Capillary electrophoretic (CE) separations were successfully coupled to time-of-flight mass spectrometric (TOFMS) detection for the analysis of three families of biological compounds that act as mediators and/or indicators of disease, namely, catecholamines (dopamine, epinephrine, norepinephrine) and their O-methoxylated metabolites (3-methoxytyramine, norepinephrine, and normetanephrine), indolamines (serotonin, tryptophan, and 5-hydroxytryptophan), and angiotensin peptides. While electrophoretic separation techniques provided high separation efficiency, mass spectrometric detection afforded specificity unsurpassed by other types of detectors. Both catecholamines and indolamines are present in body fluids at concentrations that make it possible for them to be determined by capillary zone electrophoresis coupled to TOFMS without employing any preconcentration scheme beyond sample work up by solid phase extraction (SPE). Using this hyphenated approach, submicromolar levels of catecholamines and metanephrines in normal human urine and indolamines in human plasma were detected after the removal of the analytes from their biological matrices and after preconcentration by SPE on mixed mode cation-exchange sorbents. The CE-TOFMS and SPE methods were individualized for each group of compounds. While catecholamines and metanephrines in urine samples were quantitated using 3,4-dihydroxybenzylamine as an internal standard, deuterated isotopes, considered ideal internal standards, were used for the quantitation of indolamines. Because the angiotensin peptides are present in biological fluids at much lower concentrations than the previous two families of analytes, their analysis required the application of additional preconcentration techniques. In this work, the coupling of either of two types of electrophoretic preconcentration methods - field amplified injection (FAI) and isotachophoresis (ITP) - to capillary zone electrophoresis with both UV and MS detection was evaluated. Using FAI-CE-UV, angiotensins were detected at ~1 nM concentrations. Using similar conditions but TOFMS detection, the detection limits were below 10 nM. ITP was evaluated in both single-column and two-column comprehensive arrangements. The detection limits achieved for the ITP-based techniques were approximately one order of magnitude higher than for the FAI-based preconcentration. While the potential usefulness of these techniques was demonstrated using angiotensins standards, substantial additional research would be required to allow these approaches to be applied to plasma as part of clinical assays.

v anglickém jazyce: Thesis abstract: Background and aims: Both obesity and metabolic syndrome form severe health problems in the whole world. Nevertheless the armament of pharmacotherapy for both diseases remains unsatisfactory. We aimed our work to main organs in risk of the mentioned diseases -liver and visceral fat using hepatocytes and visceral adipocytes as model. We detected 3 main metabolic and signalization activities- glycogenolysis, Nitric oxide (NO) production and transcription of inducible NO synthase (iNOS) in hepatocytes, lipolysis, NO production and iNOS transcription rate in adipocytes. We directed our interest to combination of peroxisome proliferation activator receptor γ (PPARγ) agonist, antagonist and β3 adrenergic agonist in the culture of epididymal rat adipocytes in the first part of our work. While in the second part we investigated the influence of β and α adrenergic mimetics, adrenergic blockers in the culture of rat high glycogen content hepatocytes. Methods: NO production was detected under the active agents treatments by detection of NO oxidative products NO2 and NO3 in media. Glycogenolysis was measured as free glucose rise released by hepatocytes into the media. NOS transcription level was extrapolated after comparative polymerase chain reaction with reverse...

