Dissertations / Theses on the topic 'Rat carotid body cells'

A insuficiência cardíaca (IC) é o estágio final de diversas patologias cardíacas e apresenta alta morbimortalidade. Dentre as causas, estão os efeitos cardiotóxicos em pacientes tratados com doxorrubicina (Dox). A fisiopatologia da IC apresenta aumento da atividade barorreflexa e marcada hiperatividade simpática (HS), estado compensatório à redução do débito cardíaco. Porém, a HS prolongada culmina em alterações deletérias para o sistema cardiovascular (SC) com piora do quadro de sintomas. Atualmente muito se discute sobre o papel dos corpúsculos carotídeos (CC) na fisiopatologia da IC devido ao seu reflexo simpatotônico e a melhora de pacientes portadores de IC após a remoção dos CC. O nosso objetivo foi avaliar a influência do CC na evolução da IC induzida pela DOX. Para tal, 35 ratos Wistar machos foram dispostos em 4 grupos: controle Salina (CSAL; n=7) e Controle Dox (CDOX; n=12), Desnervado Salina (DSAL; n=4) e Desnervado Doxo (DDOX; n=12). A desnervação consistiu na ressecção do nervo sinusal bilateral prévia à administração de Dox; a indução da IC ocorreu através de 6 aplicações de Dox, na dose de 2.5mg/kg, pela via IP a cada 4 dias. Após 15 dias do término da indução, os animais foram avaliados pelo ecocardiograma e canulados para registro de pressão arterial invasiva e avaliação hemodinâmica, autonômica, barorreflexa e quimiorreflexa. A análise dos resultados mostra que o grupo CDOX apresentou redução do peso corporal, da sensibilidade baro e quimiorreflexa, hiperatividade simpática acompanhada de redução vagal, redução da morfologia cardíaca associada à disfunção diastólica e sistólica e redução do peso bruto cardíaco e ventricular. A desnervação não foi capaz de reverter os efeitos deletérios causados pela Dox, inclusive a desnervação acentuou a disfunção diastólica e sistólica induzida pela Dox. Concluiu-se que a desnervação carotídea não foi eficiente em melhorar a insuficiência cardíaca induzida pela Dox no modelo experimental proposto
Heart failure (HF) is the final stage of several cardiac pathologies and results in high morbimortality. Among the causes, we can mention the cardiotoxic effects in patients treated with doxorubicin (Dox). The pathophysiology of HF has increased baroreflex activity and marked sympathetic hyperactivity (HS), a compensatory state to the reduction of cardiac output. However, prolonged HS results in worsening of the symptoms. Currently, the role of carotid corpuscles (CC) in the pathophysiology of HF is discussed due improvement of sympathetic reflex presents in patients with HF after CC removal. The objective of this study was to evaluate the influence of CC on the evolution of HF induced by DOX for this method 35 Male Wistar rats arranged in 4 groups: Salina control (CSAL; n = 7) and Dox Control (CDOX; n = 12) Salina Denerved (DSAL; n = 4) and Dox Denerved (DDOX; n = 12). A denervation consisted of bilateral sinus nerve resection prior to Dox administration, induction of HF through 6 Dox applications at a dose of 2.5mg / kg, via IP every 4 days. After 15 days of the end of the induction, the animals were evaluated by echocardiogram and cannulated to record invasive blood pressure and hemodynamic, autonomic, baroreflex and chemorreflex evaluation. Our experiment demonstrated that the CDOX group had reduction of body weight, baro and chemoreflex sensitivity, sympathetic hyperactivity accompanied by vagal reduction, reduction of cardiac morphology associated with diastolic and systolic dysfunction and reduction of gross cardiac and ventricular weight. The denervation is not able to reverse the deleterious effects caused by Dox, including denervation accentuated by Dox-induced diastolic and systolic dysfunction. Based on our results on a carotid denervation it was not effective in improving heart failure induced by Dox

The effects of a variety of agents known to stimulate chemoafferent activity were employed to define in greater detail the stimulus-secretion coupling mechanism in the rat carotid body superfused in vitro. Hypoxia, carbachol and sodium cyanide were independently able to elicit amine release. Hypoxia-evoked release was calcium dependent and was reduced by nitrendipine suggesting the involvement of voltage-dependent calcium channels in the secretory response. The effect of carbachol on catecholamine release was abolished by atropine indicating the presence of muscarinic cholinergic receptors on the Type I cells. Hypoxia-induced catecholamine release was partially blocked by atropine suggesting a possible role for muscarinic receptors in the secretory response. Hypoxia, carbachol and cyanide stimulated the release of 4 5Ca from carotid bodies pre-loaded with 4 5Ca, and the release of 4 5Ca by hypoxia or carbachol could be reduced by atropine and nitrendipine. These results suggest that the mobilization of intracellular calcium pools may also contribute to the secretory response. Carbachol was able to stimulate the efflux of [3H] inositol from pre-loaded carotid bodies and the response was abolished in the presence of atropine or lithium. Cytoplasmic concentrations of IP3 and IP3 significantly increased following stimulation with carbachol and the effect was abolished with atropine. In comparison, hypoxia was unable to induce [3H] inositol efflux and only had a moderate effect on inositol phosphate accumulation. These results suggest that carbachol may control cytosolic calcium via the formation of inositol phosphate second messengers. It is suggested that carbachol and hypoxia stimulate catecholamine secretion by altering the intracellular free calcium concentration in the Type I cells. The major effect of hypoxia was to stimulate the entry of extracellular calcium via the voltage-dependent calcium channels whereas mobilization of intracellular calcium stores was a more important event during carbachol-induced catecholamine secretion.

