dados) foram: 1) histórico da gestante; 2) exame físico do RN; 3) exames para pesquisa de infecção congênita – toxoplasmose, rubéola, ZIKV e CMV (reação de cadeia da polimerase - PCR ou sorológicos); 4) exames de imagem do SNC; 5) avaliação genética (para os casos com história familiar ou suspeita de alteração genética). As crianças foram avaliadas do nascimento até conclusão diagnóstica, seguimento perdido ou término do estudo. O período de avaliação das crianças não foi superior a quatro meses. Resultados: Noventa e cinco casos (58,6%) apresentavam microcefalia grave, resultando em uma prevalência desta complicação ao nascimento de 6.5/10.000 RN. A causa foi definida em 73 dos 162 casos. Destes eram infecções congênitas 31 casos (19.3%), síndromes genéticas 19 casos (11.7%), e malformação isolada do sistema nervoso central 20 casos (12,4%). E a causa não foi identificada em 89 (54,9%). Dos 31 casos com infecções congênitas, três (9.7%) foram diagnosticados com ZIKV, seis (19.3%) com citomegalovírus, oito (25,8%) com toxoplasmose, e 14 (45.2%) com sífilis congênita. Nenhum caso de rubeola congênita foi diagnosticado e a imunidade adquirida para rubeola das mães dos RN notificados foi de 91.6%. Destes casos 14 (45.1%) apresentaram baixo peso ao nascer e 21 (66.7%) eram pequenos para idade gestacional. A microcefalia grave foi identificada em 12 (38.7%) e 6 59.2% dos casos apresentaram alterações cerebrais, o que reforça a gravidade da ação das doenças infecciosas. Conclusão: Este é o primeiro estudo a avaliar os casos de microcefalia e/ou alterações do SNC durante o surto de ZIKV no RS. A prevalência de casos de ZIKV no RS foi inferior a estados do Nordeste do Brasil. A maioria dos casos de infecção congênita apresentaram lesões neurológicas graves, principalmente os casos de ZIKV, o que pode ocasionar atraso no desenvolvimento neurológico e sequelas nestas crianças ao longo da primeira infância. No entanto, salientamos a importância das demais infecções congênitas e causas desconhecidas associadas à microcefalia no RS, independente da presença de ZIKV. / Introduction: Microcephaly is a clinical sign associated with etiological heterogeneity. The main causes of microcephaly are congenital infections and congenital anomalies. The Ministry of Health (MOH) has instituted compulsory notification for newborns with microcephaly and / or Central Nervous System (CNS) disorders in 2015, following the zika virus (ZIKV) outbreak and the increase in cases of microcephaly. In Brazil, the geographical distribution of ZIKV occurred in a differentiated way in the northern and southern regions, and in the southernmost part of the country there were fewer infections due to this virus. Thus, the state of Rio Grande do Sul (RS) has become an important site for the systematic evaluation of the causes of microcephaly in this country, regardless of the presence of continuous transmission of ZIKV. Objectives: To evaluate and describe the causes of microcephaly of newborns notified by microcephaly and / or CNS changes in RS. Methods: Descriptive study of the 162 newborns with microcephaly reported from December 2015 to December 2016. Of these, 99 cases were retrospectively evaluated based on a database review, and 63 cases were evaluated prospectively in an outpatient clinic specific to HCPA-Brazil. The proposed steps for the evaluation (or information collected from the databases) were: 1) history of the pregnant woman; 2) physical examination of the newborn; 3) screening tests for congenital infection - toxoplasmosis, rubella, ZIKV and CMV (polymerase chain reaction - PCR or serological); 4) imaging studies of the CNS; 5) genetic evaluation (for cases with family history or suspected genetic alteration). The children were evaluated from birth to completion of diagnosis, missed follow-up or termination of the study. The evaluation of them was not more than four months. Results: Ninety-five cases (58.6%) presented severe microcephaly, resulting in a prevalence of this complication at birth of 6.5 / 10,000 newborn. A definite cause was established in 73 of the 162 causes. The leading etiology was congenital infections in 31 cases (19.3%), genetic syndromes in 19 cases (11.7%), and isolated central nervous system malformation in 20 cases (12.4%). Of the 31 cases with congenital infections, three (9.7%) were diagnosed with ZIKV, six (19.3%) with cytomegalovirus, eight (25.8%) with toxoplasmosis, and 14 (45.2%) with congenital syphilis. No case of congenital rubella was diagnosed and the acquired immunity to rubella from the mothers of the newborns was 91.6%. Of these, 14 (45.1%) had low birth 8 weight and 21 (66.7%) were small for gestational age. Severe microcephaly was identified in 12 (38.7%) and 59.2% of the cases presented cerebral alterations, which reinforces the severity of the action of infectious diseases. Conclusion: This is the first study to assess the cases of microcephaly and / or CNS changes during the outbreak of ZIKV in RS. The prevalence of ZIKV cases in RS was lower than in the northeastern states of Brazil. Most cases of congenital infection have severe neurological lesions, especially cases of ZIKV, which can cause delay in neurological development and detectable sequelae in these children throughout their first infancy. However, we emphasize the importance of other congenital infections and unknown causes associated with microcephaly in RS, regardless of the presence of ZIKV.