Cannabinoid (CB) receptors have been found in most vertebrates that have been studied. The location of various CB receptors in the body and brain are known, but their physiological functions are not fully understood. The effects CBs have on the cardiovascular system have been of growing interest in recent years. Increasing reports from emergency departments and law enforcement agencies detail acute cardiovascular and psychological effects from synthetic CB intoxication, such as JWH-018. This major health concern is substantiated by governmental agencies like the CDC and NIDA. This pilot study investigates the acute toxic effects of the synthetic CB, JWH-018, on the cardiovascular and neuroendocrine systems in Ictalurus punctatus (channel catfish). Research in organisms besides the traditional mammal models can provide new insights into CB function and physiology. Ictalurus punctatus lend multiple benefits as a model organism that permits researchers to investigate in vivo effects of both cardiovascular and neuroendocrine systems without much influence from traditional sampling methods, and further more provide ample size and tissue to perform specific cardiovascular experiments. Multiple methods were used to assess cardiovascular function and sympathetic nervous system activation. Two different doses, low (500 µg/kg) and high 1,500 µg/kg, of JWH-018 were evaluated in the study. Delivery of JWH-018, via dorsal aorta cannulation, was administered to channel catfish in order to measure cardiovascular functions and sample blood. Plasma levels of the hypothalamus-pituitary-adrenal/interrenal (HPA/I) biomarkers; ACTH, cortisol, epinephrine, and norepinephrine, were measured using ELISAs. Myocardial and neural tissue was collected after the exposures for rt-PCR analysis on β2 adrenergic and glucocorticoid receptor density change. Acute exposure of JWH-018 in undisturbed channel catfish yielded several findings: (1) High dose of JWH-018 was responsible for cardio depressor effects in catfish with a tendency to produce tachycardia, (2) rt-PCR results showed a 2.7 fold increase of glucocorticoid receptor mRNA density in catfish cardiomyocytes when exposed to each dose of JWH-018, (3) Catfish plasma ACTH levels were increased with high doses of JWH-018, while plasma cortisol was increased by low doses. Channel catfish is an excellent animal model to examine the effects of synthetic cannabinoids and cardiovascular function. Acute exposures to high levels of JWH-018 appear to produce cardiovascular dysfunction providing evidence that substantiates emergency department reports, in addition yields novel information about the interaction of CBs exposure and the increase of glucocorticoid receptors levels on cardiomyocytes. The channel catfish is a new animal model that can aid in further investigations of CB exposure and multiple physiological functions for health and toxicology studies. With relatively easy adjustments from this pilot study, the effects on CBs can be monitored on Ictalurus punctatus with confident results concerning human health.

Au cours des vingt dernières années, l’anesthésie régionale est devenue, autant en médecine vétérinaire qu’humaine, un outil essentiel à l’élaboration de protocoles analgésiques péri-opératoires. Parmi l’éventail de techniques mises au point en anesthésie canine, le bloc paravertébral du plexus vertébral (PBPB) et sa version modifiée sont d’un grand intérêt pour toute procédure du membre thoracique, dans sa portion proximale. Toutefois, l’essentiel des données publiées à ce jour provient d’études colorimétriques, sans évaluation clinique, et peu d’information est disponible sur les techniques de localisation nerveuse envisageables à ce site. Notre étude visait à décrire une approche échoguidée du PBPB modifié, puis à caractériser ses paramètres pharmacocinétiques et pharmacodynamiques après administration de lidocaïne (LI) ou lidocaïne adrénalinée (LA). Huit chiens ont été inclus dans un protocole prospectif, randomisé, en aveugle et croisé, réparti sur trois périodes. L’impact pharmacodynamique du bloc effectué avec LI ou LA a été évalué régulièrement pour 180 min suivant