The influence of count's number in myocardium in the determination of reproducible parameters in Gated-SPECT studies simulated with GATE

Myocardial Perfusion Gated Single Photon Emission Tomography (Gated-SPET) imaging is used for the combined evaluation of myocardial perfusion and left ventricular (LV). The purpose of this study is to evaluate the influence of the total number of counts acquired from myocardium, in the calculation of myocardial functional parameters using routine software procedures. Methods: Gated-SPET studies were simulated using Monte Carlo GATE package and NURBS phantom. Simulated data were reconstructed and processed using the commercial software package Quantitative Gated-SPECT. The Bland-Altman and Mann-Whitney-Wilcoxon tests were used to analyze the influence of the number of total counts in the calculation of LV myocardium functional parameters. Results: In studies simulated with 3MBq in the myocardium there were significant differences in the functional parameters: Left ventricular ejection fraction (LVEF), end-systolic volume (ESV), Motility and Thickness; between studies acquired with 15s/projection and 30s/projection. Simulations with 4.2MBq show significant differences in LVEF, end-diastolic volume (EDV) and Thickness. Meanwhile in the simulations with 5.4MBq and 8.4MBq the differences were statistically significant for Motility and Thickness. Conclusion: The total number of counts per simulation doesn't significantly interfere with the determination of Gated-SPET functional parameters using the administered average activity of 450MBq to 5.4MBq in myocardium.

Avaliação da viabilidade miocárdica pós-enfarte: Gated-SPECT versus Gated-PET

Mestrado em Medicina Nuclear - Ramo de especialização: Tomografia por Emissão de Positrões

The influence of number of counts in the myocardium in the determination of reproducible functional parameters in gated-SPECT studies simulated with GATE

Myocardial perfusion gated-single photon emission computed tomography (gated-SPECT) imaging is used for the combined evaluation of myocardial perfusion and left ventricular (LV) function. The aim of this study is to analyze the influence of counts/pixel and concomitantly the total counts in the myocardium for the calculation of myocardial functional parameters. Material and methods: Gated-SPECT studies were performed using a Monte Carlo GATE simulation package and the NCAT phantom. The simulations of these studies use the radiopharmaceutical 99mTc-labeled tracers (250, 350, 450 and 680MBq) for standard patient types, effectively corresponding to the following activities of myocardium: 3, 4.2, 5.4-8.2MBq. All studies were simulated using 15 and 30s/projection. The simulated data were reconstructed and processed by quantitative-gated-SPECT software, and the analysis of functional parameters in gated-SPECT images was done by using Bland-Altman test and Mann-Whitney-Wilcoxon test. Results: In studies simulated using different times (15 and 30s/projection), it was noted that for the activities for full body: 250 and 350MBq, there were statistically significant differences in parameters Motility and Thickness. For the left ventricular ejection fraction (LVEF), end-systolic volume (ESV) it was only for 250MBq, and 350MBq in the end-diastolic volume (EDV), while the simulated studies with 450 and 680MBq showed no statistically significant differences for global functional parameters: LVEF, EDV and ESV. Conclusion: The number of counts/pixel and, concomitantly, the total counts per simulation do not significantly interfere with the determination of gated-SPECT functional parameters, when using the administered average activity of 450MBq, corresponding to the 5.4MBq of the myocardium, for standard patient types.

Diferenças relacionadas ao sexo nos volumes ventriculares e na fração de ejeção do ventrículo esquerdo estimados por cintilografia de perfusão miocárdica: comparação entre os programas Quantitative Gated SPECT (QGS) e Segami