abstract: Stromal cell-derived factor-1α (SDF-1α) and its key receptor, CXCR4 are ubiquitously expressed in systems across the body (e.g. liver, skin, lung, etc.). This signaling axis regulates a myriad of physiological processes that range from maintaining of organ homeostasis in adults to, chemotaxis of stem/progenitor and immune cell types after injury. Given its potential role as a therapeutic target for diverse applications, surprisingly little is known about how SDF-1α mediated signaling propagates through native tissues. This limitation ultimately constrains rational design of interventional biomaterials that aim to target the SDF-1α/CXCR4 signaling axis. One application of particular interest is traumatic brain injury (TBI) for which, there are currently no means of targeting the underlying biochemical pathology to improve prognosis. Growing evidence suggests a relationship between SDF-1α/CXCR4 signaling and endogenous neural progenitor/stem cells (NPSC)-mediated regeneration after neural injury. Long-term modulation of the SDF-1α/CXCR4 signaling axis is thus hypothesized as a possible avenue for harnessing and amplifying endogenous regenerative mechanisms after TBI. In order to understand how the SDF-1α/CXCR4 signaling can be modulated in vivo, we first developed and characterized a sustained protein delivery platform in vitro. We were the first, to our knowledge, to demonstrate that protein release profiles from poly(D,L,-lactic-co-glycolic) acid (PLGA) particles can be tuned independent of particle fabrication parameters via centrifugal fractioning. This process of physically separating the particles altered the average diameter of a particle population, which is in turn was correlated to critical release characteristics. Secondly, we demonstrated sustained release of SDF-1α from PLGA/fibrin composites (particles embedded in fibrin) with tunable burst release as a function of fibrin concentration. Finally, we contrasted the spatiotemporal localization of endogenous SDF-1α and CXCR4 expression in response to either bolus or sustained release of exogenous SDF-1α. Sustained release of exogenous SDF-1α induced spatially diffuse endogenous SDF-1/CXCR4 expression relative to bolus SDF-1 administration; however, the observed effects were transient in both cases, persisting only to a maximum of 3 days post injection. These studies will inform future systematic evaluations of strategies that exploit SDF-1α/CXCR4 signaling for diverse applications. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2016

Les exosomes sont des vésicules d'origine endocytaire sécrétées par les cellules dans leur environnement après fusion des endosomes multivésiculés avec la membrane plasmique. Ils représentent un nouveau moyen de communication cellulaire par le transfert intercellulaire de protéines, de lipides et d'ARN. Dans le laboratoire, nous nous intéressons aux rôles que pourraient jouer les exosomes neuronaux dans le système nerveux central. Nous avons montré que les neurones matures sécrètent des exosomes. Nous avons mis en évidence que cette sécrétion est directement reliée à l'activité synaptique glutamatergique et à une entrée de Ca2+. Nous avons également découvert que la partie C-terminale de la chaîne lourde de la toxine du tétanos peut être sécrétée par voie exosomale. Nous avons observé que les exosomes la contenant sont repris par des neurones en culture. Un tel cargo semble d'ailleurs influencer le devenir des exosomes. De plus, pour étudier la recapture