O uso da radiação ionizante em procedimentos médicos está associado a riscos significativos à saúde dos pacientes e da equipe de saúde. Avaliar a segurança e a eficácia aguda da ablação por cateter para tratamento da fibrilação atrial (FA) e arritmias supraventriculares (SVTs), utilizando uma abordagem exclusivamente não fluoroscópica, guiada por eco intracardíaco (ICE) e mapeamento 3D. 95 pts  programados para ablação de FA  ou SVTs . Nove pacientes  tinham marcapasso definitivo ou dispositivos de ressincronização com desfibrilador. Dois sistemas de mapeamento eletroanatômico foram utilizados – CARTO (65%) e NAVx (35%), bem como cateteres de ICE disponíveis – Acunav e ViewFlex. O isolamento das veias pulmonares (VPs), bem como todos os outros alvos que precisavam de ablação em ambos os átrios, foram alcançados e adequadamente visualizados. Não foram observados derrames pericárdicos, complicações trombóticas ou outras intercorrências nesta série. Punções transseptais difíceis (19 pacientes – 20%) foram realizadas sem fluoroscopia em todos os casos. Não foi utilizada fluoroscopia de backup, e nenhum vestuário de chumbo foi necessário. Avaliações detalhadas dos marcapassos após o procedimento não mostraram nenhum dano aos eletrodos, deslocamentos ou mudanças de limiar. Uma estratégia de ablação por cateter sem uso de radiação para FA e outras arritmias atriais é segura e eficaz quando guiada pela utilização adequada do ICE e do mapeamento 3D. Diversos sítios em ambos os átrios podem ser alcançados e adequadamente ablacionados sem a necessidade de fluoroscopia de backup. Não foram observadas complicações. :1015-1026) É amplamente realizado em vários centros ao redor do mundo, dada a crescente prevalência de FA na população e a modesta resposta a medicamentos antiarrítmicos.A ablação por cateter é atualmente o método mais eficaz para o tratamento da fibrilação atrial (FA),6 Esses efeitos são cumulativos, e todos estamos continuamente expostos devido ao uso frequente em métodos de imagem com fins diagnósticos terapêuticos.7Como acontece com a maioria dos procedimentos cardíacos percutâneos, a fluoroscopia tem sido a modalidade primária de imagem para manipular cateteres no espaço vascular e câmaras cardíacas. No entanto, a radiação ionizante tem múltiplos efeitos deletérios tanto para os pacientes quanto para a equipe de saúde envolvida.ALARA ( A s L ow A s R easonably A chieveable) foi proposto para ajustar o uso de radiação ao mínimo necessário para atender aos objetivos.3 Nos últimos anos, vários avanços foram obtidos para reduzir a exposição à radiação durante os procedimentos de ablação por cateter, incluindo a redução do tempo de exposição e das doses de fluoroscopia,9 melhores métodos de barreira, e, especialmente, o uso crescente de outras modalidades de imagem não fluoroscópica – sistemas eletroanatômicos 3D (EA) e ecocardiografia intracardíaca (ICE).Neste cenário, o princípio 10 inteiramente. Relatadas pela primeira vez há cerca de 10 anos,13 estas técnicas “zero-fluoro” estão ganhando popularidade na comunidade eletrofisiológica, pois podem ser tão seguras e eficazes quanto as orientadas pela fluoroscopi.16Estas ferramentas de redução de fluoroscopia têm sido cada vez mais utilizadas no laboratório de eletrofisiologia ao longo dos anos, de tal forma que se tornou possível usá-las para orientar todo o procedimento de ablação e, assim, evitar o uso de raios-XO objetivo deste estudo foi demonstrar a viabilidade e segurança da ablação por cateter para tratamento da FA, flutter atrial e outras taquicardias supraventriculares, sem o uso de fluoroscopia, utilizando exclusivamente o mapeamento eletroanatômico e o ecocardiograma intracardíaco ecocardiografia, em uma série de 95 pacientes consecutivos.Todos os procedimentos foram realizados sob anestesia geral e os acessos venosos profundos foram guiados por ultrassom, de acordo com a necessidade específica do procedimento; usualmente, consistiu em duas ou três punções na veia femoral direita, uma punção na veia femoral esquerda (para o cateter ICE), e uma na veia jugular punção interna . O monitoramento durante o procedimento incluiu eletrocardiograma de 12 derivações e adesivos cutâneos de mapeamento EA A partir da veia femoral esquerda, um cateter ICE (ViewFlex Xtra – Abbott ou Acunav – Biosense Webster) foi avançado para o átrio direito (RA), guiado pela visualização de “espaços livres de eco” no sistema vascular.O cateter ICE foi avançado através da veia ilíaca esquerda, mantendo um “espaço livre de eco” próximo do campo proximal de imagem do ultrassom . Essa técnica permite ao operador discriminar entre o avanço livre através do lúmen vascular e uma resistência ao avanço pela parede vascular quando esse padrão de imagem não é obtido.Quando o caminho até a veia cava inferior não era tão claro ou tortuoso, a técnica de seguir um fio-guia no lúmen vascular, usando guias longas inseridas através da veia femoral esquerda, permitiu avançar o cateter ICE seguindo a imagem da guia no lúmen. É de particular valor em pacientes com veias ilíacas finas, onde o “espaço livre de eco” pode não ser muito claro.Ao chegar à veia cava inferior, é possível apreciar a junção cavo-atrial onde, ao nível do fígado, uma imagem parenquimatosa com as veias intra-hepáticas é visualizada. Neste momento, é essencial identificar e evitar a progressão inadvertida do cateter ICE através das veias hepáticas e direcioná-lo corretamente para o átrio direito (AD), o que pode ser feito com suave deflexão anterior. Uma vez no AD, todas as visualizações “padrão” podem ser obtidas usando a técnica convencional de torque no sentido horário ou anti-horário a partir da “home view”. O cateter é então prolapsado para ventrículo direito usando deflexão anterior, mantendo a válvula tricúspide visível, e imagens do espaço pericárdico são obtidas para excluir qualquer derrame pericárdico de base. De volta ao AD, uma deflexão posterior com um torque suave no sentido horário permite uma visão longitudinal da veia cava superior (VCS). Essa visão permite a visualização adequada de cateteres provenientes de cavas (superior e inferior) e é também a visão padrão para monitorar o avanço das guias e bainhas transseptais   .Um longo fio-guia foi, então, inserido através da veia femoral direita e a progressão suave para o VCS foi confirmada pelo ICE. É possível visualizar o apêndice atrial direito perto do óstio da VCS, e o posicionamento inadvertido do fio-guia nessa estrutura pode, então, ser evitado. Uma longa bainha transeptal foi avançada sobre a guia e colocada na VCS .Quando os cateteres multipolares são inseridos através das veias femorais, usando bainhas curtas, é possível ver o avanço do cateter por mapeamento EA e pelo ICE, até que potenciais elétricos apareçam nos polos distais, confirmando a “chegada” no AD.Quando o sistema CARTO foi utilizado, um cateter de ablação irrigado com sensor de força de contato foi avançado guiado pela visualização de EA e ICE, e um mapa atrial direito limitado foi construído, criando assim uma matriz , e delineando a anatomia septo e SC   . Essa eNos casos de FA, uma vez criada uma geometria rápida do AD (sistema CARTO apenas), dois fios-guia foram inseridos na SVC e o posicionamento adequado confirmado pelo ICE. Duas longas bainhas transseptais (curva fixa e deflectível) foram avançadas sobre o fio até a VCS. É importante ressaltar que a heparina foi dada assim que os acessos venosos foram obtidos, antes de qualquer inserção de cateter, visando um TCA > 350s. Estes níveis eram mantidos até o final da instrumentação do átrio esquerdo através de infusão contínua de heparina e novos bolus, quando necessário.Duas punções transseptais foram realizadas separadamente e guiadas continuamente pelo ICE, descendo até o septo a partir da VCS   . Após cA anatomia do átrio esquerdo e veias pulmonares foi reconstruída por um mapa de alta densidade usando o cateter multipolar   . Em parInfusão de alta dose de Isoproterenol a uma taxa de 20 mcg/10 min foi rotineiramente realizada em busca de focos extrapulmonares indutíveis, que foram mapeados e ablacionados quando presentes. Em pacientes com flutter atrial típico documentado, o cateter de ablação foi trazido de volta ao RA, e uma lesão linear guiada pelo ICE no istmo cavo-tricuspídeo foi realizada. A visualização detalhada ao ICE foi essencial para evitar embaraçar o cateter com os eletrodos de marcapasso, quando presentes. Em anatomias desafiadoras , o ICE é fundamental para garantir o contato adequado com o tecido ao longo da linha.Recuperar o acesso transseptal ao LA, sempre que necessário, foi fácil usando os mesmos sítios de acesso previamente marcados no mapa EA. Durante o procedimento, por segurança, o cateter ICE foi frequentemente prolapsado no ventrículo direito para verificar se havia derrame pericárdico, nos seguintes tempos: (1) no início do procedimento, (2) após as punções transseptais, (3) após o isolamento das veias esquerdas, (4) após o isolamento das veias direitas e (5) ao final do procedimento. O ICE permite também a imediata detecção de trombos, que não são detectados por nenhum outro método de imagem não baseado em ultrassom.Em pacientes com marcapasso, a avaliação do dispositivo foi realizada antes e depois do procedimento para garantir a integridade dos eletrodos.O fechamento do acesso às veias femorais foi feito com suturas em “figura de oito” com Prolene “0” para atingir a hemostasia total. Protamina era usada na dose máxima de 50 mg IV para reversão parcial da anticoagulação. A deambulação foi permitida após 6h, e a anticoagulação oral foi retomada no mesmo dia.Para os casos de SVTs, foi utilizada uma rotina semelhante aos casos de FA. Para facilitar o avanço do cateter multipolar na ausência de bainhas transseptais, foram preferidas bainhas longas que já entregam os cateteres na cava inferior, evitando assim as tortuosidades anatômicas dos vasos femorais e ilíacos.A partir desse local, a progressão para o AD foi marcada pelo aparecimento de eletrogramas atriais e visualização pelo ICE, como descrito. Marcos anatômicos como o feixe de His e SC, veias cavas e apêndice atrial direito foram marcados nos mapas EA, sob orientação do ICE   .Esta série reporta pacientes consecutivos, não selecionados, encaminhados para procedimentos de ablação por cateter para o tratamento de arritmias atriais  realizados sem uso de fluoroscopia, guiados exclusivamente pelo mapeamento eletroanatômico e ICE. O software Excel (versão 2019) foi utilizado para tabulação dos dados. Os principais objetivos são descrever a viabilidade desta estratégia inovadora e demonstrar seu perfil de segurança.2 , e 36% (25 pts) apresentavam cardiopatia estrutural, incluindo doenças valvares reumáticas (3 pts – 3%) e funcionais (8pts – 8%), doença coronária (17 pts – 17%) e pós-cirurgia cardíaca . Os pacientes e as características dos procedimentos estão resumidos na De maio/2019 a dezembro/2019, 95 pacientes  foram submetidos à abordagem “zero fluoro”, com a seguinte distribuição de