Currently, opioids are extensively used in clinical practices in order to treat pain in patients. However, prolonged administration of opioids are not feasible due to the development of side effects especially tolerance, constipation, addiction and dependence. Our drug design is mainly aimed to reduce opioid induce side effects such as development of tolerance. The first strategy examined involves design and synthesis of peptide based single molecules that have a mu agonist and delta agonist pharmacophore in combination with a COX2 inhibitory pharmacophore. A new molecule, 3-17 having good delta agonist activity, partial COX2 inhibitory activity and weak mu agonist activity was produced. Moreover, Investigation of the bioactivities of the synthesized ligands including 3-17 in terms of their ligand receptor interactions were probed using NMR conformational analysis along with docking analysis to the respective homology modeled mu and delta opioid receptors as well as the COX2 enzyme. As a further continuation of this work, instead of peptide based mu agonist and delta agonist type pharmacophore, the highly mu selective fentanyl pharmacophore was used in combination with a pyrazole based and a pyrazolone based COX pharmacophore. Based on the SAR study and docking analysis of synthesized ligands to the homology modeled mu opioid receptor, an ideal tolerant position without significant loss of mu opioid agonist activity for fentanyl were found. The second strategy involves design and synthesis of a peptide based single molecule that has a mu agonist and a delta antagonist pharmacophore in combination with a NK1 antagonist pharmacophore. A novel molecule (4-2) containing delta antagonist activity, weak mu agonist activity and NK1 antagonist activity was identified. Its homology modeled mu opioid receptor bound conformation was compared with that of reference ligands. Docking analysis of modified 4-2 to the homology modeled mu opioid receptor revealed that it can be further modified to obtain better mu agonist activity. 4-2 showed antinociception for 45 min period of time after injection in tail flick assay. In addition to studies that were directed to avoid tolerance development due to opioid administration, peptide based potential analgesics such as biphalin was modified by introducing more peptidomimetic character in order to enhance its blood brain barrier permeability and proteolytic stability. The novel molecule (6-7) was produced in this study and its antinociception lasted for 30 min period of time after injection in the tail flick assay.

Epilepsy is a common neurological disease affecting 6 million people in Europe. Early prevention and accurate diagnosis and treatment are of importance to obtain seizure freedom. In this thesis new applications of carbon-11-labelled tracers in PET and autoradiographic studies were explored in focal epilepsy. Patients with low-grade gliomas often experience epileptic seizures. A retrospective PET-study assessing seizure activity, metabolic rate measured with 11C-methionine and other known prognostic factors was performed in patients with glioma. No correlation was found between seizure activity and uptake of methionine. The presence and termination of early seizures was a favourable prognostic factor. Activation of the neurokinin-1 (NK1) receptor by substance P (SP) induces epileptic activity. PET with the NK1 receptor antagonist GR205171 was performed in patients with temporal lobe epilepsy (TLE) and healthy controls. In TLE patients an increased NK1 receptor availability was found in both hemispheres, most pronounced in anterior cingulate gyrus ipsilateral to seizure onset. A positive correlation between NK1 receptors and seizure frequency was observed in ipsilateral medial structures consistent with an intrinsic network using the NK1-SP receptor system for transmission of seizure activity. The uptake of 18F-fluoro-deoxy-glucose (FDG) is related to cerebral blood flow (CBF). Previously, methods to estimate blood flow from dynamic PET data have been described. A retrospective study was conducted in 15 patients undergoing epilepsy surgery investigation, including PET with 11C-FDG and 11C-Flumazenil (FMZ). The dynamic FMZ dataset and pharmacokinetic modeling with a multilinear reference tissue model were used to determine images of relative CBF. Agreement between data of FDG and CBF was analyzed showing a close association between interictal brain metabolism and relative CBF. Epilepsy often occurs after traumatic brain injuries. Changes in glia and inhibitory neuronal cells contribute to the chain of events leading to seizures. Autoradiography with 11C-PK11195, 11C-L-deprenyl and 11C-Flumazenil in an animal model of posttraumatic epilepsy studied the temporal and spatial distribution of microglia, astrocytes and GABAergic neurons. Results showed an instant increase in microglial activity that subsequently normalized, a late formation of astrogliosis and an instant and prolonged decease in GABA binding. The model can be used to visualize pathophysiological events during the epileptogenesis.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