son exécution. Le traitement à l’adrénaline n’a pas démontré d’impact significatif (P = 0,845) sur la durée du bloc sensitif, tel qu’évalué par un stimulus douloureux mécanique appliqué aux dermatomes ciblés. À l’opposé, l’atteinte proprioceptive évaluée par la démarche a été trouvée prolongée (P = 0,027) et le bloc moteur mesuré par le pic de force verticale (PVF) au trot sur la plaque de force s’est avéré plus marqué (PVF réduit; P = 0,007) sous LA. À l’arrêt comme au trot, le nadir de la courbe PVF-temps a été trouvé retardé (P < 0,005) et la pente ascendante de retour aux valeurs normales adoucie (P = 0,005). Parallèlement aux évaluations cliniques, des échantillons plasmatiques ont été collectés régulièrement afin de quantifier et décrire le devenir pharmacocinétique de la lidocaïne. Parmi les trois élaborés, un modèle bi-compartimental doté d’une double absorption asynchrone d’ordre zéro a finalement été sélectionné et appliqué aux données expérimentales. Sous LA, la Cmax a été trouvée significativement diminuée (P < 0,001), les phases d’absorption prolongées [P < 0,020 (Dur1) et P < 0,001 (Dur2)] et leurs constantes réduites [P = 0,046(k01) et P < 0,001 (k02)], le tout en concordance avec les effets proprioceptifs et moteurs rapportés. Bien que l’extrapolation du dosage soit maintenant théoriquement envisageable à partir du modèle mis en lumière ici, des études supplémentaires sont encore nécessaires afin d’établir un protocole de PBPB d’intérêt clinique. L’analyse sur plaque de force pourrait alors devenir un outil de choix pour évaluer l’efficacité du bloc dans un cadre expérimental. / Over the last decade, regional anaesthesia has become a gold standard for peri-surgical management in veterinary medicine. Among the many techniques developed for analgesia in dogs, the paravertebral brachial plexus block (PBPB) is of great interest when targeting the proximal half of the thoracic limb. Yet, most available data on this technique is based on colorimetric protocols rather than clinical evaluation, and there are very few published results for PBPB execution using nerve location techniques. Through this work, we wished to describe an ultrasound-guided approach of the PBPB and characterize its pharmacokinetic/ pharmacodynamic parameters when executed with either lidocaine alone (LI) or combined to adrenaline (LA). Eight dogs were included in a prospective, randomised, blinded, crossover protocol performed over three distinct periods. Pharmacodynamic impact of LI and LA was compared for 180 minutes after block administration. No significant difference (P = 0.845) was noted between treatments regarding length of the sensitive block, as evaluated regularly through a mechanical painful stimulus applied to selected dermatomes. On the opposite, gait examination showed a longer proprioceptive deficit using LA (P = 0.027). Motor block measured with dynamic force plate analysis showed a lower peak vertical force with LA than LI (P = 0.007). For both dynamic and static evaluations, nadir was clearly delayed (P < 0.005) and the ascending slope back to baseline significantly softened (P = 0.005) in the LA group. Throughout block execution and evaluation, blood samples were collected regularly in order to quantify and describe lidocaine kinetics. Models where developed and compared. A two-compartment model with dual zero-order absorption processes was selected as the best fit for our experimental data. Cmax proved to be significantly reduced with LA (P < 0.001), thus reducing potential toxicity. Absorption phase was prolonged [P < 0.020 (Dur1) and P < 0.001 (Dur2)] and zero-order absorption constant rates lowered [P = 0.046(k01) and P < 0.001 (k02)] following adrenaline addition, in accordance with the previously noted prolonged gait and motor effects. Though dosage extrapolation is now possible using the model developed and tested here, further studies would be needed to establish a PBPB protocol of more clinical interest. Then, force plate analysis could become a key tool for block quality assessment, as both dynamic and static measurements proved to be the reliable ways to collect ground reaction force (GRF) data.