procedimentos: ablação de FA  ou SVT (26 pts [27%) – 14 reentrada nodal [15%], 6 WPW , 1 taquicardia atrial [1%]). Em pts com FA, a média do volume atrial esquerdo era 36 ± 4 ml/mO protocolo incluía internação hospitalar de 24h em uma unidade com telemetria. Nenhum exame de imagem era necessariamente realizado antes da alta hospitalar. Tomografias computadorizadas não foram realizadas antes e nem no seguimento, enquanto outros métodos de imagem sem radiação foram utilizados a critério do médico responsável.Não foram observados derrames pericárdicos, complicações trombóticas ou outras complicações nesta série. Todos os alvos de ablação em ambos os átrios foram alcançados e adequadamente visualizados. Todas as aplicações planejadas foram realizadas, o que significa que a falta de imagens fluoroscópicas não impediu a entrega de RF a nenhum sítio. Esses locais incluíram o antro das VPs, parede posterior do AE, parede anterior, septo, apêndice atrial esquerdo, apêndice atrial direito, SC, istmo cavo-tricuspídeo, anel mitral e tricúspide, via lenta nodal e crista terminalis (AD). Não foi usado fluoroscopia de backup, e nenhum avental de chumbo foi necessário em qualquer paciente.Punções transseptais difíceis , observado em 19 pacientes (20%), foram administradas sem uso de fluoroscopia em todos os casos. Este é um achado significativo, uma vez que há uma crença comum de que a visualização fluoroscópica transseptal de todo o conjunto bainha-agulha é essencial tanto para a perfuração septal, a penetração no AE e a troca de bainhas sobre guias. Todos esses passos foram claramente visualizados usando o ICE de forma otimizada. O mesmo se aplica à passagem por ramos venosos tortuosos para avançar o cateter ICE – todos os casos foram gerenciados com sucesso sem fluoroscopia por visualização cuidadosa do espaço livre de eco e/ou inserção de fios-guia.Eletrodos de marcapasso definitivo estiveram presentes em 9 pacientes , 7 marcapassos de dupla câmara (DDD) e 2 dispositivos desfibriladores-ressincronizadores com 3 eletrodos . Cinco pacientes (56%) eram dependentes da estimulação devido a bloqueio AV completo sem qualquer ritmo de escape. Em 3 desses casos, foram realizados mapeamento e ablação no AD (istmo cavo-tricuspídeo e flutter relacionado à cicatriz de atriotomia), além de instrumentação do AE e isolamento das VPs. Todos esses casos também foram devidamente concluídos sem fluoroscopia. É importante ressaltar que as avaliações após o procedimento não mostraram nenhum dano, desposicionamento ou alterações de limiar dos eletrodos. Importante salientar que se deve ter cuidado para diferenciar fios-guia dos eletrodos permanentes no ICE.21Essa série de casos destaca a viabilidade, a segurança e a eficácia de uma abordagem “zero- fluoro” para tratar tanto FA quanto diferentes tipos de arritmias atriais, mesmo na presença de eletrodos de marcapasso (mesmo em pacientes dependentes). Nesse caso, é de extrema importância que o mapeamento EA e o ICE sejam usados de forma ótima.Nossa série representa uma experiência pioneira no Brasil e na América Latina usando uma abordagem livre de radiação. Resultou de uma preocupação antiga sobre a necessidade de redução da radiação e da implementação constante de etapas independentes de raios-X ao nosso protocolo de ablação. Já tínhamos experiência significativa no uso do ICE e mapeamento EA em todos os casos de FA, nos últimos 16 anos, o que certamente facilitou nossa curva de aprendizado. Nesse sentido, não foi observado aumento de custo em nossa série, pois exatamente os mesmos cateteres são utilizados nos procedimentos utilizando fluoroscopia.22 compararam, retrospectivamente, a segurança e a eficácia entre dois grupos (60 procedimentos de ablação não fluoroscópica e 60 fluoroscópicos). Não foi observado aumento significativo de complicações ou no tempo de procedimento, com eficácia comparável. O grupo fluoroscópico teve uma exposição média de raios-X de 33 minutos em casos de ablação de FA. Bulava et al.,14 relataram estudo com 80 pacientes randomizados para serem submetidos a isolamento elétrico das veias pulmonares guiado por fluoroscopia ou utilizando apenas ICE e CARTO 3 com cateter de força de contato. Não foi encontrada diferença na sobrevida livre de arritmias após 12 meses. Não foram registradas complicações graves em ambos os grupos. Nessa série, o grupo fluoroscópico teve uma exposição média de 3 min para ablações de FA, mostrando que os operadores já tinham experiência no uso de imagens não fluoroscópicas. Juntos, esses dados sugerem que a adoção de medidas de redução de radiação pode afetar drasticamente a exposição a raios-X mesmo em procedimentos fluoroscopicamente guiados, sem prejuízos na segurança.A capacidade de usar o mapa EA e o ICE para fornecer visualização adequada de cada etapa do procedimento já foi relatada. Razminia et al.,12 relataram uma série de 20 procedimentos consecutivos de ablação de FA sem o uso de fluoroscopia, contando apenas com imagens ICE e o sistema NAVx para criar geometria. Nessa série, a integração da imagem EA com uma tomografia computadorizada de átrio esquerdo adquirida previamente foi utilizada na maioria dos pacientes, exigindo acesso à artéria femoral e mapeamento da raiz aórtica para criar uma fusão confiável das imagens. Novas tecnologias, como cateteres de mapeamento com múltiplos polos de registro e softwares de reconstrução, podem gerar uma geometria com menos trauma, de forma mais rápida e confiável. Esses sistemas permitem um mapa de alta densidade com melhor delineamento de anatomia, comparável a uma reconstrução de tomografia computadorizada, sem a necessidade de exposição à radiação ou acesso arterial. Em nossa série, nenhum paciente foi submetido à tomografia pré-ablação. Além disso, os sistemas EA fornecem orientação da movimentação de cateter permitindo movimentos facilmente reprodutíveis e uma excelente correlação entre torque, deflexão e força de contato.O mapeamento EA é ferramenta fundamental no procedimento, uma vez que fornece uma geometria confiável para orientar a navegação e aplicações de RF, mas pode potencialmente fornecer informações enganosas se não for rigorosamente usada. A descrição inicial de Reddy et al.,Em nosso país, apenas duas empresas fornecem sistemas de mapeamento EA – sistema CARTO 3  e sistema Ensite-NavX . Quando esses dois sistemas foram comparados para mapeamento e ablação (4), os resultados  foram semelhantes. Em nosso estudo, o CARTO 3 foi utilizado em 67,8% dos pacientes e o sistema NavX, em 32,2% de todos os procedimentos   , com re23 relatou, recentemente, 747 punções transseptais sem uso de fluoroscopia, realizadas em 646 pacientes, em 5 centros nos EUA, usando diferentes abordagens para alcançar a fossa oval. O acesso transseptal foi associado a uma baixa taxa de complicações . Em nosso estudo, foram realizadas 142 punções transseptais sem qualquer complicação. É fato que uma curva de aprendizado relativamente curta é necessária para se tornar confortável e proficiente na manipulação do ICE. Mas, definitivamente, dá informações melhores e mais detalhadas do que a fluoroscopia.A visualização do ICE é fundamental em cada passo de uma ablação complexa não fluoroscópica. Com a varredura completa do ICE, todas as etapas podem ser adequadamente monitoradas, até mesmo quando os cateteres saem pela ponta da bainha . Nenhum passo é cego usando essa abordagem, mesmo ao avançar cateteres ou guias no sistema venoso até o coração. A visualização e a canulação do SC são melhores do que com fluoroscopia. Sem mencionar as punções transseptais, que são, indubitavelmente, melhor visualizadas no ICE. Baykaner et al.,15 relataram 5 anos de acompanhamento de ablações sem fluoroscopia em uma série de 500 pacientes. Os procedimentos foram realizados de forma segura e eficaz, com taxas semelhantes de recorrência e complicações quando comparadas com a técnica padrão. Em nossa série, também não observamos nenhuma complicação significativa. À medida que essa técnica se torna a prática padrão para procedimentos ainda mais complexos, como taquicardias ventriculares, um aumento na taxa de complicações poderia ser esperado. Por isso, relatos da segurança e eficácia para os pacientes são extremamente importantes e, associado a um treinamento mais difundido de ICE e mapeamento EA, serão vitais para a adoção em larga escala desses procedimentos na prática clínica.Razminia et al.,24 O engajamento neste campo só precisa de uma equipe motivada com uma mudança de mentalidade. Uma vez feito, é um caminho sem volta. É altamente benéfico para os pacientes que se submetem, frequentemente, a mais de um procedimento ablação e usam outras modalidades diagnósticas ou terapêuticas que utilizam, ao longo de sua vida, radiação  e que, geralmente, ou não são contabilizadas ou são negligenciadas. O risco é cumulativo ao longo do tempo. Temos que ter isso em mente, especialmente, frente ao preocupante crescimento de cânceres, nas estatísticas, e levando em consideração que o impacto pode ocorrer anos após a exposição.Todas as ferramentas necessárias para uma ablação sem radiação já estão disponíveis na maioria dos laboratórios de eletrofisiologia e são familiares para a maioria dos eletrofisiologistas.25 dão uma indicação IIa em centros experientes. Mesmo em casos convencionais de SVTs, onde procedimentos simples com sedação e uso de apenas 2 cateteres são frequentemente utilizados, há valor o uso de ICE e anestesia geral. Ambos contribuem para um procedimento seguro e confortável para pacientes e médicos e adicionam a possibilidade de induzir apneia transitória para melhorar a estabilidade dos cateteres em aplicações próximas ao nódulo AV / feixe de His.Intervenções sem radiação também permitem um tratamento seguro de pacientes grávidas. As diretrizes europeias mais recentemente publicadas pelo ESC, para o tratamento de arritmias supraventriculares,7 São preocupantes os relatos mostrando que 85% dos cânceres cerebrais em médicos intervencionistas ocorrem no hemisfério esquerdo,28 sugerindo uma relação causal entre a exposição ocupacional e os efeitos da radiação (já que o lado esquerdo é sabidamente mais exposto do que o direito). Sem mencionar o considerável benefício de evitar o uso de aventais de chumbo pesados, o que, ao longo do tempo, torna as questões ortopédicas uma ocorrência quase unânime.31 Os autores não podem enfatizar, suficientemente, o alívio que horas de pé sem uso de aventais de chumbo representa.A fluoroscopia zero também é altamente benéfica para a equipe de saúde. Em primeiro lugar, reduzir a exposição à radiação é obviamente desejado para pessoas que têm exposição diária por anos. É muito incômodo os relatos de aumento de até 1% no risco de câncer.