While much is known about the influence of ventricular afferent neurons on cardiovascular function in the dog, identification of the neurochemicals transmitting cardiac afferent signals to central neurons is lacking. Accordingly, we identified ventricular afferent neurons in canine dorsal root ganglia (DRG) and nodose ganglia by retrograde labeling after injecting horseradish peroxidase (HRP) into the anterior right and left ventricles. Primary antibodies from three host species were used in immunohistochemical experiments to simultaneously evaluate afferent somata for the presence of HRP and markers for two neurotransmitters. Only a small percentage (2%) of afferent somata were labeled with HRP. About half of the HRP-identified ventricular afferent neurons in T3 DRG also stained for substance P (SP), calcitonin gene-related peptide (CGRP), or neuronal nitric oxide synthase (nNOS), either alone or with two markers colocalized. Ventricular afferent neurons and the general population of T3 DRG neurons showed the same labeling profiles; CGRP (alone or colocalized with SP) being the most common (30-40% of ventricular afferent somata in T3 DRG). About 30% of the ventricular afferent neurons in T2 DRG displayed CGRP immunoreactivity and binding of the putative nociceptive marker IB4. Ventricular afferent neurons of the nodose ganglia were distinct from those in the DRG by having smaller size and lacking immunoreactivity for SP, CGRP, and nNOS. These findings suggest that ventricular sensory information is transferred to the central nervous system by relatively small populations of vagal and spinal afferent neurons and that spinal afferents use a variety of neurotransmitters.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

O fator de crescimento neural (NGF) está relacionado à dor e ao aumento de Substância P (SP). Ressonância magnética funcional de forma não invasiva investiga áreas cerebrais. O Anti-NGF é um tratamento para dor, porém não investigado em dor neuropática. Nós avaliamos no modelo CCI a resposta comportamental e o envolvimento do NGF e da SP após tratamento com o Anti-NGF. Nós observamos NGF aumentado a longo prazo no gânglio da coluna posterior (DRG) e na medula espinal, já a SP aumentada no DRG em ambos os tempos e apenas tardiamente na medula espinal. O tratamento com Anti-NGF diminuiu a nocicepção dos animais. O Anti-NGF reverteu os altos níveis de NGF e SP no DRG e na medula espinal. Houve aumento de Fos no grupo CCI e diminuição após tratamento com Anti-NGF no córtex cingulado anterior. Observamos modificações da conectividade do tálamo, córtex somatossensorial primário e córtex cingulado com todo o encéfalo. Esperamos que estes resultados possam ser usados para o desenvolvimento de uma estratégia terapêutica que auxilie pacientes com dor neuropática crônica. / Nerve growth factor (NGF) is related to pain and increase of Substance P (SP). Non-invasively functional magnetic resonance imaging investigates brain areas. Anti-NGF is a treatment for pain, but not investigated in neuropathic pain. We evaluated the behavioral response and the involvement of NGF and SP in the CCI model after Anti-NGF treatment. We observed increased long-term NGF in the dorsal root ganglion (DRG) and spinal cord, as well as increased SP in DRG at both time and only late in the spinal cord. Treatment with Anti-NGF decreased the nociception of the animals. Anti-NGF reversed high levels of NGF and SP in DRG and spinal cord. There was an increase in Fos in CCI group and a decrease after Anti-NGF treatment in the anterior cingulate cortex. We observed changes in the connectivity of the thalamus, primary somatosensory cortex and cingulate cortex with the entire brain. We hope that these results can be used to develop a therapeutic strategy that will help patients with chronic neuropathic pain.

