Streptococcus pneumoniae ist der häufigste Erreger bakterieller Meningitiden. Eine Pneumokokken-Meningitis führt trotz Ausschöpfung aller heute verfügbaren Behandlungsmöglichkeiten in 25 % der Fälle zum Tod. Steigende Antibiotikaresistenzen und die Limitation verfügbarer Vakzine auf einige Serotypen von S. pneumoniae erfordern neue Ansätze in der antimikrobiellen Therapie. Cholin-bindende Proteine (CBPs) sind gemeinsames Merkmal aller Pneumokokkenstämme und für die Virulenz dieses Bakteriums essenziell. Durch Zugabe potenter Anticholinergika können die CBPs von der Bakterienoberfläche abgelöst und damit inhibiert werden. In dieser Arbeit wurde untersucht, ob durch Inhibition von CBPs während des Wachstums der Pneumokokken deren Phagozytose durch Mikrogliazellen in vitro gesteigert werden kann. Während ihres Wachstums wurden die Bakterien dazu mit potenten Anticholinergika inkubiert und am Ende ihrer exponentiellen Wachstumsphase auf murine Mikrogliazellen gegeben. Nicht alle eingesetzten Anticholinergika konnten die Inhibition der CBPs – angezeigt durch die Bildung langer Kokkenketten – bewirken, obwohl für sie alle eine hohe Affinität zu den CBPs in früheren Arbeiten nachgewiesen worden war. Ipratropium, das in höheren Konzentrationen das Pneumokokkenwachstum inhibiert, induzierte in den von uns eingesetzten niedrigen Konzentrationen weder die Bildung von Ketten noch führte es zu einer erhöhten Phagozytoseleistung. Mit DMAE funktionalisiert zeigten PAMAM-Dendrimere der 1. Generation ebenfalls keine Inhibition der CBPs: Es bildeten sich weder Kokkenketten noch zeigte sich eine erhöhte Bakterienaufnahme der Mikroglia. Im Gegensatz dazu stellte sich unter Einfluss von PPI-g2-DMAE neben ausbleibender Kettenbildung ein dosisabhängiger phagozytosehemmender Effekt dar. Einzig durch Co-Inkubation mit dem mit Cholin funktionalisierten PPI-Dendrimer der 2. Generation gelang die Inhibition der CBPs mit resultierender Bildung langer Ketten. Die Phagozytoseleistung zeigte eine dosisabhängige Steigerung sowohl für eine CoInkubation während der gesamten exponentiellen Wachstumsphase als auch nach Co-Inkubation während ihrer letzen 2 Stunden. Dennoch konnte im Sepsismodell der Maus durch intraperitoneale Injektion dieses Dendrimers 15 min vor Infektion mit S. pneumoniae kein protektiver Effekt erzielt werden: Zwischen den mit Dendrimeren behandelten Tieren und denen der Kontrollgruppe zeigten sich keine Unterschiede in Überlebenszeit und Sterblichkeit, dem krankheitsbedingten Gewichtsverlust, dem klinischen Score und der durch Ausplattieren von Milzhomogenaten ermittelten Keimkonzentration im Blut infektionsbedingt verstorbener Tiere. Die von uns eingesetzten Konzentrationen von Ipratropium scheinen für eine Inhibition der CBPs nicht ausgereicht zu haben. Der bislang nicht genau geklärte wachstumsinhibitorische Effekt, der sich in unseren Versuchen bereits ab 5 mM bemerkbar machte, könnte jedoch durch Inhalation von Ipratropium gezielt zur Prophylaxe von Pneumokokken-Pneumonien genutzt werden. Bei an Dendrimere gekoppelten, eigentlich potenten Liganden der CBPs konnte beobachtet werden, dass sie als Teil des Dendrimers ihre Affinität gegenüber den CBPs nicht nur deutlich verändern, sondern auch unerwartete Effekte (Verminderung der Phagozytose) hervorrufen können. Wegen der raschen Elimination scheint die einmalige Gabe eines potenten Dendrimers zur Inhibition der CBPs in vivo nicht auszureichen und erklärt das Versagen im Sepsismodell. Neuere Untersuchungen zur Distribution im Hirnparenchym nach intraventrikulärer oder subarachnoidaler Injektion lassen hoffen, dass durch Gabe subtoxischer Dosen die von uns beobachtete Phagozytosesteigerung in vivo reproduzierbar ist. Durch Inhibition der CBPs ist es möglich, die Virulenz aller Serotypen des Pneumokokkus stark zu reduzieren. Potente Inhibitoren könnten sowohl als Therapeutikum als auch zur Infektionsprophylaxe eingesetzt werden, ohne dass es dabei zur Ausbildung von Resistenzen kommt, da zeitgleich mehrere Virulenzfaktoren inhibiert werden. Es ist daher von großem medizinischen Interesse, Inhibitoren der CBPs zu entwickeln, die in subtoxischen Dosen eine hohe Affinität zu den CBPs aufweisen und diese inhibieren.