“Zero-fluoro” é, então, altamente benéfico para os pacientes e toda a equipe de saúde. Múltiplas exposições à radiação são comuns, na era moderna, com as modalidades de imagem disponíveis. Normalmente, não percebemos a natureza cumulativa de múltiplas exposições e seus potenciais efeitos prejudiciais a longo prazo. Pacientes submetidos à ablação frequentemente tiveram ou terão exposição repetida à tomografia, à fluoroscopia, à angiografia coronária e periférica, bem como a exames nucleares. Um procedimento livre de radiação cujos custos, segurança e eficácia sejam, ao menos, semelhantes à alternativa padrão baseada em fluoroscopia, mesmo na presença de eletrodos permanentes, é, portanto, altamente valioso. Uma equipe motivada com uma mudança de mentalidade é fundamental nesse sentido. É nossa percepção que, após uma curva de aprendizado, na maioria dos casos, a visualização e a manipulação de cateteres são, de fato, mais precisas do que com a fluoroscopia, sem nenhuma parte cega.Relatamos um número, relativamente, pequeno de pacientes sem um grupo controle. Os procedimentos de fluoroscopia zero foram realizados por operadores com grande experiência em mapeamento 3D e ICE, e a reprodutibilidade de nossos resultados por operadores menos experientes pode variar devido a uma curva de aprendizado mais prolongada. No entanto, acreditamos que esses resultados são significativos e representam a base para futuras avaliações sobre a segurança e a eficácia dessas técnicas.Uma estratégia de ablação por cateter sem radiação (“zero-fluoro”) para tratamento de FA e outras arritmias atriais é agudamente segura e eficaz quando guiada pela utilização adequada do ICE e do mapeamento 3D. Diversos sítios em ambos os átrios puderam ser alcançados e adequadamente ablacionados sem a necessidade de fluoroscopia de backup. Não foram observadas complicações. 2 Atrial flutter, and supraventricular tachycardias (SVTs). It is widely performed in various centers around the world, giving the increasing prevalence of AF in the population and the modest response to anti-arrhythmic medications.Catheter ablation is currently the most effective treatment for atrial fibrillation AF,6 These effects are cumulative, and all of us are continuously exposed nowadays due to high usage in diagnostic and therapeutic imaging modalities.7As with most percutaneous cardiac procedures, fluoroscopy has been a primary imaging modality to manipulate catheters in the vascular space and cardiac chambers. However, ionizing radiation has multiple potential deleterious effects for both patients and the healthcare team.ALARA ( A s L ow A s R easonably A chievable) principle has been proposed to minimize radiation use to the minimum needed to meet the objective.3 In recent years, several efforts have succeeded in reducing radiation exposure during catheter ablation procedures, including a reduction in fluoroscopy times and doses,9 better shielding, and especially regarding other non-fluoroscopic imaging modalities – namely 3D electroanatomical systems (EA) and intracardiac echocardiography (ICE).In that regard, the 10 entirely. First reported about 10 years ago,13 those Zero-Fluoro techniques are gaining popularity in the EP community, as they are as safe and as effective as the ones guided by fluoroscopy.16Those fluoroscopy reduction tools have been increasingly used in the electrophysiology (EP) lab over the years, so that it became possible to guide the entire ablation procedure and thus avoid the use of X-rayThe purpose of this study was to demonstrate the feasibility and safety of catheter ablation of atrial fibrillation, atrial flutter, and supraventricular tachycardias without the use of fluoroscopy, using exclusively electroanatomic mapping and intracardiac echocardiography in a series of 95 consecutive patients in a single center.All procedures were performed under general anesthesia, and venous access was obtained using ultrasound guidance, according to the specific need of the procedure, but generally consisted of two or three right femoral vein punctures, one left femoral vein puncture (for the ICE catheter), and one internal jugular vein puncture . Monitoring during the procedure included 12-lead electrocardiogram and EA mapping cutaneous patches .From the left femoral vein, an ICE catheter (ViewFlex Xtra – Abbott or Acunav – Biosense Webster) was advanced to the right atrium (RA), guided by visualization of echo-free spaces in the vascular system.The ICE catheter was advanced through the left iliac vein while keeping an “echo-free space” close to the near-field of the ultrasound image (representing an absence of tissue contact at the tip of the ICE catheter). This technique allows the operator to discriminate between a free-advancement of the ICE catheter tip through the vascular lumen when echo-free space is visible and a palpable resistance to advancement when this image pattern is not obtained.Whenever the path to the inferior vena cava was not so clear, a retained guidewire technique, using long wires from the left femoral vein allowed advancing the ICE catheter following a clear wire image in the lumen. That is of particular value in patients with thin iliac veins, where echo-free space is not very clear.Upon reaching the inferior vena cava, it is possible to identify the cavoatrial junction where, at the level of the liver, and a parenchymatous image with clearly visible intrahepatic veins is appreciated. At this moment, it is essential to identify and avoid the inadvertent progression of the ICE catheter through the hepatic veins and correctly direct it to the right atrium (RA), which can be done with gentle anterior deflection. Once in the RA, all standard views can be obtained using the conventional technique of clockwise or counterclockwise torque from the home view. The catheter is then prolapsed to the right ventricle using anterior deflection, while keeping the tricuspid valve visible, and sectional views of the pericardial space were obtained to rule out any baseline pericardial effusion. Back to the RA, a posterior deflection with a gentle clockwise torque allows a longitudinal view of the superior vena cava (SVC). This view allows adequate visualization of catheters coming from superior veins and is the standard view to start advancing transseptal guidewires and sheaths   .A long guidewire was then inserted through the right femoral vein, and smooth progression to the SVC was confirmed by ICE imaging. It is possible to visualize the right atrial appendage near the SVC ostium, and the inadvertent misplacement of the guidewire in this structure can then be avoided. A long transeptal sheath was advanced over the wire and placed in the SVC .When multipolar catheters are inserted through the femoral veins using short sheaths, it is possible to see catheter advancement using EA mapping and ICE image, until electrical potentials appear in the distal poles, confirming “arrival” at the RA.When the CARTO system was used, an irrigated ablation catheter with contact force sensor was then advanced under EA and ICE visualization and a limited right atrial map was constructed, mostly to create a matrix  and to delineate the septum and coronary sinus (CS) anatomy   . This sIn AF cases, once a limited RA geometry was created (CARTO system only), two guidewires were inserted from the right femoral vein and advanced to the SVC, with adequate positioning being visualized on ICE. Two long transseptal sheaths (fixed curve and deflectable) were advanced over the wire to the SVC. Importantly, heparin was given as soon peripheral access was obtained, before any catheter insertion, aiming at an activated clotting time (ACT) > 350s. Those levels were maintained until left atrial instrumentation ended by continuous infusion and rebolus as needed.Two transseptal punctures were separately performed under ICE visualization, coming down to the septum from the SVC   . After The LA and PV anatomies were reconstructed with a high-density map using the multipolar catheter   . In parHigh-dose Isoproterenol infusion at a rate of 20 mcg/10 min was performed in search of inducible extra-pulmonary foci, which were ablated when present. In patients with documented typical atrial flutter, the ablation catheter was then pulled to the RA, and a linear, ICE guided lesion was performed in the cavotricuspid isthmus (CTI). Detailed ICE visualization was essential to avoid tangling the catheter with pacemaker leads, when present. In challenging anatomies , ICE is critical to ensure adequate tissue contact throughout the CTI.Regaining access to the LA, whenever needed, was easily accomplished using previously tagged transeptal access sites in the EA map. During the procedure, to ensure safety, the ICE catheter was frequently prolapsed to the right ventricle to check for pericardial effusion, at the following times: (1) at baseline, (2) after transeptal punctures, (3) after left PV isolation, (4) after right PV isolation, (5) and at the end of procedure. ICE also allows for immediate detection of clot formation, which cannot be seen with other non-ultrasound imaging modalities.In pacemaker patients, device interrogation was performed before and after the procedure to guarantee lead integrity.Femoral vein access care was performed with figure-of-eight sutures with Prolene “0” to achieve full hemostasis. Protamine at a maximal dose of 50 mg IV was given to allow for partial reversal of anticoagulation. Deambulation was allowed after 6h, and oral anticoagulation was resumed on the same day.For SVT cases, a routine somewhat similar to AF cases was used. To facilitate multipolar catheter advancement in the absence of transseptal sheaths, long sheaths that deliver the catheters in the inferior vena cava (IVC) were preferred, thus avoiding the anatomical tortuosity of the femoral and iliac vessels.Starting from that site, progression to the RA was marked by the appearance of atrial electrograms and ICE visualization, as described. Anatomical landmarks such as His bundle and CS, SVC, IVC, and right atrial appendage ostia were tagged in the EA maps under ICE guidance   .We report a series of consecutive, unselected cases of catheter ablation procedures for the treatment of atrial tachyarrhythmias  performed without fluoroscopy, exclusively guided by ICE and EA mapping. Excel software (2019 version) was used for data tabulation. The main goals are to describe the feasibility of this innovative approach and to show the safety profile of this technique.2 and 36% (25 pts) had structural heart disease, including rheumatic (3 pts - 3%) and other types of valvular disease (8 pts - 8%), coronary artery disease (17 pts - 17%), post-open heart surgery pts . Patients and procedural characteristics are summarized in From May/2019 to December/2019, 95 consecutive patients  referred for ablation underwent the zero fluoro approach, with the following procedure distribution: AF Ablation  or non-AF SVT  typical flutters [5%], 1 atrial tachycardia [1%]). In AF pts, the mean LA volume was 36 ± 4 ml/mThe protocol included an overnight hospital stay in a telemetry bed. No routine imaging was required before hospital discharge. CT scans were performed neither at baseline nor in the follow-up, while other radiation-free imaging methods were used at