Objetivos: Avaliar a implicação da doença periodontal (DP) avançada, e da expressão da NOS e sP dos tecidos gengivais inflamados, na intensidade de dor e na qualidade de vida de pacientes com dor crônica crânio-facial. Casuística e Métodos: foram avaliados e tratados 20 pacientes com queixas de dores crônicas crânio-faciais e DP (Grupo de Estudo), comparativamente com 20 pacientes que tinham somente DP (Grupo Controle). Todos os pacientes receberam tratamento cirúrgico periodontal. A avaliação foi realizada pré e pós-tratamento periodontal (7, 30 e 180 dias). Instrumentos de avaliação: ficha clínica EDOF-HC, Escala Visual Analógica (EVA), questionário de dor McGill, Índices de Placa (IP), Sangramento (IS), Profundidade Clínica de Sondagem (PCS) e de Inserção (PCI); questionário de qualidade de vida WHOQOL-bref e análise imunohistoquímica para a expressão da óxido nítrico sintase (NOS) e da substância P do tecido gengival inflamado. A avaliação da dor foi realizada por investigador independente. Resultados: Os grupos apresentaram evolução clínica semelhante pelos parâmetros da DP: IP (p=0,0934), IS (p=0,8657), PCS (p=0,1728) e PCI (p=0,7406) nos três momentos avaliados. Houve redução da EVA no Grupo de Estudo aos 30 dias (p<0,05) e 180 dias (p<0,05), comparativamente ao pré-operatório. Aos 180 dias houve melhora significativa das queixas iniciais (p=0,005 para o Grupo de Estudo e p = 0,027 para o Grupo Controle), comparativamente ao pré-operatório, entretanto, houve diferença significativa entre os dois grupos nos três momentos da avaliação, seja para o percentual de melhora (p<0,001), seja para os descritores de melhora Edof-HC (p=0,004 aos 30 dias e p=0,001 aos 180 dias). No Grupo de Estudo, houve redução significativa dos descritores afetivos de dor (questionário de dor McGill) aos 180 dias (p = 0,014). Os escores de qualidade de vida do Grupo de Estudo foram inferiores aos do Grupo Controle nos domínios psicológico e físico (p<0,001 e p=0,007, respectivamente). As expressões da nNOS e da iNOS foram maiores nas gengivas inflamadas do Grupo de Estudo (p<0,001 e p= 0,003, respectivamente) e a expressão da sP foi semelhante nos dois grupos (p=0,363). Conclusões: Houve redução da intensidade da dor crônica crânio-facial dos pacientes que receberam tratamento para doença periodontal avançada. A expressão da substância P foi semelhante nos tecidos gengivais inflamados dos dois grupos, mas a expressão da nNOS e iNOS foi superior na gengiva inflamada dos pacientes com dor crônica crânio-facial. Pelos dados deste estudo a Doença Periodontal avançada pode ter implicação nas queixas de dor dos pacientes com dor crônica crânio-facial. / Aims: To assess the implications of advanced periodontal disease (PD), and the expression of NOS and sP of inflamed gingival tissue, in the intensity of pain and the quality of life in patients with chronic craniofacial pain. Casuistic and Methods: Were evaluated and treated 20 patients with complaints of chronic craniofacial pain and DP (Study Group), compared with 20 patients who had only DP (Control Group). All patients received surgical periodontal treatment. The evaluation was performed at pre-operative and post-operative periodontal treatment (7, 30 and 180 days). Tools for evaluation: clinical record EDOF-HC, Visual Analogic Scale (VAS), McGill Pain Questionnaire, Scores of Plaque (IP), Bleeding (IS), Clinical Probing Depth (CPD) and Insertion (CPI), the quality of life questionnaire WHOQOL-Bref and immunohistochemical analysis for the expression of nitric oxide synthase (NOS) and substance P from inflamed gingival tissue. The assessment of pain was conducted by independent researcher Results: The groups had similar clinical evolution by parameters of the PD: IP (p=0.0934), IS (p=0.8657), PCS (p=0.1728) and PCI (p=0.7406) in the three moments evaluated. There was reduction in the VAS of Study Group at 30 days (p<0.05) and 180 days (p<0.05), compared to preoperative. At 180 days there was significant improvement of the initial complaints (p=0.005 for the Study Group and p=0.027 for the Control Group), compared to preoperative, but there was significant difference between the two groups in the three moments of assessment, for the percentage of improvement (p<0.001), and either for the improvement descriptors Edof-HC (p=0.004 at 30 days and p=0.001 for 180 days). In Study Group, there was significant reduction of pain affective descriptors (McGill Pain Questionnaire) to 180 days (p=0.014). The scores of quality of life of the Study Group were lower than the Control Group in the psychological and physical (p<0.001 and p=0.007, respectively). The nNOS and iNOS expressions were higher in inflamed gingival Study Group (p<0.001 and p=0.003, respectively) and the expression of sP was similar in the two groups (p=0.363). Conclusions: There was reduction in the intensity of chronic craniofacial pain of patients receiving treatment for advanced periodontal disease. The expression of substance P was similar in inflamed gingival tissue of the two groups, but the expression of iNOS and nNOS was higher in inflamed gingival of patients with chronic craniofacial pain. The data of this study show that advanced periodontal disease can have involvement in complaints of pain in patients with chronic craniofacial pain.