OBJETIVO: Analisar as diferenças relacionadas ao sexo nas medidas obtidas pelos programas Segami e Quantitative Gated SPECT (QGS). MÉTODOS: Cento e oitenta e um indivíduos assintomáticos sem evidência de cardiopatia foram submetidos a estudos de perfusão miocárdica. O volume diastólico final (VDF), volume sistólico final (VSF) e a fração de ejeção do ventrículo esquerdo (FEVE) foram quantificados pelos programas QGS and Segami para avaliar a influência do sexo, idade, peso, altura, freqüência cardíaca, pressão arterial sistólica, pressão arterial diastólica, índice de massa corporal e área de superfície corporal. RESULTADOS: As médias obtidas com o método QGS foram VDF (mulheres = 68 ml; homens = 95 ml; p < 0,001) e FEVE (mulheres = 66,24%; homens = 58,7%), e com o Segami, VDF (mulheres = 137 ml; homens = 174 ml) e FEVE (mulheres = 62,67%; homens = 58,52%). Foram observadas diferenças significantes entre homens e mulheres no VDF (p < 0,001) e VSF (p < 0,001), que persistiram após o ajuste em relação à área de superfície corporal. CONCLUSÃO: Os volumes ventriculares foram significantemente menores e a FEVE foi significantemente maior em mulheres, de acordo com os programas QGS e Segami.

Valor prognóstico da cintilografia miocárdica de perfusão com tetrofosmin marcado com Tecnécio-99m sincronizada com o ciclo cardíaco (" Gated SPECT" ) na avaliação de pacientes com diabete melito e suspeita clínica de doença arterial coronariana

FUNDAMENTO: A doença cardiovascular é a principal causa de morte em diabéticos, tornando-se primordial a identificação dos indivíduos sob maior risco de eventos cardiovasculares. OBJETIVO: Avaliar o valor prognóstico da cintilografia miocárdica de perfusão com " gated SPECT" em pacientes com diabete melito (DM) e suspeita clínica de doença arterial coronariana. MÉTODOS: Estudo retrospectivo envolvendo 232 pacientes diabéticos submetidos à cintilografia miocárdica com " gated SPECT" . Foram avaliados os parâmetros da cintilografia de perfusão (escores e número de segmentos alterados) e da função ventricular (fração de ejeção, volumes e contratilidade do ventrículo esquerdo). Foram considerados eventos cardiovasculares futuros ocorrência de óbito cardíaco, síndrome coronariana isquêmica aguda, procedimentos de revascularização ou acidente vascular encefálico. Foi realizada a análise uni e multivariada pelo modelo de regressão logística múltipla (p < 0,05). RESULTADOS: Estiveram associados com desfechos futuros na análise univariada: idade (p=0,02); angina de peito (p=0,01); tratamento com insulina (p=0,02); anormalidades na perfusão miocárdica (p<0,0001); número de segmentos envolvidos (p=0,0001); escores de perfusão (p=0,0001); fração de ejeção (p=0,004); volume sistólico final (p=0,03) e achado de alteração segmentar na contratilidade do VE (p<0,0001). Na análise multivariada, o sexo masculino (p=0,007), a idade (p=0,03), a angina (p=0,001), o uso de insulina (p=0,007) e o SDS > 3 (p=0,0001) e o número de segmentos alterados > 3 (p=0,0001) foram preditores de eventos. CONCLUSÃO: A cintilografia miocárdica com " gated SPECT" adiciona informações independentes para a estratificação do risco de eventos cardiovasculares futuros em pacientes com diabete melito e suspeita de doença arterial coronariana.

Fatores biológicos e superestimação da fração de ejeção do ventrículo esquerdo no gated SPECT