the discretion of the follow-up physician.No pericardial effusions, thrombotic complications, or other difficulties were seen in these series. All targets in both atria that needed ablation were reached and adequately visualized. All intended ablations were performed, meaning that the lack of fluoroscopic imaging did not hamper RF delivery. Those sites included the PV antra, LA posterior wall, anterior wall, septum, LA appendage, RA appendage, CS, CTI, mitral and tricuspid annulus, slow pathway, and crista terminalis. No backup fluoro was used, and no lead apparel was needed in any patient.Interestingly, difficult transseptal puncture , which occurred in 19 patients (20%), was managed without fluoroscopy use in all cases. This is a significant finding, since there is a common belief that transseptal fluoroscopic visualization of the entire sheath-needle assembly is essential both for septal perforation, penetration, and sheath over the wire exchange. All these steps were clearly visualized using ICE to its best advantage. The same applied to the negotiation of tortuous venous branches to advance the ICE catheter – all cases were successfully managed without fluoroscopy by careful visualization of the echo-free space and guidewire insertion.Permanent pacemaker leads were present in 9 patients , 7 dual chamber (DDD) pacemakers, and 2 CRT-D devices with 3 leads . Five patients (56%) were pacemaker-dependent due to complete AV block without any escape rhythm. In 3 of these cases, RA mapping and ablation (CTI and atriotomy scar-related flutters) were performed on top of LA instrumentation and PV isolation. All these cases were also adequately completed without fluoroscopy. Importantly, device interrogations after the procedure did not show any lead damage, dislocation, or threshold changes. Of note, care must be taken to differentiate the guidewire from lead imaging on ICE.21This series of cases highlights the feasibility, safety, and efficacy of a zero fluoro approach when treating both AF and different types of atrial arrhythmias, even in the presence of pacemaker leads (and even in pacer-dependent patients). For that matter, it is of utmost importance that ICE, and EA mapping be used to their best advantage.Our series represents a pioneer experience in Brazil and Latin America using a radiation-free approach. It resulted from a long-lasting concern about radiation reduction and steady implementation of non-X-ray steps to our ablation protocol. We already had significant expertise from using ICE and EA in every AF case for the last 16 years, which surely made our learning curve easier. In that regard, no increase in costs was seen in our series, as precisely the same catheters were used as in the procedures using fluoroscopy.22 retrospectively compared safety and efficacy between two groups (60 non-fluoroscopic and 60 fluoroscopic ablation procedures). No significant increase in complications or procedure time was observed, with comparable efficacy. The fluoroscopic group had an average X-ray exposure of 33 minutes in AF ablation cases. Bulava et al.14 reported on 80 patients randomized to either fluoroscopically-guided PVI or PVI without fluoroscopy using ICE and the CARTO 3 system with contact force ablation catheters. No difference in arrhythmia-free 12-month survival was found. No severe complications were recorded in either group. In this series, the fluoroscopy group had an average exposure of 3 min for AF ablations, showing that the operators were already experts in the use of non-fluoroscopic imaging. Taken together, these data suggest that the adoption of radiation reduction measures can dramatically affect x-ray exposure even in fluoroscopically-guided procedures, with no safety concerns.The ability to use EAM and ICE to provide adequate visualization of every step of the procedure has already been reported. Razminia et al.12 reported a series of 20 consecutive AF ablation procedures without the use of fluoroscopy, relying only on ICE images and the NavX EA system to create geometry. In this series, EA image integration with a previously acquired left atrium CT scan was used in the majority of patients, requiring femoral artery access and aortic root mapping to create a reliable fusion between aortic anatomy from EA mapping and CT image. New technologies, such as multielectrode mapping catheters and software can provide a less traumatic, fast, and reliable geometry, with a high-density map and better anatomy delineation, comparable to a CT scan reconstruction, without the need to expose the patient to radiation and avoiding arterial access during the procedure. In our series, no patient was submitted to pre-ablation CT scans. Also, the EA systems provide ablation catheter tip color-coding orientation that allows easily reproducible movements and an excellent correlation between torque, deflection, and contact force.EA mapping is a fundamental part of the procedure since it provides a reliable geometry to guide the roving catheter and RF applications but could potentially provide misleading information if not stringently used. The initial description by Reddy et al.4 that the results  from both EA mapping systems were similar. In our study, Carto 3 was used in 67.8% of patients, and the NavX system in 32.2% of all procedures  and Ensite-NavX system . When these two systems were compared for mapping and CTI catheter ablation, Macias et al. showedcedures   , with s23 recently reported on 747 zero-fluoroscopy transeptal punctures, performed in 646 patients in 5 different centers across the US, using different approaches to reach the fossa ovalis. The transseptal access was associated with a low total complication rate (0.7%). In our study, a total of 142 transseptal punctures were performed with no complications. Indeed, a somewhat short learning curve is needed to become comfortable with and proficient in ICE manipulation. But it definitively gives better and more detailed information than fluoroscopy.ICE visualization is critical in every step of a non-fluoroscopic complex ablation. With thorough ICE scanning, all the steps can be adequately monitored, even when catheters come out of the sheath tip . There is no blind step using this approach, even when advancing catheters or wires in the venous system to the heart. CS visualization and cannulation are better than with fluoroscopy, not to mention the transseptal punctures, which are undoubtedly best visualized on ICE. Baykaner et al.15 reported a 5-year follow-up of fluoroless ablations in a series of 500 patients. These procedures were safely and effectively performed, with similar rates of recurrence and complications when compared with the standard technique. In our series, we also did not observe any significant complications. As this technique becomes the standard practice for even more complex procedures, such as ventricular tachycardias, a rise in complications rate could be expected. As such, reports on the safety and effectiveness for the patient are extremely important and will, together with more widespread training in ICE and EA mapping, be vital to large-scale adoption of these procedures in clinical practice.Razminia et al.24 Engagement in this field only needs a motivated team with a change in mindset. Once one does it, there is no way back. It is highly beneficial to patients – who can frequently undergo more than one ablation, usually have other diagnostic or therapeutic modalities that use radiation  over their lifespan, which are usually unaccounted for or neglected. The risk is cumulative over time. We have to keep that in mind, especially when cancer statistics show a worrying steep rise and when the impact can occur years after exposure.All the tools needed for a successful radiation-free ablation are already available in most EP labs and familiar to most EP physicians.25 gives an IIa indication in experienced centers. Even for standard SVT cases, where simple procedures under conscious sedation and using two catheters are frequently performed, it is worthwhile using ICE and general anesthesia. They allow for safe and comfortable procedures for both pts and physicians and add the possibility of using transient apnea to enhance catheter stability when dealing with arrhythmias near the AV node / His bundle.Radiation-free interventions also allow safe ablation treatment of pregnant patients. The most recently published ESC Guidelines for the treatment of supraventricular arrhythmias7 it is uncomfortable to read reports that 85% of brain cancers in interventional physicians occur on the left hemisphere,28 suggesting a causal relation to occupational exposure to radiation effects (since the left side is known to be more exposed than the right). Not to mention the considerable benefit of avoiding using heavy lead aprons, which, over time, makes orthopedic issues an almost unanimous occurrence.31 The authors cannot stress enough the massive relief that standing hours without having to wear heavy lead aprons represents.Zero fluoroscopy is also highly beneficial to the health care team. First, reducing the radiation exposure is obviously desired for people who have been exposed daily for years. It is a matter of concern to read reports of an increase of up to 1% in one’s lifetime risk of cancer;It is also highly beneficial to patients and all the healthcare team. Multiple exposures to radiation are common in the modern era, with the readily available imaging modalities. We usually do not realize the cumulative nature of multiple exposures and their potential detrimental effects over the long term. Patients undergoing ablation not uncommonly have had or will have repeated exposure to CT, fluoroscopy, coronary and peripheral angiography, as well as nuclear scans. A radiation-free procedure with at least similar costs, safety, and effectiveness as the standard fluoro-based alternative, even when additional hardware is implanted in the heart, is thus highly valuable. A motivated team with a mindset change is critical in that regard. It is our perception that, after a learning curve, in most instances, the visualization and manipulation are indeed more precise than that with fluoroscopy, without any blind parts.We report on a relatively small number of patients and lack a control group. Zero fluoroscopy procedures were performed by operators with a large experience in ICE and 3D mapping, and the reproducibility of our results by less experienced operators may vary due to the need for a steeper learning curve. However, we believe that these results are meaningful and represent the basis for further evaluations about the safety and efficacy of these techniques.A radiation-free (fluoroless) catheter ablation strategy for AF and other atrial arrhythmias is acutely safe and effective when guided by adequate ICE and 3D mapping utilization. Multiple different bi-atrial sites could be reached and adequately ablated without the need for backup fluoroscopy. No complications were seen.