Hypoxia elicits a carotid body-mediated increase in minute ventilation, called the chemoreflex. Volatile anaesthetics depress this chemoreflex, even at sub-anaesthetic doses. The broad aim of this thesis is to explore how these anaesthetics act on carotid body mechanisms to depress the chemoreflex. The agents studied in this thesis are halothane (a potent depressant of this reflex), isoflurane (a less potent depressant) and sevoflurane (a weak depressant). Intracellular Ca2+ measurements, mitochondrial NADH and potential measurements, and single channel electrophysiology studies were performed on a primary culture of rat carotid body type-1 cells and HEK cells transiently expressing TASK channels. Cells were exposed to hypoxia and/or volatile anaesthetics. The results reveal that on all levels studied, the same order of potency of anaesthetics was observed as seen in human ventilatory studies. Volatile anaesthetics depress the hypoxia evoked Ca2+ entry in the carotid body and increase the activity of K+ background channels both in glomus and HEK cells expressing TASK. Furthermore, on all levels studied, when halothane and isoflurane were applied as a mixture, the effect evoked by the mix was of a lesser magnitude than that of the halothane alone, revealing a novel sub-additive observation, which has not been previously reported in the literature. The anaesthetic action of glomus cells was not exclusive to the TASK channels as application of all three anaesthetics evoked an increase in mitochondrial NADH and caused mitochondrial depolarization in glomus cells. These effects of anaesthetics on mitochondria mimics the effects of hypoxia, the implications of which are discussed in this thesis. Two novel TASK blocking agents A1899 and PK-THPP were able to decrease glomus cell TASK channel activity, even in the presence of a clinically relevant concentration of isoflurane. These agents may be promising as future respiratory stimulants.

Oxygen-sensing cells release neurotransmitters, including serotonin, in response to hypoxia in the blood or surrounding air/water. This stimulates the glossopharyngeal and/or vagal nerves, triggering increased ventilation via the respiratory reflex. In the adult, they are located in the carotid body (glomus cells) and lung epithelia (pulmonary neuroendocrine cells) of amniotes, and in the epithelia of the gills and orobranchial cavity (‘neuroepithelial cells’) of anamniotes. Despite their physiological importance, little is known about the molecular mechanisms of their development, while the evolutionary relationships between the various oxygen-sensing cell types are unknown. The chromaffin cells of the mammalian adrenal medulla are hypoxia-sensitive transiently during neonatal life. Both carotid body glomus cells and adrenal chromaffin cells arise from the neural crest and require the transcription factors Phox2b and Ascl1 for their development. Given these similarities, I aimed to test the hypothesis that the same molecular mechanisms underlie their development. Expression analysis of 13 sympathoadrenal pathway genes throughout chicken carotid body development revealed striking similarities with adrenal chromaffin cell development. Analysis of mouse mutants showed that the transcription factors Hand2, Sox4 and Sox11 are required for carotid body development. In addition, loss of the receptor tyrosine kinase Ret or the transcription factor AP-2β, which significantly affects sympathetic ganglion but not adrenal chromaffin cell development, has no effect on the carotid body. Adrenal chromaffin cells differentiate from neurons that migrate into the adrenal gland from ‘primary’ sympathetic ganglia at the dorsal aorta. Carotid body glomus cells were previously proposed to arise from neuronal “émigrés” from neighbouring ganglia: the superior cervical ganglion in mammals and the nodose ganglion in the chick. However, nodose neurons are considered to be nodose placode-derived. Using electroporation and grafting in the chick, I confirmed that the nodose placode does not contribute to the carotid body, identified a small population of autonomic neural crest-derived neurons in the nodose ganglion, and confirmed the existence of “bridges” of neurons between the nodose ganglion and the carotid body. My data suggest that, like adrenal chromaffin cells, carotid body glomus cells differentiate from autonomic neural