Modificações na fisiologia do organismo da mulher ocorrem durante a gravidez em conseqüência às alterações hormonais, anatômicas e metabólicas. No sistema circulatório a modificação mais significativa é o aumento do débito cardíaco a partir do primeiro trimestre da gestação. Mulheres portadoras de cardiopatias podem apresentar graves complicações durante o período gestacional devido à inapropriada adaptação à sobrecarga hemodinâmica, mesmo em pacientes consideradas em capacidade funcional favorável, no início da gestação. A literatura carece de estudos dos efeitos dos anestésicos locais com ou sem vasoconstritor utilizados nos procedimentos odontológicos, sobre os parâmetros cardiovasculares de mulheres gestantes portadoras de valvopatias e seus conceptos. A escassez científica fez deste tema nosso objetivo de estudo: avaliar e analisar parâmetros da cardiotocografia, como freqüência cardíaca, motilidade fetal e contrações uterinas e de pressão arterial e eletrocardiográficos da gestante portadora de doença valvar reumática quando submetida à anestesia local com lidocaína, com e sem vasoconstritor, durante procedimento odontológico restaurador. Para tanto, a monitorização ambulatorial da pressão arterial (MAPA) e a eletrocardiografia ambulatorial (Holter) materna, ambas obtidas durante 24 horas e a cardiotografia (CTG) de 31 portadoras de cardiopatia reumática entre a 28ª e 37ª semana de gestação, nos períodos: (1) basal - 60 minutos antes do procedimento para MAPA e Holter e 20 minutos para CTG; (2) procedimento - 56+15,5minutos (média+desvio padrão); (3) pós-procedimento - 20 minutos; e (4) média das 24 horas para freqüência cardíaca e extra-sístoles e média da vigília e do sono para pressão arterial, permitiu a análise da variação desses parâmetros, utilizando-se lidocaína 2% sem vasoconstritor e lidocaína 2% com epinefrina 1:100.000, compondo-as em dois grupos. Demonstrou-se redução significativa nos valores de freqüência cardíaca materna durante o procedimento somente quando comparado aos demais períodos (p<0,001). Quando comparados os dois grupos, não houve diferença (p>0,05). Houve ocorrência de arritmia cardíaca em 9 (29,0%) pacientes, sendo 7 (41,8%) do grupo que recebeu anestesia com adrenalina. A pressão arterial materna não apresentou diferença quando comparamos os períodos ou os grupos (p>0,05). O mesmo ocorreu (p>0,05) nas análises comparativas dos parâmetros fetais obtidos por meio da CTG - número de contrações, nível e variabilidade da linha de base, número de acelerações da freqüência cardíaca fetal e padrão de reatividade fetal. Concluiu-se que o uso da lidocaína 2% associado à adrenalina mostrou-se seguro em procedimento odontológico durante a gestação de mulheres com cardiopatia valvar reumática. / During pregnancy, the organic systems of a woman are subjected to physiological modifications consequential to hormonal, anatomic and metabolical alterations. The most significant modification in the circulatory system is an increased cardiac output from the first three months of gestation. Women with heart disease may present with severe complications during the gestational period, because of inappropriate adaptation of her body to this hemodynamic overload, even those patients who are thought to have an appropriate functional capacity during early pregnancy. There are scant studies in the literature on the effects of local anesthetics, with and without vasoconstrictor, used in dental procedures on the cardiovascular variables of pregnant women with valvar disease, as well as on their concepti. Driven by this shortage, we decided to have this subject studied, by assessing and analyzing cardiotachographic parameters, such as heart rate, fetal motility and uterine contractions, in addition to blood pressure and electrocardiographic variables, in pregnant women with rheumatic valvar disease who undergo local anesthesia with lidocaine, with and without vasoconstrictor, during restorative dental procedure. For this, 31 rheumatic heart disease patients who were in their 28th to 37th week of gestation, had 24-hour ambulatory monitoring of their blood pressure (BP) and Holter electrocardiography (Holter-ECG), and cardiotocography (CTG), performed during: (1) baseline - 60 minutes before the procedure for BP and Holter- ECG monitoring, and 20 minutes before the procedure for CTG; (2) procedure - 56±15.5 minutes (mean±SD); (3) post-procedure - 20 minutes; and (4) mean 24-hour heart rate and extrasystoles measurement, and mean wake and sleeping periods BP monitoring. Variation of the above variables was analyzed in two groups, one with infusion of a 2% solution of lidocaine with vasoconstrictor, and the other with infusion of a 2% solution of lidocaine with epinephrine 1:100.000. The maternal heart rate values obtained during the procedure showed a significant reduction only in comparison with the other time periods (P<0.001). The comparison of the two groups did not reveal any significant difference (P>0.05). Cardiac arrhythmia was detected in 9 (29.0%) patients, 7 of them (41.8%) from the group who received anesthetics with epinephrine. Maternal blood pressure did not show any significant difference neither between time periods, nor between groups (P>0.05). The same occurred in the comparative analysis of the fetal parameters obtained during CTG -number of contractions, level and variability from baseline, number of fetal heart rate accelerations and fetal reactivity pattern. Our conclusion was that the use of 2% solution of lidocaine in association with epinephrine proved safe during dental procedure in pregnant women with rheumatic valvar cardiopathy.