Pennisetum Rich. or following recent taxonomic insights Cenchrus L. is a genus with some 120 species worldwide, especially in warm areas. The genus includes some crops, some ornamentals but mostly species that are considered weedy. The name of one of the weedy species Pennisetum setaceum (Forssk.) Chiov. is also found on labels of ornamental grasses as P. setaceum “Rubrum.” It has been debated to belong to a species on its own Pennisetum advena Wipff & Veldkamp or Cenchrus advena (Wipff & Veldkamp) Morrone, only known from cultivation, whereas others still adhere to a broader species concept of P. setaceum. The recent inclusion of P. setaceum on the EU List of Union concern has revitalized the discussion on this issue for commercial reasons. Based on a morphological and molecular comparison  of the type specimen of P. advena, five of its “cultivars” in trade and collections of P. setaceum from different regions of the world we conclude that plants currently in trade in Western Europe belong to a separate species P. advena. A drooping inflorescence is consistent as is the difference in width of the leaf blade, the leaf blade being flat or involute, the central vein being swollen or not, and the length of the stipe being 0.3–1.1 mm in P. advena and 1.1–3.1 mm in P. setaceum. On the chloroplast markers rbcL and trnH‐psbA, the species consistently differ in 2 and 4 base pairs, respectively. On the nuclear ITS sequence, there is only 90% overlap between the two species. This justifies these ornamentals to be excluded from the List of Union concern of EU regulation 1143/2014. Pennisetum setaceum on the EU list of Union concern has revitalized the discussion on this issue for commercial reasons. Based on a morphological and molecular comparison  of the type specimen of Pennisetum advena, five of its “cultivars” in trade and collections of P. setaceum from different regions of the world we conclude that plants currently in trade in Western Europe belong to a separate species P. advena.The proper identity of ornamental Pennisetum plants traded as “Rubrum” has been debated. The recent inclusion of  Naturalized P. setaceum plants were collected from the Canary Islands and in Catalonia, Spain . Ornamental P. setaceum were collected in New Zealand  and cuttings of P. advena intercepted in a mislabeled commercial import from China .For the present detailed study, plants listed in Table 2.2Of all living material height of the plant including inflorescence was measured, color of the stem, width, length and color of leaves; color and length of inflorescence; length of spikelet; texture of axis of inflorescence; length of stipe; number of spikelets per fascicle; and color, length and presence of long hairs on bristles. For all herbarium specimens, macromorphological measurements were similar. All measurements are used to build an interactive image‐driven key using LUCID software .2.3Genomic DNA was isolated from approximately 100 mg plant material with the DNeasy Plant Mini Kit  using the TissueLyser procedure and eluted with 50 μl prewarmed (65°C) AE buffer. DNA was stored at −20°C until use.2.4TM Mix  and 2 μl genomic DNA. The cycle conditions for rbcL and trnH‐psbA loci were as follows: 5 min at 95°C, followed by 5 cycles of 30 s at 94°C, 30 s at 45°C, 30 s at 72°C and 35 cycles of 30 s at 94°C, 30 s at 50°C, 30 s at 72°C and a final extension for 10 min at 72°C. The cycle condition for ITS locus was as follows: 5 min at 95°C, followed by 40 cycles of 30 s at 94°C, 30 s at 52°C, 100 s at 72°C, and a final extension for 10 min at 72°C.PCRs for the chloroplast rbcL gene and trnH‐psbA intergenic spacer and nuclear ITS  loci were performed in 25 μl reaction mixes containing 200 nmol/L of either primers rbcL‐a F and rbcLa SI_Rev, trnH2, and psbAF or ITS5 and ITS4  preceding bidirectional cycle sequencing with the BigDye Terminator v1.1 Cycle Sequencing Kit  using amplification primers as sequencing primers in separate reactions according to the manufacturer's instructions. Cycle sequence products were purified with the DyeEx 2.0 Spin Kit  and sequenced using a 3500 Genetic Analyzer . Consensus sequences were generated from an assembly with trace files from both Sanger sequencing runs in Geneious R10 . Amplification primer sequences were trimmed in the assembly, and when needed, additional trimming was performed to obtain high‐quality (PHRED >30) consensus sequences.2.5® Ultra DNA Library Prep Kit for Illumina  was used to process the samples. Fragmentation of the DNA using the Bioruptor Pico , ligation of sequencing adapters, and PCR amplification of the resulting product were performed according to the procedure described in the NEBNext Ultra DNA Library Prep Kit for Illumina Instruction Manual. Reads were reference assemble to the rbcL, trnH‐psbA spacer, and ITS sequences of a Pennisetum orientale specimen (NPPO‐NL 6148331) using CLC genomic workbench v10 (Qiagen) with default settings. Variants were detected by using the Basic Variant Detection tool in CLC genomic workbench v10 with default settings.Genomic DNA was Illumina sequenced (PE150) with the NextSeq 500 V2 platform with minimal 2 Gb output per sample. The NEBNext33.1https://keys.lucidcentral.org/keys/v3/pennisetum/en/index.html.The analysis of all living material and the herbarium collections of the wild samples and cultivars belonging to the 10 species can be found in Costerus  and CostP. advena and P. setaceum.For this paper, we focus on the distinguishing characters between P. setaceum being rarely branched as opposed to P. advena and the inflorescence of the latter being flexuous and drooping as opposed to rigid in P. setaceum. Both characters are added to the attractiveness of the species. Leaf characters in addition to the color of P. setaceum never being purplish are the consistently larger width and flatness of the blade in P. advena as well as the more prominent thickened central vein and involute leaf in P. setaceum. These character differences also apply to the ornamental P. setaceum collected in New Zealand. More detailed measurements on the inflorescence and leaves of P. setaceum. P. advena and cultivars are given in Table Most obvious morphological characters when judging its ornamental value are the culm of 3.2P. advena and P. setaceum can be distinguished from each other. For rbcL, there are two nucleotide (nt) differences, and for trnH‐psbA region, there are four nt differences between P. advena and P. setaceum. All five cultivars could be matched to the type specimen of P. advena. Interestingly, the trnH‐psbA region of P. setaceum sample “Simons 2006” could not be bidirectionally sequenced by Sanger technology. Illumina sequence data revealed that this problem was caused by the presence of two different variants with a single indel. Each of these variants was present in equal amounts.The results of the sequence analysis for the chloroplast rbcL gene and trnH‐psbA intergenic spacer as well as for the nuclear ITS  sequence are summarized in Table P. advena (n = 3) and P. setaceum (n = 2) specimens. Illumina sequencing revealed that in each of the specimens, two variants of the ITS sequence were present. For P. advena, the ratio between these two variants was for each specimen approximately 55% for variant aI and 45% for variant aII. For P. setaceum sample “Simons 2006,” the ratio between variants sI and sII was approximately 70% and 30%, while for P. setaceum sample “Mooney 9419,” this was the other way around (30% sI and 70% sII). Nevertheless, P. advena and P. setaceum could be distinguished based on their ITS sequences as they are only 90% identical. The cultivars “Fireworks,” “Rubrum,” and “Summer Samba” contained both variants I and II of the P. advena ITS sequences. Based on all three loci , these three cultivars could therefore be matched to P. advena.Sanger sequencing of the ITS region revealed the presence of SNPs in both P. advena (aI and aII) and variant I of P. setaceum (sI). For “Cherry Sparkler,” the ratio between the different variants was approx. 45% (aI), 15% (aII), and 40% (sI) and for “Sky Rocket” 20% (aI), 50% (aII), and 30% (sI).The ITS sequences of the cultivars “Cherry Sparkler” and “Sky Rocket” could not be obtained by Sanger sequencing. Illumina sequencing of these cultivars revealed that both cultivars contained ITS variants I and II of 4Pennisetum advena and P. setaceum should be addressed as Cenchrus species, following recent molecular and phylogenetic studies on the genera Cenchrus and Pennisetum . Descriptive aspects and a comprehensive bibliography of relevant cultivars are already dealt with in the recommendation for Pennisetum by Val’Hor  and wild plants in Ethiopia (P.C.M. Jansen 3946). Drooping inflorescence is consistent as is the difference in width of the leaf blade, the leaf blade being flat or involute, the central vein being swollen or not, and the length of the stipe being 0.3–1.1 mm in P. advena and 1.1–3.1 mm in P. setaceum.Distinguishing morphological characters for Veldkamp . Some reP. advena compared with P. setaceum would be a major criterion to justify it not to be regulated. Simpson and Bashaw (P. advena (purple P. setaceum) rarely sets seed under field conditions and does not behave as an apomictic species nor is the plant capable of selfing as opposed to P. setaceum. Chilling injury studies point to hardiness issues for P. advena when temperatures drop below 5°C for 2 weeks  with other Pennisetum, Cenchrus, and Setaria species, which makes identification based on these two loci alone difficult. Furthermore, GenBank contains a few accessions for P. setaceum (or Cenchrus setaceus), but none for P. advena. In addition, the rbcL and trnH‐psbA sequences (GenBank acc. GU135184 and GU135350) for the Cenchrus setaceus voucher J.R. Abbott 24732 (FLAS) are identical to the rbcl and trnH‐psbA sequences of P. advena from this study, suggesting a misidentification.Sequences from the chloroplast loci rbcL and trH‐psbA as well as the nuclear loci ITS showed enough resolution to distinguish P. advena and P. setaceum each contained two variants of the ITS sequences with 2 and 3 SNP, respectively. Most likely these variants are caused by their polyploid nature, as P. setaceum for instance is known to be hexaploid ; Data curation ; Formal analysis (supporting); Investigation ; Supervision (lead); Writing‐original draft ; Writing‐review & editing . Maarten Costerus: Conceptualization ; Data curation ; Formal analysis (lead); Writing‐original draft ; Writing‐review & editing . Marcel Westenberg: Data curation ; Formal analysis ; Writing‐original draft ; Writing‐review & editing .