crest-derived neurons in nearby ganglia, which migrate into the carotid body primordium and down-regulate neuronal markers. The proposed evolutionary relationship between the carotid body glomus cells and the serotonin-positive neuroepithelial cells of anamniote gills has never been tested. Using vital dye labelling, neural fold grafts, genetic lineage-tracing in zebrafish and analysis of zebrafish mutants lacking all neural crest cells, I found that serotonin-positive cells in the gills and orobranchial epithelia of lamprey (jawless fish), zebrafish (ray-finned bony fish) and frog (anamniote tetrapod) are not neural crest-derived, and hence are not homologous to carotid body glomus cells. Genetic lineage-tracing in mouse and neural fold grafts in chick also confirmed that serotonin-positive neuroendocrine cells in the lung are not neural crest- derived, hence must have an endodermal origin (since the lungs are out-pocketings of the gut). My results suggest that the neuroepithelial cells of anamniotes are not related to carotid body glomus cells, but rather are homologous to the oxygen-sensing cells of the lung. Consistent with this hypothesis, I found that many genes expressed during carotid body development are not expressed by the epithelia of either chick lungs or lamprey gills. Taken together, my data suggest that as air-breathing evolved, gut endoderm- derived cells that originally responded to hypoxia in water were maintained in the lungs to monitor oxygen levels in air, while a population of neural crest-derived chromaffin cells near the pharyngeal arch arteries was recruited to monitor oxygen levels in blood.

This study was designed to examine the influence of Cu deficiency on the binding, uptake, and degradation of apolipoprotein E-free high density lipoproteins (apo E-free HDL) in cultured rat hepatic parenchymal cells. The binding of apo E-free HDL during time course studies was slightly but significantly increased in cells derived from Cu-deficient rats. In saturation studies, the amount of surface-bound apo E-free HDL appeared to be saturable, although no difference was observed between Cu-deficient and adequate animals. The amount of total and specific cell-associated uptake of apo E-free HDL was significantly increased in hepatic parenchymal cells of Cu-deficient animals. The present data suggest that hepatic uptake of the HDL protein moiety may be increased in rats fed a diet deficient in copper.

RESUMO: O corpo carotídeo (CB) é um pequeno órgão sensível a variações na PaO2, PaCO2 e pH. As células tipo I (células glómicas) do corpo carotídeo, as unidades sensoriais deste órgão, libertam neurotransmissores em resposta às variações dos gases arteriais. Estes neurotransmissores atuam quer em recetores pré-sinápticos, localizados nas células tipo I, quer em recetores póssinápticos, localizados nas terminações do nervo do seio carotídeo, ou em ambos. A activação dos recetores pré-sinápticos modula a atividade do corpo carotídeo, enquanto que, a activação dos recetores pós-sinápticos, de carater excitatório, desencadeia um aumento da frequência de descarga das fibras do CSN, com subsequente despolarização dos neurónios do gânglio petroso, e posterior despolarização de um grupo específico de neurónios do centro respiratório central, desencadeando, como resposta final, hiperventilação. Estes recetores pré- e pós-sinápticos podem ser classificados em ionotrópicos ou metabotrópicos, estando os últimos acoplados a adenilatos ciclases transmembranares (tmAC). O mecanismo exato pelo qual as variações dos gases arteriais são detetadas pelo CB não se encontra ainda completamente elucidado, mas tem sido sugerido que alterações nos níveis de cAMP estejam associadas ao mecanismo de deteção de variações de O2 e CO2. Os níveis de cAMP podem ser regulados através da sua via de síntese, mediada por dois tipos de adenilatos ciclases: tmAC sensível aos eurotransmissores e adenilato ciclase solúvel (sAC)sensível a variações de HCO3/CO2, e pela sua via de degradação mediada por fosfodiesterases. A via de degradação do cAMP pode ser manipulada farmacologicamente, funcionando enquanto alvo terapêutico para o tratamento de patologias do foro respiratório (e.g. asma, hipertensão pulmonar, doença pulmonar obstructiva crónica e apneia do sono), que induzem um aumento da actividade do CB.O trabalho descrito nesta dissertação partiu da hipótese