Epoxy fatty acids (EXA) are valuable to industry as they are used in synthesizing plasticizers such as of poly vinyl chloride, resins, adhesives, coating materials such as paint, lubricant, lubricant additives, insecticides, insect repellants, crop oil concentrates and formulations of carriers for slow release pesticides and herbicides. There is interest in developing commercial oilseeds accumulating epoxy fatty acids to at least 50% of the seed oil. Soybeans are the most widely cultivated oilseed and its oil has high levels of linoleic acid which can be a substrate for epoxygenase enzymes. Cahoon et al., expressed a cytochrome P450 enzyme (CYP726A1) from Euphorbia lagascae in soybean somatic embryos and found that the epoxy fatty acid, vernolic acid, reached ~8% of the total fatty acids in transgenic somatic embryos. Rabbit Livers possess a cytochrome P450, CYP2C2, which catalyzes the same epoxidation reaction as the E. lagascae enzyme but might be less likely to be influenced by regulatory machinery in plant cells. This CYP2C2 gene was placed in a plant expression vector under a seed-specific promoter and used to transform soybean, Glycine max, somatic embryos. The ten putative transgenic clones observed after 4-5 weeks were separated and proliferated under selection. glucuronidase (GUS) assays and PCR analyses performed on selected clones were positive. However vernolic acid in total lipids and specific lipid classes was not detected as analyzed by GC. In vitro enzyme assay performed on microsomes isolated from mature somatic embryos at three weeks of maturation using [14C] 18:2 PC as substrate showed presence of [14C] methyl vernoleate. Preliminary analyses on toxicity of epoxy fatty acids and corresponding diols in bacteria, yeast and caco-2 cells showed that leukotoxin diol (LD) most toxic.

A few abnormal cells found in a small piece of prostate tissue are most consequential for a man’s future. The prevalence of prostate cancer (PCa) is increasing globally. The main instigating factor for this cancer is not yet known, but it appears to be the consequence of many variables such as an increasingly older population, more frequent PSA-testing, and factors involving lifestyle. Prostate cancer screening, as an equivalent for breast cancer screening, has been suggested but unfortunately there are no accurate diagnostic tools available for this type of screening. The reason for this is simply that the prostate is one of the most difficult organs to diagnose and, consequently, PCa screening would generate far too many false-positive and false-negative results.  The prostate is not easily accessible as it is deeply-seated in the male pelvic area, wrapped around the urethra and surrounded by sensitive vital organs.  Furthermore, PCa is frequently multi-focal, and the cancer cells have a tendency of assimilating among normal cells and, thus, do not always form solid lumps.  Therefore, prostate tumors are often not felt by digital rectal examination (DRE) or identified by imaging.  The PSA-test is not reliable as it is more prostate-specific than cancer-specific.  Due to increasing prostate awareness, more early-stage and locally confined PCa are being detected. This is saving lives, although there is a high risk of over treatment and unnecessary side-effects.  The increased detection of PCa requires sophisticated diagnostic methods and highly skilled clinicians who can discern between indolent and aggressive cancers.  The current “gold-standard” for PCa diagnosis is biopsy grading by pathologists using the Gleason score system, which is a difficult task.  Therefore, innovative methods to improve the precision of prostate diagnosis, by increased biopsy sensitivity and tumor localization, are of essence. In light of these difficulties, the metabolomic approach using 1D and 2D high-resolution magic angle spinning (HRMAS) NMR spectroscopy combined with histopathology on intact prostatectomy specimens was evaluated in this research project.  The non-destructive nature of HRMAS NMR enables spectroscopic analysis of intact tissue samples with consecutive histological examinations under light microscope. Metabolomics aids in the unraveling and the discovery of organ-specific endogenous metabolites that have the potential to be reliable indicators of organ function and viability, extrinsic and intrinsic perturbations, as well as valuable markers for treatment response. The results may, therefore, be applied clinically to characterize an organ by utilizing biomarkers that have the capacity to distinguish between disease and health. The aim was to characterize the human and the rat prostate in terms of its intermediary metabolism, which I show here to differ between species and anatomical regions.  Furthermore, the aim is to seek the verification of HRMAS NMR derived metabolites which are known to be a part of the prostate metabolome such as, citrate, choline, and the polyamines which were performed, but also the identification of metabolites not previously identified as part of the local prostate metabolism, such as Omega-6, which was detected in tumors.  The extended aim was to elucidate novel bio-markers with clinical potential. In this study, the common phyto-nutrient, inositol, which appears to possess protective properties, was identified as being a potentially important PCa bio-marker for the distinction between the more indolent Gleason score 6 and the more aggressive Gleason score 7 in non-malignant prostate tissues with tumors elsewhere in the organ. Further studies in this area of PCa research are therefore warranted.