des exosomes, nous avons utilisé des exosomes de cellules N2a exprimant la tétraspanine CD63 fusionnée à la GFP. En incubant des neurones d'hippocampe avec des exosomes GFP-CD63, nous sommes parvenus à démontrer qu'ils étaient endocytés par les neurones receveurs. Cependant, bien que les exosomes semblent avoir été internalisés, nos résultats suggèrent que leur trafic serait indépendant de la voie endocytaire classique. Enfin, nous nous sommes intéressé au contenu en ARN des exosomes de N2a et de neurones. Nous avons démontré qu'ils contenaient majoritairement des ARN courts (≤ 200 nucléotides) parmi lesquels, les microARN 132 et 138. Les microARN sont de puissants régulateurs de l'expression génique. Leur transfert, via les exosomes, représenterait une nouvelle voie de régulation très fine et avec un impact conséquent sur le fonctionnement du système nerveux. Les exosomes neuronaux pourraient donc jouer un rôle dans la physiologie normale de la synapse, en permettant l'échange d'ARN et de récepteurs aux neurotransmetteurs entre neurones. Ils pourraient également être impliqués dans la propagation de protéines pathogènes comme la toxine du tétanos et la propagation de certaines maladies neurodégénératives comme Alzheimer et Creutzfeldt-Jacob. L'ensemble de nos résultats suggère que les exosomes joueraient un rôle-clé dans le système nerveux central, de par leur implication dans des processus physiologiques et pathologiques. / Exosomes are vesicles of endocytic origin released by cells into their environment on fusion of multivesicular endosomes with the plasma membrane. They represent a novel mechanism of cell communication by intercellular transfer of proteins, lipids and RNAs. In our laboratory, we are interested in the roles neuronal exosomes could play in the central nervous system. We first showed that mature neurons secrete exosomes and that this is regulated by synaptic glutamatergic activity and by Ca2+ influx. We next demonstrated that the C-terminal part of the tetanus toxin heavy chain can be released in association with neuronal exosomes which can then be taken up by other neurons. Moreover, such a cargo seems to influence the actual fate of the exosome. In order to further examine exosome reuptake, we used exosomes from N2a cells expressing the tetraspanin CD63 fused to the green fluorescent protein, GFP. By incubating cultured hippocampal neurons with GFP-CD63 exosomes, we succeeded in proving that they were found inside the recipient neurons. However, although exosomes are internalized, our results suggest that their traffic is independent of the classical endosomal pathway. We also studied the RNAs contained in the N2a and neuronal exosomes. These were mainly short RNAs (≤ 200 nucleotides) including microRNAs 132 and 138. MicroRNAs are key regulators of gene expression. Their transfer by exosomes could represent a new way for fine regulation with a potentially powerful impact on the nervous system. Neuronal exosomes could play a crucial role in the normal physiology of synapses, by allowing the exchange of RNAs and neurotransmitter receptors between neurons. They could also propagate pathogenic proteins such as tetanus toxin and be involved in neurodegenerative disorders such as Alzheimer's and Creutzfeldt-Jacob's diseases. Altogether, our results pave the way towards the demonstration that exosomes play an important part in the functioning of the central nervous system via their involvement in physiological and pathological processes.

Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2015-06-09T18:38:57Z No. of bitstreams: 0 / Made available in DSpace on 2015-06-09T18:38:57Z (GMT). No. of bitstreams: 0 / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Fibromyalgia (FM) is characterized by chronic non-inflammatory widespread pain (CWP) and changes in sympathetic function. In attempt to elucidate the pathophysiological mechanisms of FM we used a well-established CWP animal model. We aimed to evaluate changes in cardiac autonomic balance and baroreflex function in response to CWP induction in rats. Therefore, it was used 30 male Wistar rats (250-350 g) with 2 to 3 months. CWP was induced by two injections of acidic saline (pH 4.0, n=16) five days apart into the left gastrocnemius muscle, whereas control animals were injected twice with normal saline (pH 7.2, n = 14). To evaluate changes in cardiovascular responses, one day after the second injection of acidic saline (n=8) or normal saline (n=6) animals were instrumentated to blood pressure recordings and posterior analysis of pulse interval (PI) and systolic arterial pressure (SAP) variability, and spontaneous baroreflex sensitivity (BRS) was performed. To determine the involvement of areas of the CNS (NTS , RVLM and CVLM ), two days after the second injection of acidic saline (n=8) or normal saline (n=8) animals were anesthetized, perfused, the brains removed and cut in a cryostat. The brain sections were subjected to immunofluorescence protocol for Fos protein. Results are expressed as mean ± SEM. Differences between groups were analyzed using by t test, paired and unpaired. Values of p < 0.05 were considered statistically significant. After induction of CWP, there was an increase of power in the low frequency (LF) band of PI spectrum (12.75±1.04 nu), a decrease in the high frequency (HF) band (87.25±1.04 nu) and an increase of LF/HF ratio (0.16±0.01), when compared to control animals (7.83±1.13 nu LF; 92.16±1.13 nu HF; 0.08±0.01 LF/HF). In addition, there was an increase of power in the LF band of SAP spectrum (7.93±1.39 mmHg2) when compared to control animals (2.97±0.61 mmHg2). BRS was lower in acidic saline injected rats (0.59±0.06 ms/mmHg) when compared to control animals (0.71±0.03 ms/mmHg). By immunofluorescence , we found that in all investigated areas the percentage of Fos immunoreactive cells was significantly higher ( p < 0.001) in saline acidic compared to neutral saline group. Our results showed that induction of CWP in rats shifts cardiac sympathovagal balance towards sympathetic predominance and decreases BRS, data which corroborate findings in humans with FM, and also that activation of NTS, CVLM and RVLM appears to indicate the involvement of these areas in an attempt to control the cardiac autonomic dysfunction. / A fibromialgia (FM) é caracterizada por dor muscular crônica difusa não-inflamatória (DMCD) e mudanças na função simpática. No intuito de elucidar os mecanismos fisiopatológicos da FM, foi utilizado um modelo animal de dor crônica difusa bem estabelecido na literatura. Desta forma, o objetivo do estudo foi avaliar a modulação autonômica cardíaca e a função do barorreflexo, em resposta à indução de dor muscular crônica difusa em ratos. Para tanto, foram utilizados 30 ratos Wistar machos (250 a 350g) com 2 a 3 meses. A DMCD foi induzida por duas injeções de solução salina ácida (pH 4,0, n=16) com cinco dias de intervalo, no músculo gastrocnêmio esquerdo, enquanto que os animais controle foram injetados duas vezes com solução salina neutra (pH 7,2, n=14). Para avaliar as alterações nas respostas cardiovasculares, um dia após a segunda injeção de solução salina ácida (n=8) ou salina neutra (n=6) os animais foram instrumentados para gravações da pressão arterial, e posterior análise da variabilidade do intervalo de pulso (IP) e da pressão arterial sistólica (PAS), como também da sensibilidade espontânea do barorreflexo (SBR) foi realizada. Para avaliar a ativação de neurônios em núcleos do Bulbo envolvidos com a modulação autonômica cardíaca (NTS, RVL e CVL), dois dias após a segunda injeção de salina ácida (n = 8) ou de solução salina normal (n=8), os animais foram anestesiados, perfundidos, o cérebro removido e cortado num criostato. As secções do cérebro foram submetidas ao protocolo de imunofluorescência para a proteína Fos. Os resultados são expressos como média ± EPM. Diferenças entre os grupos foram analisados pelo teste t, pareado e não-pareado. Valores de p < 0,05 foram considerados estatisticamente significativos. Após a indução de dor crônica difusa nos ratos (n=8), as variações do intervalo de pulso apresentaram maior oscilação em baixa frequência (LF) (12,75±1,04 un), menor oscilação em alta frequência (HF) (87,25±1,04 un) e maior valor da relação LF/HF (0,16±0,01), quando comparadas ao grupo controle (n=6) (7,83±1,13 un LF; 92,16±1,13 nu HF; 0,08±0,01 LF/HF). Em adição, houve maior oscilação em LF da pressão arterial sistólica (PAS) (7,93±1,39 mmHg), comparado ao grupo controle (2,97±0,61 mmHg). A sensibilidade espontânea do barorreflexo foi menor nos ratos injetados com salina ácida (0,59±0,06 ms/mmHg) quando comparada ao grupo controle (0,71±0,03 ms/mmHg). Através da imunofluorescência, observou-se que em todas as regiões investigadas a porcentagem de células imunorreativas à Fos foi significativamente maior (p<0,001) no grupo salina ácida, em comparação ao grupo salina neutra. Nossos resultados mostraram que a indução da DCMD em ratos desloca o balanço simpatovagal cardíaco em direção a uma predominância simpática e diminui SBR, dados que corroboram achados em seres humanos com FM, e ainda, que a ativação de neurônios do NTS, CVL e RVL parece indicar o envolvimento dessas áreas na modulação da disfunção autonômica cardíaca.