de que a actividade do CB é manipulada por fármacos, que interferem com a via de sinalização do cAMP, tendo sido nosso objectivo geral, investigar o papel do cAMP na quimiotransdução do CB de rato, e determinar se a actividade dos enzimas responsáveis pela via de sinalização do cAMP é ou não regulada por variações de O2/CO2. Assim, a relevância deste trabalho é a de estudar e identificar possíveis alvos moleculares (sAC, isoformas de tmAC e PDE) com potencial para serem usados no tratamento de patologias relacionadas com o controlo respiratório. A primeira parte do presente trabalho, centrou-se na caracterização farmacológica da PDE4 no CB e em tecidos não quimiorecetores (e.g. gânglio cervical superior e artérias carótidas), e na observação do efeito de hipóxia aguda na acumulação dos níveis de cAMP, induzidos pelos inibidores de PDE, nestes tecidos. A quantificação de cAMP foi efectuada por técnica imunoenzimática (EIA), tendo sido elaboradas curvas de dose-resposta para os efeitos de inibidores, não específicos (IBMX) e específicos para a PDE2 e PDE4 (EHNA, Rolipram e Ro 20-1724), nos níveis de cAMP acumulados, em situações de normóxia (20%O2/5%CO2) e hipóxia (5%O2/5%CO2). A caracterização das PDE no gânglio cervical superior foi aprofundada, utilizando-se a técnica de transferência de energia de ressonância por fluorescência (FRET) em culturas primárias de neurónios, na presença de inibidores não específicos (IBMX) e específicos para a PDE3 e PDE4 (milrinone e rolipram, respetivamente). Foram igualmente estudadas, através de RT-qPCR, as alterações na expressão de PDE3A-B e PDE4A-D, no gânglio cervical superior, em resposta a diferentes percentagens de oxigénio. Na segunda parte do trabalho investigou-se a via de síntese do cAMP no CB em resposta a variações na concentração de HCO3/CO2. Em concreto, o protocolo experimental centrou-se na caracterização da sAC, dado que a sua actividade é regulada por variações de HCO3/CO2. A caracterização da expressão e regulação da sAC, em resposta a variações de HCO3/CO2 ,foi efectuada no CB e em tecidos não quimioreceptores periféricos (e.g. gânglio cervical superior, petroso e nodoso) por qRT-PCR. A actividade deste enzima foi caracterizada indirectamente através da quantificação dos níveis de cAMP (quantificação por EIA), induzidos por diferentes concentrações de HCO3/CO2, na presença de MDL-12,33-A, um inibidore da tmAC. A expressão das isoformas da tmAC no CB e gânglio petroso foi determinada por RT-qPCR. Adicionalmente, estudámos a contribuição relativa da tmAC e sAC no mecanismo de sensibilidade ao CO2 no CB. Para o efeito foram estudadas as alterações: 1) nos níveis de cAMP (quantificado por EIA) na presença de diferentes concentrações de HCO3/CO2 e ao longo do tempo (5-30 min); 2) na ativação da proteína cinase A (PKA, FRET baseado em sensores) em células tipo I do CB; e 3) na frequência de descarga do CSN (registos) na presença e ausência de ativadores e inibidores da sAC,tmAC e PKA. Por último, foi caracterizada a expressão e actividade da sAC nos quimioreceptors centrais (locus ceruleus, rafe e medula ventro-lateral) através de técnicas de RT-qPCR e EIA. A expressão das isoformas da tmAC foi aprofundada no locus coeruleus através de RT-qPCR. Por fim, comparámos a contribuição da tmAC e sAC nos níveis de cAMP no locus coeruleus em condições de normocapnia e hipercapnia.O nosso trabalho teve os seguintes resultados principais: 1) PDE4 está funcional no corpo carotídeo, artérias carótidas e gânglio cervical superior de rato, embora a PDE2 só se encontre funcional neste último; 2) Os efeitos dos inibidores de PDE nos níveis de acumulação de cAMP foram exacerbados em situações de hipóxia aguda no CB e artérias carótidas, mas foram atenuados no gânglio cervical superior; 3) No gânglio cervical superior, diferentes tipos de células apresentaram uma caracterização específica de PDEs, sugerindo uma subpopulação de células neste gânglio com funções fisiológicas distintas; 4) Embora todas as isoformas de PDE4 e PDE3 estivessem presentes no gânglio, a PDE3a, PDE4b e a PDE4d foram as isoformas mais expressas. Por outro lado, incubações de gânglio cervical superior, em diferentes percentagens de oxigénio, não alteraram (não regularam) significativamente a expressão das diferentes isoformas de PDE neste órgão; 5) a sAC encontra-se expressa e funcional no CB e nos quimiorecetores centrais