Within the Late Pleistocene, terminations describe the major transitions marking the end of glacial cycles. While it is established that abrupt shifts in the ocean/atmosphere system are a ubiquitous component of deglaciation, significant uncertainties remain concerning their specific role and the likelihood that terminations may be interrupted by large-amplitude abrupt oscillations. In this perspective we address these uncertainties in the light of recent developments in the understanding of glacial terminations as the ultimate interaction between millennial and orbital timescale variability. Innovations in numerical climate simulation and new geologic records allow us to highlight new avenues of research and identify key remaining uncertainties such as sea-level variability. One of the most remarkable observations made by Hays et al. was that the dominant period of climate variability over the last half million years or so  is close to 100 kyr  the question remains as to why glacial terminations should occur every ~100 kyr and not more frequently as they did during the early Pleistocene6. A clue to this question lies in the asymmetric nature of records of benthic foraminiferal δ18O  forcing to which they respond in a non-linear way  variability have called on the long-term build-up of continental ice sheets, and their propensity to collapse beyond some critical threshold, in order to explain the long-lived nature of late Pleistocene glacial cycles e.g. refs. 14  and augmented to include a link to atmospheric CO2 by Broecker and Denton16. The most recent incarnation of this idea was summarised by Denton et al.17 as a sequence of events initiated by the inflow of freshwater to the surface North Atlantic  suppressing ocean circulation in the Atlantic and ultimately causing the release of CO2 to the atmosphere. At some critical point, the rise in CO2 would pass a threshold beyond which interglacial conditions could be stabilized and maintained19.The specific suggestion that abrupt reorganisations of the ocean-atmosphere system might play an active role in the mechanism of termination was introduced by Mix et al.16 original proposition, with several independent lines of proxy evidence emphasising the ubiquitous association between glacial termination and millennial-scale oscillations in ocean circulation, in particular the Atlantic Meridional Overturning Circulation, AMOC27. Moreover, ice-core measurements and quantitative carbon cycle models support an active role for abrupt shifts in ocean circulation within the mechanism of glacial termination. For example atmospheric CO2 measurements in Antarctic ice cores combined with proxy reconstructions in marine sediments suggest that CO2 rises on a millennial timescale whenever Atlantic Ocean circulation is in a substantially weakened state30 with additional century-scale increases  associated with abrupt transitions between weak and strong modes of ocean circulation32. Modelling studies further suggest that such changes in CO2 may be associated mechanistically with the corresponding variations in ocean circulation, either directly or indirectly through e.g. biological activity39.A number of recent studies provide evidence to support Broecker and Denton’s17 provides an appealing explanation for the magnification of insolation forcing related to deglaciation it leaves open several questions related to the interplay between millennial and orbital-timescale changes and the anatomy of terminations themselves, for example:Is the temporal correspondence between abrupt shifts in ocean circulation and glacial termination merely coincidence or are there systematic interactions between variations on these timescales that necessarily come into play during deglaciation?How do we explain the occurrence of terminal interruptions like the Bølling-Allerød/Younger Dryas (B/A-YD) oscillation during Termination 1 (T1) while other terminations proceed apparently without such interruption?2 while others (notably the most recent) do not?Why do some terminations end with such high levels of atmospheric COHowever, while the simple model described by Denton et al.2.Below we address each of these questions in turn, highlighting new results and outstanding uncertainties requiring future research focus. We begin by investigating the occurrence of millennial-scale climate variability with respect to orbital-timescale changes in ice volume and atmospheric COBox 1 Glossary of key termsLinear system (Panel a): The equilibrated system response will be directly proportional to a change in the control parameter Non-linear system (Panel b): The equilibrated system response includes non-linear behaviour i.e. the response to a finite change in the control parameter depends on the location within the parameter space.Hysteresis (Panel c): The response to a finite change in the control parameter depends on the location within the parameter space and different solutions can coexist (e.g. bistability) for a single value of the control parameter. Hence depending on the starting point different states can be attained for the same forcing. In this case a transient change in the control parameter, or e.g. a one-time input of freshwater, can have a lasting impact.Stability (Panel c): A system may undergo changes in structural stability within the parameter space and alter e.g. from monostability to bistability (e.g. if hysteresis is present). In the case of monostability the system recovers to the initial state after a transient perturbation, while in the bistable phase space a transient change in forcing can have a lasting impact.Sensitivity : Increasing sensitivity will produce a stronger system response to a given change in the control parameter. In the case of non-linear systems and those with hysteresis the sensitivity to a given perturbation is dependent on the location within the phase space i.e. non-linearities and changes in stability behaviour can produce a stronger or weaker system response to a given perturbation.Window of opportunity (Panel d): Conditions, which give rise to more frequent and larger amplitude millennial-scale climate variability. Specifically, the window of opportunity arises when both ice volume and atmospheric CO2 are at levels intermediate between their full glacial and full interglacial values. By definition, climate moves through the window of opportunity during glacial termination.Sweet spot (Panel g): A maximum in the activity of millennial-scale climate variability within the window of opportunity.Moveable window of bistability : The stability properties of a system with respect to one parameter (e.g. atmospheric CO2) can vary as function of another parameter (e.g. ice volume). This can shift the region of bimodality in systems with hysteresis.40 first described the pervasive nature of millennial-scale climate variability  stability of AMOC during full glacial periods, while high  concentrations of atmospheric CO2 seem to be responsible for an equivalent stability during interglacial periods, a suggestion also supported by more recent studies47. When both parameters occupy the space between full glacial and interglacial conditions a ‘window of opportunity’ arises  and possibly sea level , highlighting the possibility that millennial-scale variability may be considered both slave and master to orbital-timescale changes, an idea we develop below.Thus, millennial-scale variability seems to be dependent on orbital-timescale changes. Indeed, previous studies implied and demonstrated that gradual global warming can directly trigger abrupt transitions in the AMOC30 we use variations in North Atlantic SST to infer changes in the AMOC. We quantify millennial power using a Hilbert transform (amplitude envelope) of the 0.5–7 kyr bandpass filtered %NPS  record from ODP Site 98325 . We suggest that these minima are manifestations of the reciprocal influence of millennial activity on the boundary parameters themselves. Specifically we propose that the feedbacks between abrupt changes in the AMOC and the global parameters of benthic δ18O and CO2 are sufficiently strong when millennial variability is at its maximum that the climate system is effectively forced away from the sweet spot and towards a more stable climate regime. If this is case then the occurrence and active role of abrupt climate shifts during glacial termination (and inception for that matter) is no mere coincidence, but rather represents the necessary and ubiquitous interaction between these two distinct modes of climate variability.Less expected is the observation that the peak in millennial power  towards the LGM  and vice versa36. Thus, the effect of accentuated millennial-scale activity at that time was to continually drive CO2 in one direction or another depending on the mode of ocean circulation (i.e. feedbacks could be both negative and positive). On the other hand, the resulting changes in CO2 may themselves have reversed the oceanic conditions that produced them in the first place (e.g. ref. 45) thus forcing CO2 to remain within range of the sweet spot throughout MIS 3. Note that we are not suggesting that all D-O events were caused by changes in CO2 (or vice versa) and while this may have been the case for some of the larger events, we believe that high frequency D-O variability represents the inherent tendency of the AMOC to oscillate from one mode to another as an inevitable consequence of an intermediate climate background63.For example, atmospheric CO2 declined further throughout MIS 3 and eventually ice sheets attained their maximum configuration during the LGM , inducing a period of reduced millennial-scale activity associated with a monostable strong AMOC. Subsequently, during glacial termination, when climate again passed through the sweet spot, the effect of accentuated millennial-scale activity was to help push CO2 straight through the window of opportunity  but the inevitable return to a weak mode of circulation during the YD (see sub-section: ‘solutions in the time domain’) enabled CO2 to increase beyond the window of opportunity with the onset of interglacial conditions, eventually returning to a monostable strong mode of AMOC during the Holocene (and MIS 5e). Notably, the occurrence of high amplitude millennial-scale variability associated with high  CO2 is a peculiar feature of deglacial periods .COods Fig. , which w2 and benthic δ18O could be mere coincidence and needs to be confirmed using alternative proxies and locations. Furthermore, the minima themselves are not particularly prominent, and could be a result of controlling factors other than millennial-scale AMOC variability. On the other hand, we observe a maximum in the absolute rate of change of Antarctic temperature  aligned with the maximum in millennial power at Site 983  represents a convolution of ice volume and deep ocean temperature this could mean that the local minimum in the histogram of benthic δ18O reflects the existence of two distinct modes in deep ocean temperature, which could correspond to the two equivalent modes in atmospheric CO2  