[Truncated abstract] Hepatocellular carcinoma (HCC) is the major malignancy complicating chronic liver disease. New therapies for the prevention of HCC are required due to the limited success and high tumour recurrence rates of existing treatments. Emerging evidence suggests that HCC arise from the transformation of adult liver progenitor cells (LPCs), which have the capacity to differentiate into hepatocytes and biliary cells during liver regeneration. LPC activation precedes neoplasia in experimental hepatocarcinogenesis. LPCs share antigenic epitopes with HCCs, including α-fetoprotein (AFP) and M2- pyruvate kinase (M2PK). In animal models of hepatocarcinogenesis, attenuation of the LPC response reduces the incidence of HCC following prolonged liver injury via a tumour necrosis factor (TNF) dependent mechanism. As TNF is a pro-inflammatory cytokine, these data suggest that anti-inflammatory agents may be effective in inhibiting LPC activation and hepatocarcinogenesis. Cyclo-oxygenase-2 (COX-2) is an inducible enzyme that mediates the production of many prostaglandins during inflammation and carcinogenesis. Recent investigations show that the administration of selective COX-2 inhibitors (SC2Is) may reduce the incidence of a variety of tumours including breast, colon and skin. The broad aim of this thesis was to conduct a series of detailed studies on the effects of a SC2I on LPC activation and the hepatic pathologies associated with hepatocarcinogenesis in order to test the hypothesis that S2CIs may be a beneficial therapy that can reduce liver injury and pre-neoplastic changes in the choline-deficient, ethionine supplemented (CDE) murine model of hepatocarcinogenesis. Administration of a SC2I (SC-236) significantly inhibited a variety of hepatic cell populations that expand during the first month of the CDE mouse model of hepatocarcinogenesis (a choline deficient, ethionine supplemented diet). Numbers of M2PK-positive LPCs (which are more hepatocytic in morphology and are also COX-2 positive) and inflammatory cells were all significantly reduced by SC-236. In contrast, numbers of A6-positive LPCs (which are more biliary cell-like in morphology and do not express COX-2) were unchanged. ... In summary, these data suggest that COX-2 inhibitors such as SC-236 inhibit LPC activation and a variety of pre-neoplastic liver pathologies as a result of COX-2 dependent and independent mechanisms that may be mediated through inhibition of Akt phosphorylation and induction of apoptosis. Moreover, SC2Is may be useful as preventative treatment strategies for HCC in patients with chronic liver disease.