estudados (locus coeruleus, rafe e medula ventrolateral). A sAC apresenta maior expressão no CB comparativamente aos restantes orgãos estudados, exceptuando os testículos, orgão controlo. Variações de HCO3/CO2 de 0/0 para 24/5 aumentaram os níveis de cAMP no CB e quimiorecetores centrais, tendo sido o aumento mais significativo observado no CB. Concentrações acima dos 24mM HCO3/5%CO2 não induziram alterações nos níveis de cAMP, sugerindo que a actividade da sAC se encontra saturada em condições fisiológicas (normocapnia) e que este enzima não desempenha qualquer papel na deteção de situações de hipercapnia; 6) No CB, a expressão das isoformas tmAC1, tmAC4, tmAC6 e tmAC9 é mais elevada comparativamente à expressão da sAC; 7) Utilizamos diferentes inibidores da tmAC (MDL 12-330A, 500μM, 2’5’-ddADO, 30-300μM, SQ 22536, 200μM) e da sAC (KH7, 10-100μM) para estudar a contribuição relativa destes enzimas na acumulação do cAMP no CB. Tanto a tmAC como a sAC contribuem para a acumulação dos níveis de cAMP em condições de hipercapnia. Contudo, existe um maior efeito destes inibidores nas condições de 12 mM HCO3/2.5%CO2 do que em condições de normocapnia e hipercapnia, sugerindo um papel relevante destes enzimas na atividade do CB em situações de hipocapnia; 8) Não se observaram variações nos níveis de cAMP em resposta a diferentes concentrações de HCO3/CO2 ao longo do tempo (5-30 min). O efeito inibitório induzido por ddADO e KH7 foi sobreponível após 5 ou 30 minutos de incubação em todas as concentrações de HCO3/CO2 estudadas; 9) Por último, verificou-se um aumento na frequência da descarga do nervo do seio carotídeo entre as condições de normocapnia e hipercapnia acídica. Ao contrário do KH7 (10μM), o 2’5’-ddADO reduziu significativamente a frequência de descarga do nervo, quer em condições de normocapnia quer de hipercapnia acídica. Contudo, não se verificou aumento na frequência de descarga do nervo entre normocapnia e hipercapnia isohídrica, sugerindo que a sensibilidade à hipercapnia no CB é mediada por variações de pH. Em conclusão, os resultados decorrentes deste trabalho permitiram demonstrar que, embora os enzimas que medeiam a via de sinalização do cAMP possam ser bons alvos terapêuticos em condições particulares, a sua actividade não é específica para o CB. Os resultados sugerem ainda que o cAMP não é um mediador específico da transdução à hipercapnia neste orgão. Contudo, os nossos resultados demonstraram que os níveis de cAMP são mais elevados em condições fisiológicas, o que sugere que o cAMP possa ter uma função homeostática neste orgão. Por último, o presente trabalho demonstrou que os aumentos de cAMP descritos por outros em condições de hipercapnia, não são observáveis quando o pH se encontra controlado. ------------------ ABSTRACT: The work presented in this dissertation was aimed to establish how specific is cAMP-signaling pathways in the CB mainly in different CO2 conditions and how O2 concentrations alter/drives the manipulation of cAMP signaling in the CB. The experimental studies included in this thesis sought to investigate the role of cAMP in the rat CB chemotransduction mechanisms and to determine whether the enzymes that participate in cAMP signal transduction in the CB are regulated by O2/CO2. We characterized the enzymes involved in the cAMP-signaling pathway in the CB (sAC, tmAC, PDE) under different O2/CO2 conditions. Our results demonstrated that many of these enzymes are involved in CO2/O2 sensing and while they may be useful in treating conditions with alterations in CO2/O2 sensing,they will not be specific to chemoreception within the CB: 1) PDE4 is ubiquitously expressed in CB and non-chemoreceptor related tissues and their affinity to inhibitors change with O2 tensions in both CB and carotid arteries, and 2) sAC and tmAC are expressed in peripheral and central chemo- and non-chemoreceptor tissues and their effect on cAMP levels do not change between normocapnic and isohydric hypercapnic conditions. Our results provide evidence against a specific role of cAMP as a mediator for O2 and CO2 chemotransduction in the rat CB and emphasized the role of pH in CO2 sensitivity of the CB. Furthermore, our results demonstrate that cAMP levels are maintained higher under physiological conditions, supporting recent finding from our lab, which all together suggests that cAMP has a homeostatic function in this organ.