It is a major clinical problem that survival rates after out-of-hospital cardiac arrest have not markedly improved during the last decades, despite extensive research and the introduction of new interventions. However, recent studies have demonstrated promising treatments such as targeted temperature management (TTM) and methylene blue (MB). In our first study, we investigated the effect of MB administered during experi-mental cardiopulmonary resuscitation (CPR) in the setting of postponed hypother-mia in piglets. We set out to study if MB could compensate for a delay to establish targeted TTM. The study demonstrated that MB more than compensated for 30 min delay in induction of TTM. The effect of MB added to that of TTM. The second study examined the effects of TTM and S-PBN on the endothelin system and nitric oxide synthases (NOS) after prolonged CA in a porcine CPR mod-el. The study was designed to understand the cardioprotective mechanism of S-PBN and TTM by their influence on the endothelin system and NOS regulation. We veri-fied for the first time, that these two cardioprotective postresuscitative interventions activate endothelin-1 and its receptors concomitantly with eNOS and nNOS in the myocardium. We concluded that nitric oxide and endothelin pathways are implicated in the postresuscitative cardioprotective effects of TTM. The third study compared survival and hemodynamic effects of low-dose amio-darone and vasopressin to vasopressin in a porcine hypovolemic CA model. The study was designed to evaluate whether resuscitation with amiodarone and vasopressin compared to vasopressin alone would have an impact on resuscitation success, survival, and hemodynamic parameters after hemorrhagic CA. We found that combined resuscitation with amiodarone and vasopressin after hemorrhagic circulatory arrest resulted in greater 3-hour survival, better preserved hemodynamic parameters and smaller myocardial injury compared to resuscitation with vasopressin only. In our fourth study we planned to compare hemodynamic parameters between the treatment group (milrinone, esmolol and vasopressin; MEV) and control group (vasopressin only) during resuscitation from prolonged cardiac arrest in piglets. The study was designed to demonstrate if MEV treatment improved hemodynamics or cardiac damage compared to controls. We demonstrated that MEV treatment reduced cardiac injury compared with vasopressin alone.

Modificações na fisiologia do organismo da mulher ocorrem durante a gravidez em conseqüência às alterações hormonais, anatômicas e metabólicas. No sistema circulatório a modificação mais significativa é o aumento do débito cardíaco a partir do primeiro trimestre da gestação. Mulheres portadoras de cardiopatias podem apresentar graves complicações durante o período gestacional devido à inapropriada adaptação à sobrecarga hemodinâmica, mesmo em pacientes consideradas em capacidade funcional favorável, no início da gestação. A literatura carece de estudos dos efeitos dos anestésicos locais com ou sem vasoconstritor utilizados nos procedimentos odontológicos, sobre os parâmetros cardiovasculares de mulheres gestantes portadoras de valvopatias e seus conceptos. A escassez científica fez deste tema nosso objetivo de estudo: avaliar e analisar parâmetros da cardiotocografia, como freqüência cardíaca, motilidade fetal e contrações uterinas e de pressão arterial e eletrocardiográficos da gestante portadora de doença valvar reumática quando submetida à anestesia local com lidocaína, com e sem vasoconstritor, durante procedimento odontológico restaurador. Para tanto, a monitorização ambulatorial da pressão arterial (MAPA) e a eletrocardiografia ambulatorial (Holter) materna, ambas obtidas durante 24 horas e a cardiotografia (CTG) de 31 portadoras de cardiopatia reumática entre a 28ª e 37ª semana de gestação, nos períodos: (1) basal - 60 minutos antes do procedimento para MAPA e Holter e 20 minutos para CTG; (2) procedimento - 56+15,5minutos (média+desvio padrão); (3) pós-procedimento - 20 minutos; e (4) média das 24 horas para freqüência cardíaca e extra-sístoles e média da vigília e do sono para pressão arterial, permitiu a análise da variação desses parâmetros, utilizando-se lidocaína 2% sem vasoconstritor e lidocaína 2% com epinefrina 1:100.000, compondo-as em dois grupos. Demonstrou-se redução significativa nos valores de freqüência cardíaca materna durante o procedimento somente quando comparado aos demais períodos (p<0,001). Quando comparados os dois grupos, não houve diferença (p>0,05). Houve ocorrência de arritmia cardíaca em 9 (29,0%) pacientes, sendo 7 (41,8%) do grupo que recebeu anestesia com adrenalina. A pressão arterial materna não apresentou diferença quando comparamos os períodos ou os grupos (p>0,05). O mesmo ocorreu (p>0,05) nas análises comparativas dos parâmetros fetais obtidos por meio da CTG - número de contrações, nível e variabilidade da linha de base, número de acelerações da freqüência cardíaca fetal e padrão de reatividade fetal. Concluiu-se que o uso da lidocaína 2% associado à adrenalina mostrou-se seguro em procedimento odontológico durante a gestação de mulheres com cardiopatia valvar reumática. / During pregnancy, the organic systems of a woman are subjected to physiological modifications consequential to hormonal, anatomic and metabolical alterations. The most significant modification in the circulatory system is an increased cardiac output from the first three months of gestation. Women with heart disease may present with severe complications during the gestational period, because of inappropriate adaptation of her body to this hemodynamic overload, even those patients who are thought to have an appropriate functional capacity during early pregnancy. There are scant studies in the literature on the effects of local anesthetics, with and without vasoconstrictor, used in dental procedures on the cardiovascular variables of pregnant women with valvar disease, as well as on their concepti. Driven by this shortage, we decided to have this subject studied, by assessing and analyzing cardiotachographic parameters, such as heart rate, fetal motility and uterine contractions, in addition to blood pressure and electrocardiographic variables, in pregnant women with rheumatic valvar disease who undergo local anesthesia with lidocaine, with and without vasoconstrictor, during restorative dental procedure. For this, 31 rheumatic heart disease patients who were in their 28th to 37th week of gestation, had 24-hour ambulatory monitoring of their blood pressure (BP) and Holter electrocardiography (Holter-ECG), and cardiotocography (CTG), performed during: (1) baseline - 60 minutes before the procedure for BP and Holter- ECG monitoring, and 20 minutes before the procedure for CTG; (2) procedure - 56±15.5 minutes (mean±SD); (3) post-procedure - 20 minutes; and (4) mean 24-hour heart rate and extrasystoles measurement, and mean wake and sleeping periods BP monitoring. Variation of the above variables was analyzed in two groups, one with infusion of a 2% solution of lidocaine with vasoconstrictor, and the other with infusion of a 2% solution of lidocaine with epinephrine 1:100.000. The maternal heart rate values obtained during the procedure showed a significant reduction only in comparison with the other time periods (P<0.001). The comparison of the two groups did not reveal any significant difference (P>0.05). Cardiac arrhythmia was detected in 9 (29.0%) patients, 7 of them (41.8%) from the group who received anesthetics with epinephrine. Maternal blood pressure did not show any significant difference neither between time periods, nor between groups (P>0.05). The same occurred in the comparative analysis of the fetal parameters obtained during CTG -number of contractions, level and variability from baseline, number of fetal heart rate accelerations and fetal reactivity pattern. Our conclusion was that the use of 2% solution of lidocaine in association with epinephrine proved safe during dental procedure in pregnant women with rheumatic valvar cardiopathy.