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2018-07-31T12:54:31Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Ana Cláudia Dalmolin.pdf: 4507654 bytes, checksum: 94b0e5d7e5b5768e1913e17076d5138e (MD5) / Made available in DSpace on 2018-07-31T12:54:31Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Ana Cláudia Dalmolin.pdf: 4507654 bytes, checksum: 94b0e5d7e5b5768e1913e17076d5138e (MD5) Previous issue date: 2018-02-22 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A hipersensibilidade dentinária cervical (HSDC) ocorre devido à exposição dentinária da região cervical por fatores como perda de minerais do esmalte ou da própria dentina, ou por recessão gengival. A exposição dos túbulos dentinários, conforme a teoria hidrodinâmica, facilita a transmissão de estímulos desde a superfície dentinária até a polpa. Os biovidros são biomateriais que inicialmente foram desenvolvidos para reparo e regeneração óssea, e que passaram a ser empregados também no tratamento da HSDC. Considerando a falta de compreensão quanto a condição pulpar, e a lacuna existente entre os biomateriais e a eficácia no tratamento, foi proposta a utilização de um modelo in vivo em ratos, em que, através da exposição dentinária cervical, pudesse demonstrar o processo neuroinflamatório no tecido pulpar, bem como testar e avaliar os efeitos de duas formulações de biovidros experimentais para a obliteração de túbulos dentinários. Desta forma, foram utilizados 29 ratos Wistar machos, os quais receberam cirurgias de indução de recessão gengival, com instalação de dispositivo de permanência temporária, assim promovendo a manutenção da condição de túbulos dentinários cervicais expostos. Prosseguiu-se com a aplicação dos tratamentos de acordo com os grupos de estudo: Naive (N) - sem recessão gengival e sem tratamento; Sham (S) – com recessão gengival e sem tratamento; Verniz cavitário (V) - recessão gengival e tratamento com verniz cavitário; Biosilicato® (BV-BS) - recessão gengival e tratamento com Biosilicato®; Biovidro de estrôncio (BV-Sr) - recessão gengival e tratamento com biovidro de estrôncio; Biovidro de potássio (BV-K) - recessão gengival e tratamento com biovidro de potássio. Os tratamentos foram aplicados sobre a superfície dentinária exposta dos primeiros molares superiores esquerdos, a cada 4 dias durante 28 dias, totalizando 7 aplicações. Realizou-se processamento histológico das hemimaxilas esquerda e direita, com posterior coloração de hematoxilina e eosina (HE) para avaliar alterações no complexo dentino-pulpar, além de coloração imunohistoquímica (IHQ) para identificação de substância P (SP). Os resultados apontaram que não houve diferenças significativas (p>0,05, teste de Kruskal-Wallis) entre os grupos, considerando os critérios para alterações dentino-pulpares; tampouco foram observadas diferenças na imuno-marcação para SP. Concluiu-se que não há alterações no complexo dentino-pulpar ou aumento de substância P em dentes com exposição radicular e túbulos expostos em ratos. Nas condições em que foram aplicados, os biovidros experimentais não são capazes de causar danos na polpa dental. No entanto, sugere-se a continuidade das pesquisas, com a avaliação de outros parâmetros no modelo animal para a melhor compreensão do mecanismo da HSDC. / Cervical dentin hypersensitivity (CDH) occurs due to dentin exposure of the teeth’s cervical region by factors such as loss in enamel or dentin minerals, or gingival recession. Dentin tubules exposure, according to the hydrodynamic theory, facilitates the transmission of stimuli from the dentin surface to the pulp. Bioactive glasses are biomaterials that were initially developed for bone repair and regeneration, but they could also be used for CDH treatment. There is no understanding about pulp conditions when dentin is exposed, neither about the efficacy of biomaterials used to CDH treatment. It was proposed an in vivo model of cervical dentin exposure in rats. This in vivo model demonstrates the pulp neuroinflammatory process and tests the efficacy of Biosilicate® and of two others experimental formulations of bioglasses in dentinal tubules obliteration. Thus, 29 male Wistar rats were submitted to exposure of the dentinal tubules, through gingival recession surgery. A temporary device was installed to keep the dentinal tubules exposure. Then, the treatments were applied over the exposed dentin surface of the upper left first molars every 4 days during a 28 days period, with the total of 7 applications. The treatments were applied according to the study groups: Naive (N) – no recession and no treatment; Sham (S) – recession but no treatment; Cavity varnish (V) – gingival recession and treatment with varnish; Biosilicate® (BV-BS) – gingival recession and treatment with Biosilicate®; Strontium bioglass (BV-Sr) – gingival recession and treatment with strontium bioglass; Potassium bioglass (BV-K) – gingival recession and treatment with potassium bioglass. The dentin-pulp complex changes and investigation of substance P (SP) were evaluated through hematoxylin and eosin (HE) and immunohistochemical (IHC) stainings after histological processing and paraffin sectioning of the left and right hemi-maxilae. The results showed that there were no significant differences (p>0.05, Kruskal-Wallis test) among the groups considering the conditions of the dentin-pulp complex. No differences were observed in immunostaining for SP. It was concluded that teeth with cervical root exposition and opened tubules in rats do not have changes in the dentinpulp complex or in the pattern of immunotracing for SP. Experimental bioactive glasses are not capable of causing damage to the dental pulp under the conditions in which it was applied. Nevertheless, new animal model studies could be conducted using others histological and immunohistochemical parameters in order to understand the mecanisms of the CDH.