Les corps carotidiens sont des structures bilatérales situées au niveau de la bifurcation de l'artère carotide. Ils sont capables de détecter divers stimuli physiologiques tels que la concentration de gaz dans le sang et la pression sanguine. Suite à la variation de ces paramètres au-delà d’une valeur consigne, le corps carotidien, via un nerf afférent appelé nerf sinuso-carotidien (NSC), déclenche une réponse riposte par des voies nerveuses ou hormonales efférentes. De récentes études ont suggéré l’existence d’une relation entre le système immunitaire et le corps carotidien. Il a en particulier été démontré que le corps carotidien est capable de détecter des effecteurs de l’inflammation tels que les cytokines. En outre, des données in vivo démontrent que la résection bilatérale du NSC diminue la survie suite à un choc endotoxémique. Notre étude a porté sur les capacités immunomodulatrices du corps carotidien. Pour mener à bien cette étude, nous avons utilisé une approche d’électrostimulation du NSC plutôt qu’une une approche pharmacologique moins spécifique. Nous avons en particulier développé une méthode chirurgicale pour isoler le NSC, implanter des électrodes puis stimuler le nerf. Nous avons pu constater que la stimulation électrique du NSC atténuait l'expression des cytokines pro-inflammatoires induites par injection de lipopolysaccharide (LPS), en particuliers le Tumor Necrosis Factor (TNF). Par la suite nous avons pu montrer que l'effet de la stimulation du nerf sinusal de la carotide n’était médié ni par des nerfs sympathiques, ni par des nerfs parasympathiques efférents. En revanche, nous avons découvert que la stimulation du NSC augmentait le taux de corticostérone, une hormone anti-inflammatoire qui active le récepteur des glucocorticoïdes. L’antagoniste du récepteur des glucocorticoïdes, abolit les effets de l’atténuation des cytokines proinflammatoires par l’électrostimulation du NSC. De plus, l’utilisation de souris transgéniques ayant des cellules immunitaires myéloïdes dépourvues de récepteurs aux glucocorticoïdes, a montré que l’effet de la stimulation nerveuse du NSC était également perdu indiquant le rôle crucial joué par ces cellules et ce récepteur. Enfin, nous avons pu montrer que la stimulation du NSC conduisait à l’augmentation de l’activité de décharge spontanée des neurones hypothalamiques évoquant un lien entre le NSC et l’axe hypotalamo-hypophysaire. Au final, ces résultats indiquent que l’électrostimulation du NSC atténue l’inflammation en agissant sur l’hypothalamus qui, en augmentant la sécrétion de glucocorticoïdes, conduit à une inhibition de la sécrétion de cytokines inflammatoires par les cellules myéloïdes. D’un point de vu thérapeutique, nous avons pu mettre en évidence que les souris qui avaient reçu une électrostimulation du NSC avaient un taux de survive plus important après injection d’une dose élevée de LPS. Ces résultats suggèrent que l’électrostimulation du NSC pourrait être un traitement potentiel considéré par la médecine bioélectronique pour les maladies inflammatoires immunomédiées en agissant sur l’axe hypotalamo-hypophyso-surrénalien
The carotid bodies (CB) are located bilaterally at the carotid artery bifurcation. They are polymodal sensors capable of detecting various physiological stimuli – blood gas concentration and blood pressure. The carotid body, via its innervating nerve – the carotid sinus nerve (CSN), signals to the brain to modulate these physiological stimuli through efferent activity. Recent evidence has suggested that there is a relationship between the immune system and the carotid body. It has been shown that the carotid body detects inflammation and functionally responds. Additionally, there is in vivo data, demonstrating that bilateral removal of the CSN decreases survival to endotoxemic shock. We hypothesized that activation of the carotid body would attenuate inflammation. To carry out this study, we electrostimulated the CSN instead