FUNDAMENTO: Alguns pacientes apresentam fração de ejeção do ventrículo esquerdo (FEVE) superestimada na cintilografia miocárdica com sincronização eletrocardiográfica (gated SPECT). OBJETIVO: Estabelecer a relação entre fatores biológicos e FEVE superestimada. MÉTODOS: Selecionamos 3.838 pacientes que realizaram gated SPECT entre 20/5/2000 e 16/9/2005, com imagens normais de perfusão e FEVE >50%. Analisamos as variáveis: sexo (29,4% feminino e 70,6% masculino), idade (de 20 a 94 anos - média: 56 anos), peso (de 33,5 a 150 kg - média: 79,6 kg), altura (de 138 a 220 cm - média: 171 cm) e IMC (de 13,9 a 54 - média: 27,2). Em um subgrupo de 1.002 pacientes que realizaram ecocardiograma, incluímos as variáveis diâmetros diastólico (de 36 a 68 mm - média 47,5 mm) e sistólico (de 22 a 41 mm - média 29,8 mm). Dividimos os pacientes em dois grupos: FEVE normal (<80%) e superestimada (>80%). A Razão de Chances (RC) para apresentar FEVE superestimada foi calculada para cada variável por regressão logística. RESULTADOS: Encontramos as seguintes Razões de Chances (p < 0,005): sexo feminino RC = 3,585 (IC95%: 2,745 a 4,683), idade em anos RC = 1,020 (IC95%: 1,011 a 1,029) e altura em cm RC = 0,893 (IC95%: 0,829 a 0,962). O peso e o IMC não se associaram a FEVE significativamente (p>0,2). No subgrupo de 1.002 pacientes, encontramos influência estatisticamente significativa na obtenção da FEVE superestimada para as variáveis diâmetro sistólico, sexo e altura. CONCLUSÃO: Apesar de o diâmetro sistólico influenciar na obtenção da FEVE superestimada, as variáveis sexo e altura apresentam influência independente na superestimação da FEVE pelo gated SPECT.

Função ventricular após cirurgia de revascularização: Gated SPECT comparado à ressonância cardíaca

FUNDAMENTO: A avaliação da função ventricular esquerda pode ser limitada pela movimentação anômala do septo interventricular, freqüentemente encontrada após cirurgia de revascularização miocárdica (CRM). A validação do Gated SPECT como ferramenta para avaliação da função ventricular nesse grupo de pacientes é escassa. OBJETIVO: Investigamos a concordância e a correlação entre a fração de ejeção do ventrículo esquerdo (FEVE), o volume diastólico final (VDF) e o volume sistólico final (VDF), obtidos pela cintilografia de perfusão miocárdica tomográfica sincronizada pelo eletrocardiograma (Gated SPECT), com os mesmos parâmetros medidos pela ressonância magnética cardíaca em 20 pacientes submetidos à revascularização miocárdica. MÉTODOS: A correlação foi medida pelo coeficiente de correlação de Spearman (ρ), enquanto a concordância foi avaliada por meio da análise de Bland e Altman. RESULTADOS: Houve uma correlação boa entre o Gated SPECT e a ressonância magnética cardíaca nos pacientes após CRM em relação à fração de ejeção do ventrículo esquerdo (ρ = 0,85; p = 0,0001), uma correlação moderada para o volume sistólico final (ρ = 0,51; p = 0,02) e uma correlação insignificante para o volume diastólico final (ρ = 0,13; p = 0,5). Os limites de concordância para FEVE, VSF e VDF foram: de -20% a 12%; de -38 a 54ml e de -96 a 100ml, respectivamente. CONCLUSÃO: A fração de ejeção do ventrículo esquerdo obtida pelo Gated SPECT correlaciona-se de modo confiável com a da ressonância magnética em pacientes submetidos à CRM. Os volumes ventriculares, entretanto, não apresentam uma correlação adequada.

Avaliação da perfusão e função miocárdicas em vítimas de escorpionismo utilizando o Gated-SPECT