O nervo depressor aórtico (NDA) é, primariamente, um conjunto de fibras aferentes que transmitem informações oriundas de alterações da pressão arterial (PA) a partir dos barorreceptores arteriais (mecanorreceptores localizados no arco da aorta ou seio carótico) aos centros de controle cardiovascular localizados no sistema nervoso central (SNC). Este mecanismo é responsável pela regulação reflexa da função cardíaca e vascular, promovendo ajustes nos centros vasoconstritor e vasodilatador, atuando simultaneamente sobre os sistemas simpático e parassimpático. Fato este que, contribui para o aumento da atividade vagal cardíaca e inibição de descargas simpáticas para vasos e coração, garantindo a manutenção dos níveis pressóricos dentro de uma faixa de normalidade. Diversos neurotransmissores foram descritos atuando nos centros de controle cardiocirculatório localizados no tronco encefálico, mais especificamente no bulbo, participando da regulação da PA. Nestas regiões centrais, os neurotransmissores glutamato, GABA (Àcido Gama Aminobutírico) e substância P (SP) foram amplamente investigados. Entretanto, em nenhum destes trabalhos foi realizado um estudo detalhado, investigando a presença da SP em nervos depressores aórticos de forma direta, sendo esta informação ainda desconhecida. Acredita-se que a SP seja um transmissor do reflexo barorreceptor, atuando na modulação deste circuito, na tentativa de atenuar elevações da pressão sanguínea. Existe portanto a necessidade de uma investigação morfológica e imunohistoquímica com o intuito de promover o esclarecimento sobre os neurotransmissores presentes no NDA. Os nervos frênicos foram utilizados como controle positivo, já que neste território a SP já se encontra caracterizada. Inúmeros são os estudos que descrevem a existência da SP em nervos frênicos, fato este que justifica a aplicação do referido nervo como controle do NDA, foco de estudo deste projeto. Baseados nestas necessidades, o objetivo do presente estudo foi primeiramente o de promover a padronização da técnica imunohistoquímica (IHQ), bem como a verificação da viabilidade de utilização do glutaraldeído à 2,5% como um fixativo primário, auxiliando na identificação de neurotransmissores dentro do sistema nervoso periférico. Em seguida, a identificação e quantificação da SP em NDA de ratos normotensos através do método imunohistoquímico indireto (3,3\'- Diaminobenzidina \"DAB\") foram realizados. O referido estudo foi desenvolvido em duas etapas. A primeira parte corresponde a padronização e otimização da técnica de imunohistoquímica em nervos frênicos de ratos Wistar através da localização e caracterização da SP e da enzima colina acetiltransferase (CAT). A segunda fase, trata-se da identificação e quantificação da SP no NDA, sendo este, um possível neurotransmissor ou neuromodulador do reflexo barorreceptor. Para este estudo foram utilizados no total 38 ratos da linhagem Wistar (Rattus Norvegicus), normotensos, com 20 semanas de idade, machos e fêmeas. Deste total, 16 animais machos foram destinados à padronização da técnica de IHQ em nervos frênicos. E para a caracterização e quantificação da SP no NDA foram utilizados 22 ratos Wistar, sendo 12 machos e 10 fêmeas. Nossos resultados demonstram de forma inédita a presença da SP em fibras amielínicas (tipo C) e fibras de pequeno diâmetro (A-delta) no NDA de forma bastante pontualizada em segmentos proximais e difusa distalmente, sugerindo a existência de subpopulações de fibras amielínicas do tipo C. Estes achados confirmam inúmeras suposições de que a SP atue como um dos neurotransmissores de aferências barorreceptoras, podendo participar na modulação do Sistema Nervoso Autônomo (SNA), uma vez que encontra-se localizada em centros responsáveis pela regulação reflexa da PA. Adicionalmente, a análise do percentual de marcação positiva à SP entre os gêneros apresentou um aparente predomínio da SP em machos mas sem diferença significativa entre os grupos. De forma semelhante, a padronização imunohistoquímica em cortes transversais e longitudinais de nervos frênicos apresentaram uma imunomarcação positiva e aleatória da SP em conjuntos de fibras amielínicas (tipo C) e em fibras de pequeno diâmetro localizadas próximo a periferia do espaço endoneural, corroborando com a localização relatada em estudos morfológicos e