Resumo: Os confrontos dos animais com situações que induzem medo e ansiedade resultam em uma série de respostas comportamentais defensivas (ex. luta, fuga, imobilidade, vocalização, etc.), ativação neurovegetativa (ex. taquicardia, hipertensão, defecação, etc.), antinocicepção, além de influenciar o comportamento agressivo e aumentar a vulnerabilidade à dependência e recaída ao uso de drogas. Com base no potencial efeito ansiogênico dos neurotransmissores glutamato (via ativação do complexo receptor NMDA-óxido nítrico) e fator liberador de corticotrofina (via receptores CRF1 e CRF2), este estudo investigou o papel desses mediadores, através de injeções sistêmicas, na matéria cinzenta periaquedutal (MCP) ou no núcleo dorsal da rafe (NDR), nas respostas apontadas acima. Os seguintes modelos foram utilizados: labirinto em cruz elevado (LCE, ansiedade), injeção de formalina a 2,5% (nocicepção), conflito intruso-residente (agressão) e estresse de derrota social (dependência à cocaína). Os resultados indicaram: a) o efeito ansiogênico do agonista de receptores NMDA (N-metil-D-aspartato; NMDA) na MCP foi antagonizado pela inibição da enzima de síntese de NO, b) os efeitos ansiogênico e antinociceptivo do CRF na MCP foram via ativação de receptores CRF1 (mas não CRF2) e independentes de NO, c) os efeitos aversivo e antinociceptivo do NO (via administração de um doador de NO) na MCP mostraram-se sensíveis ao bloqueio de receptores CRF1, d) a ativação de receptores CRF2 intra-NDR reduziu o comportamento agressivo induzido pelo conflito intruso-residente, e) o tratamento sistêmico com antagonista CRF1 bloqueou a sensibilização comportamental à cocaína e atenuou o aumento do consumo da mesma induzidos pelo estresse da derrota social / Abstract: Animal confrontation against fear/anxiety-induced situations results in a repertory of behavioral defensive responses (e.g., fight, flight, immobility, vocalization), neurovegetative activation (e.g., tachycardia, hypertension, defecation), antinociception, as well as affects aggressive behavior and increases animals vulnerability to addiction and relapse to drug take. Based on the potential anxiogenic effect elicited by glutamate (via activation of NMDA-nitric oxide receptor complex) and corticotropin releasing factor (via CRF1 and CRF2 receptors), this study investigated the effect of systemic, intra-periaqueductal gray (PAG) or intradorsal raphe nucleus (DRN) injections of these mediators on the above described responses. The following animal models were used: elevated plus maze (EPM, anxiety test), formalin 2.5% injection (nociceptive test), resident-intruder conflict (aggression test) and social defeat stress (to induce cocaine addiction). Results indicated that: a) the anxiogenic effect elicited by intra-PAG injection of glutamate NMDA (N-methyl-D-aspartate; NMDA) receptor agonist was antagonized by prior local infusion an NO synthase inhibitor, b) the anxiogenic and antinociceptive effects elicited by intra-PAG CRF were mediated by CRF1 (but not CRF2) receptor activation and did not depend on NO synthesis, c) the aversive and antinociceptive effects of NO production (induced by intra-PAG injection of a NO donor) were sensitive to CRF1 blockade, d) activation of the CRF2 receptor within the DRN attenuated aggressive behavior elicited by resident-intruder conflict, e) systemic treatment with CRF1 receptor antagonist inhibited cocaine behavioral sensitization and social-defeat stress-induced cocaine consumption / Orientador: Ricardo Luiz Nunes de Souza / Coorientador: Klaus A. Miczek / Banca: Cleopatra da Silva Planeta / Banca: Fabrício Moreira / Banca: Hélio Zangrossi Júnior / Banca: Marcus Lira Brandão / Doutor