FUNDAMENTO: O choque cardiogênico e o edema agudo de pulmão são as principais causas de óbito em pacientes com escorpionismo, cujo mecanismo fisiopatológico ainda é controverso. OBJETIVOS: Investigar a correlação entre os distúrbios da perfusão miocárdica e a função contrátil do ventrículo esquerdo, em vítimas de escorpionismo. MÉTODOS: Quinze pacientes submeteram-se à cintilografia de perfusão miocárdica sincronizada com ECG (Gated SPECT), dentro de 72 horas e 15 dias após o acidente escorpiônico. As imagens foram analisadas visualmente por escore semiquantitativo de perfusão (0 = normal, 4 = ausente) e mobilidade (0 = normal, 4 = acinético), utilizando modelo de 17 segmentos. Para cada paciente foram calculados escores somados de perfusão (ESP) e mobilidade (ESM). A fração de ejeção (FEVE) foi calculada por software comercialmente disponível. RESULTADOS: Na avaliação inicial, 12 dos 15 pacientes apresentaram alterações da contratilidade e da perfusão miocárdica. O ESP foi de 12,5 ± 7,3, o ESM de 17,0 ± 12, 8 e a FEVE de 44,6 ± 16,0%. Houve correlação positiva entre o ESP e o ESM (r = 0,68; p = 0,005) e negativa entre o ESP e a FEVE (r = -0,75; p = 0,0021). Os estudos de seguimento mostraram recuperação da contratilidade global (FEVE de 68,9 ± 9,5, p = 0,0002), segmentar (ESM 2,6 ± 3,1, p = 0,0009) e da perfusão (ESP 3,7 ± 3,3, p = 0,0003). A melhora da FEVE correlacionou-se positivamente com a melhora do ESP (r = 0,72; p = 0,0035). CONCLUSÕES: Alterações perfusionais miocárdicas são comuns no envenenamento escorpiônico e correlacionam-se topograficamente com a disfunção contrátil. A recuperação da contratilidade correlaciona-se com a reversibilidade dos defeitos perfusionais. Estes achados sugerem a participação de alterações perfusionais miocárdicas na fisiopatologia desta forma de insuficiência ventricular aguda. (Arq Bras Cardiol 2010;94(4): 444-451)

Effect of rufinamide on gating properties of voltage-gated sodium channel Na(v)1.7

Background: Voltage-gated sodium channels (Nav1.x) are important players in chronic pain. A particular interest has grown in Nav1.7, expressed in nociceptors, since mutations in its gene are associated to two inherited pain syndromes or insensitivity to pain. Rufinamide, a drug used to treat refractory epilepsy such as the Lennox-Gastaut syndrome, has been shown to reduce the number of action potentials in cortical neurons without completely blocking Na channels. Aim: The goal of this study was to investigate the effect of rufinamide on Nav1.7 current. Methods and results: Whole-cell patch clamp experiments were performed using HEK293 cells stably expressing Nav1.7. Rufinamide significantly decreased peak sodium current by 28.3, 21.2 and 12.5% at concentrations of 500, 100 and 50μM respectively (precise EC50 could not be calculated since higher rufinamide concentrations could not be achieved in physiological buffer solution). No significant difference on the V1/2 of voltage-dependence of activation was seen; however a shift in the steady-state inactivation curve was observed (-82.6 mV to -88.8 mV and -81.8 to -87.6 mV for 50 and 100 μM rufinamide respectively, p <0.005). Frequency-dependent inhibition of Nav1.7 was also influenced by the drug. One hundred μM rufinamide reduced the peak sodium current (in % of the peak current taken at the first sweep of a train of 50) from 90.8 to 80.8% (5Hz), 88.7 to 71.8% (10 Hz), 69.1 to 49.2% (25 Hz) and 22.3 to 9.8% (50 Hz) (all p <0.05). Onset of fast inactivation was not influenced by the drug since no difference in the time constant of current decay was observed. Conclusion: In the concentration range of plasma level in human treated for epilepsy, 15 μM, rufinamide only minimally blocks Nav1.7. However, it stabilizes the inactivated state and exerts frequencydependent inhibition of Nav1.7. These pharmacological properties may be of use in reducing ectopic discharges as a causal and symptom related contributor of neuropathic pain syndrome.