ultraestruturais, assegurando a especificidade e a reprodutibilidade do método. Distintamente, as fibras de grande e médio diâmetro (A-alfa, beta e gama), consideradas fibras mielinizadas de condução rápida, foram imunorreativas à CAT em nervos frênicos. Por fim, espera-se que a identificação deste neuropeptídeo sirva de gatilho para que futuras pesquisas envolvendo a liberação de neurotransmissores em aferências barorreceptoras sejam explorados. Fato este, que contribuirá para a agregação de informações pertinentes à modulação ou transmissão da informação neural, propiciando desta forma melhor entendimento da comunicação e atividades barorreflexas associadas a mecanismos cardiovasculares. / The aortic depressor nerve (ADN) is primarily a set of afferent fibers that transmit derived information of changes in arterial blood pressure (BP) from arterial baroreceptors (mechanoreceptors located in the aortic arch and carotid sinus) to sites of cardiovascular control located into central nervous system (CNS). This mechanism is responsible for the reflex regulation of cardiac and vascular function, promoting adjustments of vasoconstrictor and vasodilator centers, simultaneously acting on the sympathetic and parasympathetic systems. In addition, contributes to increased cardiac vagal activity and inhibition of sympathetic discharges to vessels and heart, ensuring the maintenance of blood pressure levels within the normal range. Many neurotransmitters have been described operating in cardio-circulatory control centers located in the brainstem, more specifically in the bulb, participating in the regulation of BP. In these central regions, the neurotransmitters glutamate, GABA (Gamma Aminobutyric Acid) and substance P (SP) have been widely investigated. However, none of these works was carried out a detailed study, investigating the presence of SP in aortic depressor nerves directly, and this information is still unknown. It is believed that SP can be a transmitter at the synapse of the baroreceptor reflex, operating in the modulation of this circuit in an attempt to attenuate elevation of blood pressure. Therefore, there is a need to investigate a morphological and immunohistochemical approach in order to promote the clarification on the present neurotransmitters into ADN. The phrenic nerves were used as a positive control, already as substance P (SP) is characterized in this territory. There have been numerous studies describing the existence of SP in phrenic nerves, a fact that justifies the application of the nerve as control of the ADN, study focus of this project. Based on these requirements, the aim of the present study is two-fold. Firstly, it attempts to promote the standardization of the immunohistochemical (IHC) technique as well as the verification of the feasibility of using glutaraldehyde fixative as a primary, assisting in the identification of neurotransmitters in the peripheral nervous system (PNS). Subsequently, the identification and quantification of SP immunoreactivity in the ADN of normotensive rats by indirect immunohistochemical method (3,3\'-Diaminobenzidine \"DAB\") were done. The study was developed in two stages. The first part corresponds to standardization and optimization of immunohistochemical technique in phrenic nerves of Wistar rats through location and characterization of the SP and enzyme choline acetyltransferase (ChAT). The second phase is about the identification and quantification of the SP into ADN, being a possible neurotransmitter or neuromodulator from the baroreceptor reflex. For this study we used a total of 38 Wistar rats (Rattus norvegicus), normotensive, 20 weeks old, male and female. From this total, 16 male animals were used for standardization of IHC technique in the phrenic nerves. Nonetheless, for the characterization and quantification of SP in ADN were used 22 Wistar rats, 12 males and 10 females. Our results showed an unprecedented manner the presence of SP in unmyelinated fibers (type C) and small diameter fibers (A-delta) into ADN, being quite focused on proximal segments and diffuse distally, suggesting the existence of subsets of unmyelinated fibers. These findings confirm numerous assumptions that the SP acts as a neurotransmitter from afferent baroreceptor and may participate in the modulation of the Autonomic Nervous System (ANS), since it is located in centers responsible for regulating reflex of BP. Further, an analysis of the percentage of positive SP staining between genders, presented an apparent predominance of SP in males but no significant difference between the groups were found. Similarly, IHC standardization in transverse and longitudinal sections of phrenic nerves showed a positive random and immunostaining of SP in sets of unmyelinated fibers (type C) and small diameter fibers located near the periphery of endoneural space, corroborating location reported on morphological and ultrastructural studies, ensuring the specificity and reproducibility of the method. Distinctly, the fibers of large and medium diameters (A-alpha, beta and gamma), considered myelinated fibers of fast conducting, were immunoreactive to ChAT in phrenic nerves. Finally, it is expected that the identification of neuropeptide serve as a trigger for that future studies involving the release of neurotransmitters into afferent baroreceptors be explored. These results could contribute to the aggregation of relevant information for the modulation and transmission of neural information, thus providing better understanding of communication and baroreflex activities associated with cardiovascular mechanisms.

A técnica de laserterapia é um método não invasivo utilizado por diversas áreas da saúde, e tem se mostrado eficaz no tratamento da diminuição da sensibilidade a dor. O presente estudo visa elucidar os efeitos desta terapia nas alterações moleculares induzida pela constrição crônica (CCI) do nervo isquiático de ratos. Esta técnica é aplicada em dias intercalados e tem a duração de 10 sessões. Ao finalizarmos o tratamento com laserterapia os animais foram eutanasiados e os gânglios das raízes posteriores (DRG L4L6) e o nervo isquiático foram retirados para posterior análise. Os DRGs foram processados para detecção da Substância P (SP) e do receptor de potencial transitório vanilóide tipo I (TRPV1). Ainda submetemos o nervo isquiático para a detecção do fator de crescimento neural (NGF) e proteína zero (P0). Nossos resultados demonstraram uma reversão da hipernocicepção dos animais tratados com LASER. Ainda, observamos um aumento da expressão de SP e TRPV1 nos animais com lesão e uma diminuição após o tratamento com LASER. Com relação aos ensaios referente ao nervo isquiático, podemos observar que houve um aumento da densidade óptica para o NGF e para a P0 após tratamento. Esses resultados podem ser observados de modo qualitativo por meio da técnica de imunohistoquímica de fluorescência. Nossos resultados em conjunto, demonstram a eficácia da laserterapia para reversão da hipernocicepção dos animais com lesão. Ainda, esta técnica é capaz de modular liberação de mediadores que participação do processo álgico, além de induzir a regeneração do nervo isquiático. / The laser therapy technique is a noninvasive method used by several health areas, and has been shown to be effective in treat pain sensitivity. The present study aims to elucidate the effects of this therapy on molecular changes induced by chronic constriction (CCI) in rats. This technique was applied every other day, during 10 sessions. At the end of treatment, the animals were euthanized and the posterior root ganglia (DRG L4-L6) and sciatic nerve were removed for further analysis. The DRG\'s were processed for the detection of Substance P (SP) and transient potential receptor type I (TRPV1). In addition, we submit the sciatic nerve for the detection of neural growth factor (NGF) and zero protein (P0). Our results demonstrated a reversal of hypernociception in animals treated with LASER. Furthermore, we observed an increase in SP and TRPV1 expression in animals with lesion and a decrease after LASER treatment. With respect to the tests referring to the sciatic nerve, we could observe that there was an increase in the optical density for NGF and P0 after treatment. These results can be observed qualitatively by fluorescence immunohistochemistry. Our results together demonstrate the efficacy of laser therapy for the reversal of hypernociception of animals with lesions. Furthermore, this technique is able to modulate the release of mediators that participate in the algic process; in addition, this technique is also able to induce regeneration of sciatic nerve in rats.