we note that ice rafting increases away from the sweet spot of millennial activity, reaching its maximum within the glacial mode of CO2 and δ18O and when ice volume exceeds −50 m SLE. While this observation does not negate the potentially significant influence of icebergs on SST (and ocean circulation), the fact that millennial variability in SST reaches its maximum power (maximum amplitude in the 0.5–7 kyr band) when ice rafting is still relatively subdued suggests that iceberg calving is not the main influence on millennial-timescale climate variability.Finally, our analysis can inform us about the interactions between calving ice sheets around the NE Atlantic and millennial-scale climate variability. There has been some debate over whether or not iceberg calving events cause or amplify abrupt changes in ocean circulation (which can be inferred from changes in high northern latitude sea surface temperature)Box 2 Millennial-scale variability and abrupt climate change99  and Heinrich events  associated with extended cold periods known as Heinrich stadials, HS  3 and much more subdued behaviour during interglacial (e.g. the Holocene) and full glacial periods .Aside from glacial cycles, Earth’s climate also varies strongly on millennial timescales in the form of Dansgaard-Oeschger (D-O) eventsBox Fig. . The amp30. Of particular relevance to this perspective are the abrupt climate shifts associated with glacial termination  and are thought to involve abrupt changes in the state of Atlantic Meridional Overturning Circulation (AMOC)nge Fig. .Box Figure 2 Millennial-scale climate variability as documented by Greenland ice-core δ18O99  reveals abrupt fluctuations between cold  and warmer  conditions. YD Younger Dryas, B/A Bølling-Allerød, HS1 Heinrich Stadial 1, LGM Last Glacial Maximum. Black triangles represent Heinrich events (e.g. HE1).73. Based on the notion that abrupt oscillations in the AMOC are more pronounced when ice volume is intermediate between full glacial and interglacial conditions , Sima et al.71 argued that such oscillations represent a change in the underlying dynamical behaviour of the AMOC and are an intrinsic feature of deglacial transitions which by definition must pass through the D-O window. Indeed, based on an array of evidence69 we are now confident that abrupt mode switches in the AMOC really are a characteristic feature of glacial terminations. However, such a strong feature as the B/A-YD transition  seems to be a rare occurrence25, such that it is still not clear whether it really reflects a systematic change in AMOC stability or e.g. a chance rerouting of meltwater runoff specific to T174. Here we investigate the changing stability of the AMOC with respect to key boundary parameters during deglaciation, and how this might interact with freshwater perturbations to give rise to the various permutations represented by previous terminations.The abrupt climatic oscillation represented by the HS1-B/A-YD triptych during Termination 1 Box ; Fig. 4 76. Moreover, it has been shown that the region of bistability with respect to one parameter can vary, depending on the value of another  then the region of bistability no longer exists for the observed range of G-IG CO2 variability and the AMOC enters a region of monostability. Likewise, for increasing values of CO2, the window of bistability with respect to ice sheet height shifts towards smaller and smaller ice sheets until it disappears (monostability) as CO2 approaches interglacial values  until we cross the grey square to reach 1b. At this point the system is bistable and a transition to a stable weak mode could be invoked by a transient forcing, for example a one-time input of freshwater. As ice sheet height continues to decrease beyond point 1d we leave the window of bistability as the white square is crossed and the AMOC (if still in a strong mode) will experience an abrupt transition to a weak mode (point 2a). All else being equal, ice sheets would then have to grow again by ~18m (SLE)57 in order to overcome the inherent hysteresis and transition back into a monostable strong mode (i.e. to cross the white triangle back to point 1a). Alternatively, if CO2 were to increase simultaneously  even if ice sheets were kept constant.In Fig. 2 and ice sheet size). As discussed, the region of AMOC bistability with respect to either CO2 or ice sheet height is sensitive to changes in the other parameter45. This means for example that starting in a weak mode , resulting in a shift to monostable strong (2d→1b). Likewise, a strong to weak mode transition could be triggered by e.g. the combination of ice decline (1c→1d) plus a CO2 decrease (1d →2b). Hence a mixture of ice volume and CO2 changes that could come about as a result of millennial-scale changes in the AMOC could themselves increase the likelihood of further abrupt changes in the mode of AMOC and thence further changes in CO2 and or ice volume  might help to amplify the pace of deglaciation as climate passes through the sweet spot for millennial-scale variability. But the notion of a moveable window of AMOC bistability has more fundamental implications for the structure of glacial terminations in general, which we discuss below.We have already discussed the possibility that exaggerated feedbacks between ocean circulation and atmospheric CO20. This event is thought to reflect (at least in part) the addition of freshwater to the surface North Atlantic by decaying ice sheets and is temporally related to the ice rafting event known as Heinrich Event 1 (HE1). Equivalent ice sheet wasting events accompanied every deglaciation of the past 1Myr or so25, and various studies have attempted to demonstrate that these were also related to a weakening of AMOC77. A large freshwater event could perturb the AMOC into a weak mode even from its glacial monostable strong state but this would only be transient and the AMOC would recover following the end of freshwater input. However, the decrease in ice sheet height that might accompany such a freshwater event could also push the system into a bistable regime or even toward monostable weak . This would require an external forcing such as a negative freshwater  event, which is also unexpected given the tendency for meltwater generation during the deglacial period.2 pushes the bistable window to the right and forces the system toward monostable strong (e.g. 2d→1b for CO2+). This would require that the rate of CO2 increase effectively outpaced the rate of ice sheet decline, which is possible but probably unlikely again given that ice sheets tend to diminish throughout deglaciation. We suggest that this mechanism could explain AMOC recovery during the Bølling-Allerød.Increasing atmospheric CO2 eventually reaches near-interglacial levels and the system is forced into a monostable strong regime. This scenario is inevitable once CO2 reaches the desired threshold. The result would be an AMOC recovery towards the end of deglaciation.COThe last deglaciation T1; Fig.  was char25 as we might expect from option (3) (see discussion below).We suggest that the last option (4) is the most likely scenario given the reasoning above for options (1) and (2) and the fact that most terminations are not interrupted by a strong oscillation2 are at intermediate values (as apparent during MIS 3). Furthermore, if enough freshwater is added, this could induce a weak mode even when the AMOC is in a monostable strong regime (while ice sheets are still very large) and this could modulate the precise timing or occurrence of abrupt transitions during deglaciation beyond those predicted by stability changes alone.In Fig. 2 pushes the window of bistability to the right fast enough to outpace the decline in ice sheet height, triggering a recovery of AMOC as the system is forced back toward monostable strong (B/A): t3; 2d→1b (CO2+). Note that recovery of the AMOC in this scenario can occur even in the presence of continued freshwater input45. Within Termination 1, the deglacial rise in atmospheric CO2 stagnates during the B/A such that the return to a weak mode of AMOC becomes inevitable as ice sheets continue to decline, pushing the system back towards bistability and possibly to monostable weak (YD): t4; 1b→c2(→2a). Atmospheric CO2 then starts to rise again and eventually reaches a level sufficient to induce an interglacial monostable strong mode of AMOC  and recovery is achieved (t5). Brief cold events such as the 8.2ka event and the cold event following HS11 recorded at ODP Site 983  and are therefore transient by nature i.e. solely driven by e.g. freshwater fluxes.Scenario B: ‘Standard’ termination . However, while CO2 reconstructions are available back to 800ka, reliable sea-level reconstructions are limited to the last deglaciation . Of relevance here is a newly published study5 in which the authors posit a direct link between insolation forcing and the duration of glacial terminations. Quantifying the duration of a glacial termination using benthic δ18O is not ideal  but nevertheless the results are instructive since they suggest that higher levels of insolation might lead to a shorter termination by driving faster ice sheet decay. According to our arguments, in these cases we should probably not expect termination to be interrupted by an early recovery of the AMOC (SL rises too fast with respect to CO2). Furthermore, by their5 calculations Termination 1 is not only relatively long in duration  but is also longer than might be predicted from its insolation (in this case using integrated summer energy as the relevant quantity). Perhaps its longer duration (i.e. slower SL rise with respect to CO2) made Termination 1 particularly prone to an early recovery of the AMOC.A test of these predictions requires, at the very least, robust records of atmospheric CO25 these interactions are likely to have lasting effects during the period immediately following termination . This becomes important when making comparisons between interglacials whose preceding terminations may have been very different. Such comparisons are important in light of ongoing changes in e.g. greenhouse gas concentrations, which need to be viewed within the context of natural variability to allow robust differentiation of potential human influences on the climate system79. For example the rise in atmospheric CH4 and CO2 over last ~5 and ~8 kyr (respectively) of the Holocene81 has sparked debate over whether or not humans have been influencing climate for many thousands of years85.So far we have described the interplay between millennial and orbital-timescale variations with respect to glacial terminations but as discussed recently4 and CO2 over the course of the Holocene (which experienced upwards trends in both gases) as compared with previous interglacials 86. In a recent study25 we made the case that the earliest portion (first few kyr) of several previous interglacials should not be included when making these comparisons if quasi-equilibrium is a prerequisite for such comparisons to be valid. We defined (quasi-)equilibrium as a  situation