Intraobserver and interobserver variability of ascending aorta diameter measurements as assessed by ECG-gated MDCT : automatic versus manual measurements : P15

Purpose: Revolutionary endovascular treatments are on the verge of being available for management of ascending aortic diseases. Morphometric measurements of the ascending aorta have already been done with ECG-gated MDCT to help such therapeutic development. However the reliability of these measurements remains unknown. The objective of this work was to compare the intraobserver and interobserver variability of CAD (computer aided diagnosis) versus manual measurements in the ascending aorta. Methods and materials: Twenty-six consecutive patients referred for ECG-gated CT thoracic angiography (64-row CT scanner) were evaluated. Measurements of the maximum and minimum ascending aorta diameters at mid-distance between the brachiocephalic artery and the aortic valve were obtained automatically with a commercially available CAD and manually by two observers separately. Both observers repeated the measurements during a different session at least one month after the first measurements. Intraclass coefficients as well the Bland and Altman method were used for comparison between measurements. Two-paired t-test was used to determine the significance of intraobserver and interobserver differences (alpha = 0.05). Results: There is a significant difference between CAD and manual measurements in the maximum diameter (p = 0.004) for the first observer, whereas the difference was significant for minimum diameter between the second observer and the CAD (p <0.001). Interobserver variability showed a weak agreement when measurements were done manually. Intraobserver variability was lower with the CAD compared to the manual measurements (limits of variability: from -0.7 to 0.9 mm for the former and from -1.2 to 1.3 mm for the latter). Conclusion: In order to improve reproductibility of measurements whenever needed, pre- and post-therapeutic management of the ascending aorta may benefit from follow-up done by a unique observer with the help of CAD.

Regulation of the voltage-gated sodium channel Nav1.7 by ubiquitin ligase Nedd4-2

Background and aim: Neuropathic pain (NP) is a frequent and disabling disorder occurring as a consequence of a direct lesion of the nervous system and recurrently associated with a positive shift toward nervous system excitability. Peripheral nerve activity is mainly carried by voltage-gated sodium channels (VGSC), with Nav1.7 isoform being an important candidate since loss of function mutations of its gene is associated with congenital inability to experience pain. Interestingly, ubiquitin ligases from the Nedd4 family are well known proteins that regulate the turnover of many membrane proteins such as VGSC and we showed Nedd2-2 is downregualted in experimental models of chronic pain. The aim of this study was to investigate the importance of Nedd4-2 in the modulation of Nav1.7 at the membrane. Methods: In vitro: whole cell patch clamp on HEK293 cell line stably expressing Nav1.7 was used to record sodium currents (INa), where the peak current of INa reflects the quantity of functional Nav1.7 expressed at the membrane. The possibility that Nedd4-2 modulates the currents was assessed by investigating the effect of its cotransfection on INa. Biotinylation of cell surface was used to isolate membrane-targeted Nav1.7. Furthermore, as the interaction between Nedd4-2 and Nav isoforms was previously reported to rely on an xPPxYx sequence (PY-motif), we mutated this latter to study its impact in the specific interaction between Nav1.7 and Nedd4-2. GST-fusion proteins composed of the Nav1.7 c terminal 66 amino acids (wild-type or PY mutated) and GST were used to pull-down Nedd4-2 from lysates. Results: Co-transfection of Nav1.7 with Nedd4-2 reduced the Nav1.7 current amplitude by ~80% (n = 36, p <0.001), without modifying the biophysical properties of INa. In addition, we show that the quantity of Nav1.7 at the membrane was decreased when Nedd4-2 was present. This effect was dependent on the PY-motif since mutations in this sequence abolished the down-regulatory effect of Nedd4-2. The importance of this motif was further confirmed by pull down experiments since the PY mutant completely eliminate the interaction with Nedd4-2. Perspectives: Altogether, these results point to the importance of Nedd4-2 as a Nav1.7 regulator through cell surface modulation of this sodium channel. Further experiments in freshly dissociated neurons from wild type and Scn1bflox/Nedd4-2Cre mice are needed to confirm in vivo these preliminary data.