of using a non-specific pharmacological approach. A surgical method was developed to isolate the CSN, implant electrodes and then stimulate the nerve. We found that electrical stimulation of CSN attenuated the expression of proinflammatory cytokines induced by lipopolysaccharide (LPS) injection, including Tumor Necrosis Factor (TNF). The mechanism by which electrical activation of the CSN attenuated inflammation was unknown. It was investigated if the effect of CSN stimulation was mediated via efferent parasympathetic or sympathetic nerves - it was found that it was not mediated via these nerves. In contrast, it was discovered, that CSN stimulation increased corticosterone – an anti-inflammatory hormone, which activates the glucocorticoid receptor. An antagonist for the glucocorticoid receptor abolished the attenuation of pro-inflammatory cytokines by CSN electrostimulation. This indicated that corticosterone is the mediator of the effect of CSN stimulation. In addition, we found that the stimulation of the CSN led to the increase of the spontaneous discharge activity of the hypothalamic paraventricular nucleus likening the CSN activity to the hypothalamic-pituitary adrenal axis. Using transgenic mice with no glucocorticoid receptor on their myeloid immune cell, it was found that effect of CSN stimulation was additionally prevented. Finally, we were able to show that the stimulation of the CSN led to the increase of the spontaneous discharge activity of the hypothalamic neurons suggesting a link between the CSN and the hypothalamus-pituitary axis. Overall, these results indicate that electrostimulation of CSN attenuates inflammation by acting on the hypothalamus which, by increasing the secretion of glucocorticoids, leads to an inhibition of the secretion of inflammatory cytokines by myeloid cells. From a clinical perspective, using a high dose of lipopolysaccharide, it was found that mice who had received electrostimulation of the CSN were more likely to survive than control mice. This result is particularly interesting as it demonstrates that electrostimulation of the CSN may be a potential therapeutic in bioelectronic medicine for immune-mediated inflammatory diseases

The carotid body (CB) is a major arterial chemoreceptor containing glomus cells that are activated by changes in arterial blood contents including oxygen. Despite significant advancement in the characterization of their physiological properties, our understanding on the underlying molecular machinery and signaling pathway in CB glomus cells is still limited.

To overcome these limitations, in chapter 1, I demonstrated the first transcriptome profile of CB glomus cells using single cell sequencing technology, which allowed us to uncover a set of abundantly expressed genes, including novel glomus cell-specific transcripts. These results revealed involvement of G protein-coupled receptor (GPCR) signaling pathway, various types of ion channels, as well as atypical mitochondrial subunits in CB function. I also identified ligands for the mostly highly expressed GPCR (Olfr78) in CB glomus cells and examined this receptor’s role in CB mediated hypoxic ventilatory response.

Current knowledge of CB suggest glomus cells rely on unusual mitochondria for their sensitivity to hypoxia. I previously identified the atypical mitochondrial subunit Ndufa4l2 as a highly over-represented gene in CB glomus cells. In chapter 2, to investigate the functional significance of Ndufa4l2 in CB function, I phenotyped both Ndufa4l2 knockout mice and mice with conditional Ndufa4l2 deletion in CB glomus cells. I found that Ndufa4l2 is essential to the establishment of regular breathing after birth. Ablating Ndufa4l2 in postnatal CB glomus cells resulted in defective CB sensitivity to hypoxia as well as CB mediated hypoxic ventilatory response. Together, our data showed that Ndufa4l2 is critical to respiratory control and the oxygen sensitivity of CB glomus cells.