Thesis (Ph. D.)--University of Oklahoma. / Includes bibliographical references.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The transition period is the most critical phase of the dairy production system. The large drop in dry matter intake peripartum and rising energy costs in early lactation result in negative energy balance (BEN). Dairy cows under BEN obtain energy from their fat reserves through mobilization of non-esterified fatty acids (NEFA). The elevation of this metabolite and its products in the bloodstream has drastic consequences to health, production and reproduction of dairy cows. The search for strategies to reduce this imbalance and improve the transition cow performance is a challenge to producers and experts in the field. Thus, the aim of this study was to evaluate the effect of the choline protected in production, uterine health, metabolism and reproduction in cows during the transition period with different supplementation times. In the first study were used 15 cows in the transition period. The treated group was supplemented with 80 grams of choline protected for 21 days before calving to 40 days. Blood samples were collected on days 21 and 10 before delivery and on days 10, 20 and 30 for postpartum evaluation of metabolic, liver and oxidative profile. The supplementation with choline did not change the fructosamine, cholesterol, NEFA hydroxybutyrate beta (BHB), growth factor similar to insulin (IGF-I), aspartate aminotransferase (AST), gamma glutamyltransferase (GGT) and total oxidants (TOS) levels. Weighing of milk was performed on days 10, 20 and 30 of lactation, and no treatment effect on production. At 60 days postpartum was conducted gynecological examination. After 60 days, the control group had more cases of endometritis, but no effect of treatment on the calving interval. Progesterone levels were larger in the control group than in the group treated at 60 days postpartum. Choline supplementation so it did not change the metabolic profile, but reduced the number of cases of endometritis at 60 days postpartum. In second study were used 54 cows, divided into three groups: control, cows supplemented by 10 antepartum days with protected choline (T10) and cows supplemented by 20 antepartum days with protected choline (T20). Blood samples were collected on days 10, 20 and 30 postpartum for determination of fructosamine, cholesterol, NEFA, BHB, AST, GGT and TOS levels. There was no difference between groups for any of the blood parameters. Milk production was evaluated on days 10, 20 and 30 of lactation, with no effect of treatments on the volume of milk produced. The interval between delivery and conception was lower in animals T20 group than the other groups. The protected choline did not change the metabolic profile of dairy cows under moderate negative energy balance. Prolonged use of choline reduced the number of cases of endometritis, while supplementation for 20 days in antepartum reduced the interval between parturition and conception. / O período de transição é a fase mais crítica do sistema de produção leiteira. As drásticas quedas do consumo de matéria seca no periparto e o aumento dos gastos energéticos no início da lactação resultam em balanço energético negativo (BEN). Vacas leiteiras sob BEN obtêm energia de suas reservas de gordura através de mobilização de ácidos graxos não esterificados (AGNE). A elevação desse metabólito e de seus produtos na corrente sanguínea possui consequências drásticas à saúde, produção e reprodução de vacas leiteiras. A busca por estratégias para reduzir esse desbalanço e melhorar o desempenho de vacas em transição é um desafio aos produtores e técnicos da área. Diante disso, o objetivo deste estudo foi avaliar o efeito da colina protegida na produção, saúde uterina, metabolismo e reprodução em vacas no período de transição com diferentes tempos de suplementação. No primeiro estudo foram utilizadas 15 vacas no período de transição. O grupo tratado foi suplementado com 80 gramas de colina protegida por 21 dias antes do parto e 40 dias após. Amostras de sangue foram coletadas nos dias 21 e 10 antes do parto e nos dias 10, 20 e 30 do pós-parto para avaliação do perfil metabólico, hepático e oxidativo. A suplementação com colina não alterou os teores de frutosamina, colesterol, AGNE, beta hidroxibutirato (BHB), fator de crescimento semelhante à insulina I (IGF-I), aspartato aminotransferase (AST), gama glutamiltransferase (GGT) e total de oxidantes (TOS). A pesagem do leite foi realizada nos dias 10, 20 e 30 de lactação, não havendo efeito do tratamento na produção. Aos 60 dias pós-parto foi realizado exame ginecológico. Aos 60 dias o grupo controle apresentou maior número de casos de endometrite, não havendo efeito do tratamento sobre o intervalo entre partos. Os teores de progesterona apresentaram-se maiores no grupo controle que no grupo tratado aos 60 dias pós-parto. A suplementação de colina de forma não alterou o perfil metabólico, mas reduziu o número de casos de endometrite aos 60 dias pós-parto. No segundo estudo, foram utilizadas 54 vacas, divididas em três grupos: controle, vacas suplementadas por 10 dias pré-parto com colina protegida (T10) e vacas suplementadas por 20 dias pré-parto com colina protegida (T20). Amostras de sangue foram coletadas nos dias 10, 20 e 30 pós-parto para determinação dos teores de frutosamina, colesterol, AGNE, BHB, AST, GGT e TOS. Não houve diferença entre grupos para nenhum dos parâmetros sanguíneos. A produção de leite foi avaliada nos dias 10, 20 e 30 de lactação, não havendo efeito dos tratamentos sobre o volume de leite produzido. O intervalo entre parto e concepção foi menor em animais do grupo T20 em relação aos demais grupos. A colina protegida não alterou o perfil metabólico de vacas leiteiras sob balanço energético negativo moderado. O uso prolongado de colina reduziu o número de casos de endometrite, enquanto que sua suplementação por 20 dias no pré-parto reduziu o intervalo entre parto e concepção.