Intraobserver and interobserver variability of ascending aorta diameter as assessed with ECG-gated MDCT: automatic versus manual measurements

Purpose: Recently morphometric measurements of the ascending aorta have been done with ECG-gated MDCT to help the development of future endovascular therapies (TCT) [1]. However, the variability of these measurements remains unknown. It will be interesting to know the impact of CAD (computer aided diagnosis) with automated segmentation of the vessel and automatic measurements of diameter on the management of ascending aorta aneurysms. Methods and Materials: Thirty patients referred for ECG-gated CT thoracic angiography (64-row CT scanner) were evaluated. Measurements of the maximum and minimum ascending aorta diameters were obtained automatically with a commercially available CAD and semi-manually by two observers separately. The CAD algorithms segment the iv-enhanced lumen of the ascending aorta into perpendicular planes along the centreline. The CAD then determines the largest and the smallest diameters. Both observers repeated the automatic measurements and the semimanual measurements during a different session at least one month after the first measurements. The Bland and Altman method was used to study the inter/intraobserver variability. A Wilcoxon signed-rank test was also used to analyse differences between observers. Results: Interobserver variability for semi-manual measurements between the first and second observers was between 1.2 to 1.0 mm for maximal and minimal diameter, respectively. Intraobserver variability of each observer ranged from 0.8 to 1.2 mm, the lowest variability being produced by the more experienced observer. CAD variability could be as low as 0.3 mm, showing that it can perform better than human observers. However, when used in nonoptimal conditions (streak artefacts from contrast in the superior vena cava or weak lumen enhancement), CAD has a variability that can be as high as 0.9 mm, reaching variability of semi-manual measurements. Furthermore, there were significant differences between both observers for maximal and minimal diameter measurements (p<0.001). There was also a significant difference between the first observer and CAD for maximal diameter measurements with the former underestimating the diameter compared to the latter (p<0.001). As for minimal diameters, they were higher when measured by the second observer than when measured by CAD (p<0.001). Neither the difference of mean minimal diameter between the first observer and CAD nor the difference of mean maximal diameter between the second observer and CAD was significant (p=0.20 and 0.06, respectively). Conclusion: CAD algorithms can lessen the variability of diameter measurements in the follow-up of ascending aorta aneurysms. Nevertheless, in non-optimal conditions, it may be necessary to correct manually the measurements. Improvements of the algorithms will help to avoid such a situation.

Defectos paradójicos en regiones con necrosis miocárdica. análisis mediante gated-spect de perfusión miocárdica.

Objectius: Valorar el significat dels defectes paradoxals (DP) segmentaris (captació en esforç més gran que en repòs) a la gated-SPECT (Single Photon Emission Computer Tomography) de perfusió miocàrdica amb compostos tecneciats en regions amb necrosi miocàrdica. Material i mètodes: Es van revisar 1764 SPECT de perfusió miocàrdica estrès-repòs consecutives de pacients amb infart (IM) previ i es van identificar 117 pacients (6,6%) en els quals els DPs corresponien a una regió amb necrosi miocàrdica. Es van valorar els criteris gammagràfics de viabilitat (intensitat del defecte, valor d'aquesta intensitat ponderat pel nombre de segments compromesos-VIP-, la motilitat i l'engrossiment miocàrdic sistòlic) en els segments d'aquestes regions i les característiques de l'artèria responsable en els 96 pacients en els quals s'havia practicat una coronariografia. Resultats: Es van analitzar 160 necrosis, 125 (75%) paradoxals (67 (54%) en regió AnteroSeptoApical i 58 (46%) en regió InferoLateral) i 35 (25%) no paradoxals (19 (54%) ASA i 16 ( 46%) IL). Els valors de severitat de la necrosi i del VIP van ser inferiors en l'estudi d'estrès respecte al repòs i la diferència d'aquests va ser sempre negativa, evidenciant un comportament gammagràfic paradoxal. Per contra, aquests valors van ser inferiors al repòs respecte a l'estrès en les necrosi no paradoxals posant de manifest un comportament gammagràfic de mínima o nul reversibilitat. Així mateix, es van trobar alteracions lleus en la motilitat i engrossiment de les necrosi paradoxals i moderats en les necrosi no paradoxals. Es van trobar 102 necrosis amb DP i coronariografia dels quals, 84 (88%) tenien permeabilitat a l'artèria responsable de la necrosi i 12 pacients (12%) tenien circulació colateral per compensar l'oclusió de l'artèria responsable. Conclusions: Tots els segments amb DP en regions amb necrosi complien criteris gammagràfics de viabilitat en les imatges d'estrès, considerant aquestes imatges millors per a l'avaluació d'aquest tipus de lesions. El vas responsable de la regió amb DP es trobava permeable o bé presentava circulació colateral evident en els casos en què la coronària es trobava closa.