Dissertation

碩士
中原大學
生物醫學工程研究所
106
Photodynamic therapy (PDT) has been widely investigated in recent years in the treatment of cancer and non-cancer diseases. The basis of PDT is the accumulation of photosensitizers in the lesion site and activated by light of suitable wavelength which produced cytotoxic reactive oxygen species to achieve target toxic cytotoxicity. In this study, we first demonstrated that PDT combined with Indocyanine Green (ICG) using 780 nm light emitting diode irradiations could effectively inhibit carotid endothelial hyperplasia. Next, we studied the PDT effect on rat aortic smooth muscle cells (A-10). A-10 cells were incudated with different concentrations of ICG. The inverted microscope observation showed accumulation of green color increased when increasing the concentration of ICG. Highest accumulation was found of 8 h incubated at a concentration of 20 μM. Different concentrations of ICG were incubated with A-10 cells for 8 hours and then submitted to light irradiation (4 J / cm2). The results of cell viability assay showed that the half-inhibitory dose was 7.73 μM. Lactate dehydrogenase (LDH) release analysis revealed that A-10 cells incubated with 9 μM ICG without light irradiation resulted in less than 5% LDH release, which indicted no dark toxicity. While PDT resulted in more than 50% LDH release. LDH release result corresponded to cell viability result. Malondialdehyde test after PDT showed the degree of lipid peroxidation increased. Hochest 33258 fluorescence staining revealed PDT caused chromatin condensation 2 J/cm2 of light irradiation and was more pronounced with increasing light exposure. Forty-five minutes after PDT, Annexin V/Propidium iodide fluorescent staining found that both neciosis and apoptosis of A-10 cells occurred after PDT. Flow cytometry showed that most of the cells were late apoptotic cells. At 12 hours after PDT, a rise in the sub-diploid (Sub-G1) ratio in the cell cycle was observed. Western blotting studies of PARP and Caspase-3 performance revealed that PARP with a molecular weight of 25 kDa had a clear band after PDT; Caspase-3 expression decreased in the PDT group.The results of this study show that ICG mediated PDT can induce apoptosis of A-10 cells and further inhibit carotid artery stenosis in rats.

碩士
國立臺灣大學
藥理學研究所
98
Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) play an important role in the development of restenosis after percutaneous transluminal angioplasty (PTCA). PDGF has been recognized as one of the most important growth factors involved in this process. Compound 5185 [1-benzyl-2-(5-methyl-2-furyl) benzimidazole] is a benzimidazole derivative with anti-thrombotic activities. In this study, we investigated the effects of 5185 on VSMC proliferation, migration in vitro and neointima formation in rat balloon injury model in vivo. Our results demonstrated that 5185 could inhibit serum, PDGF, and U46619-induced VSMC proliferation, and serum or PDGF-induced migration in a concentration-dependent manner. We also confirmed that these effects of 5185 on VSMCs were not due to its cytotoxicity by LDH release assay. In addition, we analyzed the effects of 5185 on cell cycle distribution by PI staining, and a slight accumulation of sub-G1 was observed. Cell cycle associated Cdk2 and Cdk4 activities were inhibited by 5185 in the presence of PDGF stimulation. Furthermore, our data showed that 5185 inhibited PDGF-induced phosphorylation of PDGFRβ, PI3K, P38, PLCγ1 and c-Src in RASMCs, but phosphorylations of ERK1/2, JNK and Akt were not obviously affected. We suggest that the suppression of PDGFRβ downstream signaling transduction pathway may be responsible for the inhibitory effect of 5185 of PDGF-induced VSMC migration and proliferation. Finally, our in vivo results showed that 5185 could significantly inhibit neointima formation of rat carotid artery at a dose of 2mg/kg daily, indicating the therapeutic potential for treating restenosis after arterial injury.

碩士
南臺科技大學
生物科技系
106
About 20% of the global population is affected by allergy, type 1 hypersensitivity, including atopic dermatitis, allergic rhinitis, allergic asthma, anaphylaxis, and food allergy. Cordyceps militaris is a popular traditional Chinese medicinal fungus. Cordyceps militaris has numerous therapeutic values, such as anti-tumor, anti-oxidation and immunomodulatory effects. The proteomics method to search for the ethanol extract of Cordyceps militaris fruit body (CM_EE)-affected genes that might clarify the molecular mechanism underlying IgE-mediated allergic response. We underwent two-dimensional gel electrophoreses (2-DE) to acquire the proteomic profiles of CM_EE-treated and naive rat basophilic leukemia cells (RBL-2H3). The proteome maps of treated basophilic leukemia cell were compared with those of un-treated cell to find the differentially express proteins. Comparative proteomics indicated that 13 dys-regulated proteins were present in CM_EE-incubated RBL-2H3 cells with 10 down-regulated proteins and 3 up- regulated proteins. Ingenuity Pathways Analysis (IPA) analysis indicate that the main functions of these proteins belong to Transcription Regulator, Enzyme, peptidase, and Translation Regulator respectively.