Para avaliar o efeito do gadolínio sobre os metabólitos Colina (CO), Creatina (CRE) e N-acetil-aspartato (NAA) foi realizada espectroscopia por ressonância magnética em 25 pacientes com tumores intracranianos antes e após a injeção venosa do meio de contraste. Foram quantificadas e comparadas as relações CO/CRE, CO/NAA e NAA/CRE bem como a largura a meia altura dos picos dos metabólitos CO, CRE e NAA nos espectros pré e pós-contraste. Verificou-se redução das relações CO/CRE e CO/NAA e da largura a meia altura do pico do NAA nos espectros pós-contraste / In order to evaluate the gadolinium effect on the metabolites choline (CHO), creatine (CRE) and N-acetyl-aspartate (NAA) we performed multivoxel spectroscopy in 25 patients with intracranial tumors before and after the injection of the contrast material. The metabolite ratios CHO/CRE, CHO/NAA and NAA/CRE and the peak width at half height of the metabolites were calculated and compared between the pre and post contrast spectra. Measurements showed reduction of the CO/CRE and CO/NAA ratios and of the NAA width after the administration of the contrast material

Introduction: Central cholinergic system plays a key role in control of different brain functions such as learning, memory, attention, locomotion and rewards. Disrupted integrity, regulation or capacity of cholinergic signalling is closely connected with cognitive symptoms of several neurodegenerative and neuropsychiatric diseases, as Alzheimer disease, Parkinson disease, attention deficit hyperactivity disorder (ADHD), depression, schizophrenia and increased distractibility. The major neurotransmitter of cholinergic neurons is acetylcholine (ACh) and regulation of ACh levels is main pharmacotherapeutic approach to the treatment of diseases associated with central cholinergic system. The aim of the thesis was to study the changes of central cholinergic neurotransmission with respect to various aspects of modulation of ACh levels in the brain by controlling its release through M4 muscarinic receptors (MR), its hydrolysis by acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) and after hydrolysis in the synapse, regulation of the uptake of metabolite choline by high affinity choline transporter (CHT). Methods: Here we used telemetry to measure locomotor activity and body temperature in mice with selective deletion of M4 MR (M4KO) and their wild type (M4WT) controls under the basal conditions...

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.

Objective: Previous studies have used acetylcholinesterase (AChE) histochemistry to identify cholinergic nerves in the heart, but this enzyme is not a selective marker for cholinergic neurons. This study maps cholinergic innervation of guinea pig heart using a new antibody to the human high-affinity choline transporter (CHT), which is present only in cholinergic nerves. Methods: Immunohistochemistry was used to localize CHTs in frozen and paraffin sections of heart and whole mount preparations of atrial ganglionated nerve plexus. AChE-positive nerve fibers were identified in sections from separate hearts for comparison. Results: Control experiments established that the antibody to human CHT selectively labeled cholinergic neurons in the guinea pig. CHT-immunoreactive nerve fibers and AChE-positive nerves were very abundant in the sinus and AV nodes, bundle of His, and bundle branches. Both markers also delineated a distinct nerve tract in the posterior wall of the right atrium. AChE-positive nerve fibers were more abundant than CHT-immunoreactive nerves in working atrial and ventricular myocardium. CHT-immunoreactive nerves were rarely observed in left ventricular free wall. Both markers were associated with numerous parasympathetic ganglia that were distributed along the posterior atrial walls and within the interatrial septum, including the region of the AV node. Conclusions: Comparison of labeling patterns for CHT and AChE suggests that AChE histochemistry overestimates the density of cholinergic innervation in the heart. The distribution of CHT-immunoreactive nerve fibers and parasympathetic ganglia in the guinea pig heart suggests that heart rate, conduction velocity, and automaticity are precisely regulated by cholinergic innervation. In contrast, the paucity of CHT-immunoreactive nerve fibers in left ventricular myocardium implies that vagal efferent input has little or no direct influence on ventricular contractile function in the guinea pig.