On the function of proton-gated ion channels ASICs and TRPV1 : a patch-clamp study

AbstractAcidosis is encountered during tissue inflammation and triggers pain in humans. H+-gated ion channels are expressed at high levels in sensory neurons of the peripheral nervous system. Ion channels from two different families present the required pH sensitivity to detect the acidosis associated with peripheral inflammation: Acid-Sensing Ion Channels (ASICs) and the Transient Receptor Potential Vanilloid-1 (TRPV1) channel.ASICs are members of the Degenerin/Epithelial Na+ Channel family of ion channels. Six ASIC subunits have been identified in mammals (ASICla, -lb, -2a, -2b, -3 and -4). ASICs form In-activated voltage-insensitive homo- or heterotrimeric Na+ channels. TRPV1 is a member of the TRP family of ion channels and forms non-selective cation channels that mediate a sustained current. TRPV1 is activated by H+, heat (T>43°C), lipids, capsaicin, voltage and other stimuli. A stimulus can increase TRPV1 response to a different stimulus. For example H+ can shift the capsaicin concentration dependence of TRPV1 to lower values. ASICs and TRPV1 have been shown to be involved in inflammatory pain. Using the patch-clamp technique, we studied different aspects of the function of ASICs and TRPV1 in the physiological context of pain.In the first part of this thesis, we characterize the effect of a temperature increase from 25 to 35°C on the function of ASICs and TRPV1 in transfected CHO cells and primary cultures of rat DRG sensory neurons. ASICs give rise to transient currents while TRPV1 mediates a sustained current. In addition, ASICs and TRPV1 respond to H+ with distinct pH dependences. We assess the relative contribution of ASICs and TRPV1 to H+-evoked electrical signaling in rat DRG neurons and we conclude that ASICs are the most important pH sensors in the pH range 7.4 to 6.0 at 35°C in sensory neurons.ASICs and TRPV1 are expressed in the epithelium lining the lumen of the bladder (urothelium). The Bladder Pain Syndrome/Interstitial Cystitis (BPS/IC) is a painful condition associated with a dysfunction of the urothelial barrier and with inflammation. In the second part of this thesis, we show that human urothelial cells -the cell line TEU2 and primary cultures of human bladder urothelium- express functional ASICs but no functional TRPV1 channels. In addition, we show that the levels of ASIC2 and ASIC3 mRNA are increased in the urothelium of patients suffering from BPS/IC. These data suggest that ASICs are involved in the pathology of BPS/IC.Finally, we demonstrate that APETx2 inhibits the sensory neuron specific voltage-dependent Na+ channel Nav1.8. APETx2 was previously shown to inhibit homo- or heterotrimeric ASIC3- containing channels with IC5o from 0.08 to 1 μΜ. We show that APETx2 also inhibits Nav1.8 with an ICsoof «2.6 μΜ. APETx2 reduces the maximal conductance and induces a depolarizing shift in the voltage dependence of activation of Nav1.8. In current-clamp experiments, APETx2 reduces the number of action potentials (APs) evoked by a current ramp. Nav1.8 mediates most of the current during the AP upstroke and has been shown to be an important mediator of inflammatory pain. The fact that APETx2 inhibits two ion channels involved in inflammatory pain suggests that APETx2 or derivatives may represent novel analgesic compounds.RésuméL'acidose tissulaire est observée durant l'inflammation et entraine la douleur chez l'humain. Des canaux ioniques activés par les protons (H+) sont fortement exprimés dans les neurones sensoriels du système nerveux périphérique. De ceux-ci, les Acid-Sensing Ion Channels [ASICs) et Transient Receptor Potential Vanilloid-1 (TRPV1) présentent une sensibilité adéquate à l'acidité pour servir de détecteurs d'acidose.Les ASICs sont membres de la famille Degenerin/Epithelial Na* Channel. Six sous-unités ASIC ont été identifiées chez les mammifères (ASICla, -lb, -2a, -2b, -3 et -4). Les ASICs forment des canaux sélectifs au Na\ insensibles au voltage et activés par les H+. Les canaux fonctionnels sont des homo- ou hétérotrimères de sous-unités ASIC. TRPV1 est un membre de la famille TRP de canaux ioniques. Les canaux TRPV1 sont activés par les H+, la chaleur (T>43°Ç), les lipides, la capsaicine, le voltage et d'autres stimulus. L'activation de TRPV1 entraine un courant soutenu non-sélectif. Un stimulus peut augmenter la réponse de TRPV1 à un autre stimulus. Les H+ peuvent, par exemple, induire un décalage vers des valeurs plus faibles de la courbe de dépendance à la concentration de TRPV1 pour la capsaicine. Il a été démontré que les ASICs et TRPV1 sont impliqués dans la douleur inflammatoire. En utilisant la technique du patch-clamp, nous avons étudié différents aspects de la fonction des ASICs et de TRPV1 dans des contextes associés à la douleur.Dans la première partie de cette thèse, nous caractérisons l'effet d'une augmentation de température de 25 à 35°C sur la fonction des canaux ASICs et TRPV1, dans des cellules CHO transfectées et dans des cultures primaires de neurones sensoriels (DRG) de rat. L'activation des ASICs entraine l'apparition d'un courant transitoire tandis que l'activation de TRPV1 entraine un courant soutenu. De plus, les ASICs et TRPV1 possèdent des dépendances au pH différentes. Nous évaluons la contribution relative des ASICs et de TRPV1 au signalement électrique induit par les H+ et nous concluons que les ASICs sont les senseurs d'acidité les plus importants dans les neurones sensoriels, dans le domaine de pH de 7.4 à 6.0, à température corporelle.Les ASICs et TRPV1 sont exprimés dans l'épithélium recouvrant l'intérieur de la vessie (l'urothélium). Le Bladder Pain Syndrome/Interstitial Cystitis (BPS/IC) est une condition médicale douloureuse associée à une dysfonction de la barrière urothéliale et à une inflammation. Dans la seconde partie de cette thèse, nous démontrons que des cellules urothéliales (de la lignée cellulaire TEU2) et des cellules provenant de cultures primaires d'épithéliums de vessies humaines expriment des canaux ASIC fonctionnels mais pas de TRPV1 fonctionnels. De plus, nous montrons que le niveau d'expression de ASIC2 et -3 est augmenté dans l'urothélium de la vessie de patients souffrant de BPS/IC. Ces données suggèrent que les ASICs sont impliqués dans la pathologie BPS/IC.Pour finir, nous démontrons que la toxine APETx2 inhibe le canal spécifique aux neurones sensoriels Nav1.8, un membre de la famille des canaux sodiques dépendants du potentiel. Il a été démontré précédemment que la toxine APETx2 inhibe les canaux contenant une ou plusieurs sous-unités ASIC3 avec un ICso entre 0.08 et 1 μΜ. Nous montrons que la toxine APETx2 inhibe Nav1.8 avec un IC50 de «2.6 μΜ. La toxine APETx2 réduit la conductance maximale et induit un décalage de la dépendance au potentiel de Nav1.8 vers des valeurs plus positives. Dans des expériences de courant imposé sur des neurones sensoriels, la toxine APETx2 réduit le nombre de potentiels d'action induits par une rampe de courant. Nav1.8 est responsable de la majeure partie du courant durant la phase ascendante du potentiel d'action et a été démontré comme étant un médiateur important de la douleur inflammatoire. L'inhibition de deux types de canaux, impliqués dans la douleurs inflammatoire, par la toxine APETx2, suggère que cette dernière ou ses dérivés représentent des composés analgésiques prometteurs.