in which Earth’s climate is equilibrated with respect to key boundary conditions including orbital configuration, ice volume and atmospheric CO2 concentration and we argued that the earliest parts of many previous interglacials were not at equilibrium thanks to continued adjustment of ocean circulation patterns following deglacial oscillations of the AMOC  and subsequent ~20 ppmv jump in CO2 associated with recovery of the AMOC ~129 ka resulted in a larger net change in CO2 across T2 than the corresponding change across T1. Thus, atmospheric CO2 was effectively lower at the onset of the Holocene than it might otherwise have been if the B/A had not occurred . This logic was borne out in a modelling study by Ganopolski and Brovkin38 in which they found that a pronounced overshoot in CO2 occurs at the beginning of an interglacial when recovery of AMOC happens only at the end of termination. This is followed by a rather constant or even decreasing trend in CO2 during the subsequent interglacial. In contrast  if a termination is interrupted by an early recovery of the AMOC, CO2 will tend to be lower at the start of the subsequent interglacial and proceed to increase throughout the next several thousand years.Deaney et al.2 also increased throughout MIS 11, which has been used as an analogy for MIS 1 because of the similarity in insolation forcing88. Indeed, insolation forcing across T1 was much weaker than e.g. T2 because of lower eccentricity and this has been suggested as a possible reason why an early AMOC recovery did not occur during T272, However, more recently MIS 19 has been advocated as a better insolation analogue for MIS 186 and it is notable that T9 (into MIS 19) did not experience an early AMOC recovery25 and CO2 decreased throughout MIS 19, following an initial overshoot25.We note that CO2 to coincide during deglaciation, together with the ubiquitous input of freshwater, we argue that the most likely case for a typical termination is Scenario B: essentially an uninterrupted period of weakened AMOC and consequently a late recovery of AMOC (as compared with T1). Following this logic and the studies mentioned above38 we conclude that the relatively low concentration of CO2 observed at the onset of MIS 1 , and its subsequent increase throughout the Holocene, may also be considered as atypical with respect to other interglacials, regardless of any influence that humans may or may not have had. This should be taken into account when making comparisons between the Holocene and more typical interglacials such as MIS 5.Earlier we made a case that Termination 1 (T1) was unusual in the sense that it was interrupted by an early recovery of the AMOC (associated with the Bølling/Allerød). Given the tendency for changes in ice volume and CO2 throughout the Holocene, which itself may therefore be considered unusual a priori .In this perspective we have highlighted progress in our understanding of glacial terminations as the ultimate expression of the interaction between millennial and orbital-timescale variations in Earth’s climate. Specifically we discussed the importance of feedbacks, which appear most accentuated within the sweet spot of millennial activity, and in particular during glacial termination when they help to explain the magnitude and rapidity of these events. We then made the case that structural changes in the stability of the AMOC can explain the occurrence of terminal AMOC oscillations such as the HS1-B/A-YD triptych. Finally we argued that the unusual AMOC oscillation of Termination 1 led to the low initial concentration and subsequent rise of atmospheric CO61 and across MIS 5a/490 and Termination 294; Fig. 2 rise and ice sheet decline during previous terminations and assess our assertions for example about the uniqueness of the B/A-YD oscillation.However, there are still major gaps in our knowledge, which need to be filled before we can test some of the hypotheses put forward here. For example, sea-level reconstructions beyond the last glacial maximum are uncertain and inconsistent  in proposed mechanisms as will efforts to compare unforced (e.g. without freshwater forcing) AMOC oscillations in a variety of different model setups. To date most simulations of abrupt climate variability have been forced with freshwater but a growing number of models display forms of ‘unforced’ oscillations . Given the apparent importance of factors other than freshwater forcing we advocate a community effort to simulate abrupt transitions through other means. Ultimately, the incorporation of an interactive carbon cycle and ice sheet dynamics (including e.g. solid earth and ice shelf cavity processes) will allow a fuller representation of the mechanisms involved in the coupled earth system. In this way we will be able to vastly improve our understanding of how internal climate system dynamics respond to external forcing across deglacial transitions and finally reach a turning point in our understanding of glacial terminations. This knowledge is not merely of academic interest; uncertainties over the location of stability thresholds with respect to our current climate mean that we cannot rule out the possibility that the AMOC is already within a regime of multiple equilibria with respect to North Atlantic freshwater forcing96.An area of gathering momentum is model intercomparison. Many of the exciting theoretical discoveries made over the past few years have been obtained using a single model, leaving their results vulnerable to possible inadequacies in a particular model. Projects such as PMIP


Rationale: Enhanced proliferation and distal migration of human pulmonary arterial smooth muscle cells (hPASMCs) both contribute to the progressive increases in pulmonary vascular remodeling and resistance in pulmonary arterial hypertension (PAH). Our previous studies revealed that Rictor deletion, to disrupt mTOR Complex 2 (mTORC2), over longer periods result in a paradoxical rise in platelet-derived growth factor receptor (PDGFR) expression in PASMCs. Thus, the purpose of this study was to evaluate the role of combination therapy targeting both mTOR signaling with PDGFR inhibition to attenuate the development and progression of PAH.Methods and Results: Immunoblotting analyses revealed that short-term exposure to rapamycin (6h) significantly reduced phosphorylation of p70S6K (mTORC1-specific) in hPASMCs but had no effect on the phosphorylation of AKT . In contrast, longer rapamycin exposure (>24 h), resulted in differential AKT (T308) and AKT (S473) phosphorylation with increases in phosphorylation of AKT at T308 and decreased phosphorylation at S473. Phosphorylation of both PDGFRα and PDGFRβ was increased in hPASMCs after treatment with rapamycin for 48 and 72 h. Based on co-immunoprecipitation studies, longer exposure to rapamycin (24–72 h) significantly inhibited the binding of mTOR to Rictor, mechanistically suggesting mTORC2 inhibition by rapamycin. Combined exposure of rapamycin with the PDGFR inhibitor, imatinib significantly reduced the proliferation and migration of hPASMCs compared to either agent alone. Pre-clinical studies validated increased therapeutic efficacy of rapamycin combined with imatinib in attenuating PAH over either drug alone. Specifically, combination therapy further attenuated the development of monocrotaline (MCT)- or Hypoxia/Sugen-induced pulmonary hypertension (PH) in rats as demonstrated by further reductions in the Fulton index, right ventricular systolic pressure (RVSP), pulmonary vascular wall thickness and vessel muscularization, and decreased proliferating cell nuclear antigen (PCNA) staining in PASMCs.Conclusion: Prolonged rapamycin treatment activates PDGFR signaling, in part, via mTORC2 inhibition. Combination therapy with rapamycin and imatinib may be a more effective strategy for the treatment of PAH. Pulmonary arterial hypertension (PAH) is characterized by progressive increases in pulmonary vascular resistance (PVR) and pressure, which can lead to deterioration of right ventricular function . The majmTOR is a key serine/threonine protein kinase that responds to a variety of extracellular stimuli including growth factors, insulin, nutrients , and hypoxic stress . mTOR fuPrior studies have shown that mTORC1 inhibition with rapamycin attenuates pre-clinical PAH development . FurtherReceptor tyrosine kinase (RTK) signaling, including PDGF, mediates lung vascular remodeling in PAH . RTK sig2 10%) for 3 weeks after given single dose of SU5416 . They were transitioned to normoxia for 2 weeks. Rats were randomly assigned to four groups: rapamycin alone , imatinib alone , rapamycin (2 mg/kg/d) combined with imatinib (10 mg/kg/d), or vehicle only. All drugs were given once daily by intraperitoneal injection for the last 2 weeks of each model. Terminal measurements were recorded. Right ventricular systolic pressure (RVSP) was measured as previously published  of Guangzhou Medical University. For the MCT model, male Sprague-Dawley (SD) rats (200–250 g) were studied after a single intraperitoneal monocrotaline (MCT) injection . For the hypoxia-SU5416 model, SD rats (200–250 g) placed under hypoxic condition . After cell attachment, inserts were removed using sterilized forceps. Cells were cultured in serum-free medium containing rapamycin (100 nM), imatinib (5 μM) or vehicle and pictures recorded every 4 h. The number of cells migrating out of the wound edge was scored using Image J image analysis software. Cell viability was assessed by CCK8 kit . hPASMCs were seeded into 96-well plates at a density of 5,000 cells per well with six replicates per group. Cells were cultured in 2% serum-containing medium exposed to either rapamycin (100 nM), imatinib (5 μM), or vehicle. After addition of 10ul CCK8 per well, the plate was cultured for 4 h at 37°C. The absorbance value of each well was measured and collected at 450 nm. Cell proliferation assays were performed using BrdU incorporation kit . hPASMCs were seeded into 96-well plates at a density of 5,000 cells per well with four replicates per group. Cells were cultured in 2% serum-containing medium exposed to either rapamycin (100 nM), imatinib (5 μM), or vehicle. BrdU assay was conducted according to the standard protocol of the manufacturer and BrdU uptake was measured in each well at dual wavelengths of 450–540 nm.Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1). Cells were maintained in SMC medium  with 2% fetal bovine serum and SMC growth supplement . For migration assay, hPASMCs suspension was diluted to 2 × 10Total cellular and isolated pulmonary arterial (PA) proteins were extracted using RIPA lysis buffer containing proteinase and phosphatase inhibitor. Western blotting was performed using the following antibodies: anti-mTOR (#2983), anti-Rictor (#5379) anti-phospho-PDGFRα/β (#3170), anti-PDGFRα (#3174), anti-PDGFRβ (#3169), anti-phospho-AKT (T308) (#13038), anti-phospho-AKT (S473) (#4060), anti-AKT (Pan) (#4691), anti-phospho-p70S6k (#9204), anti-p70S6k (9202), anti-phospho-S6 (#4858), and anti-S6 (#2317) . For Co-immunoprecipitation, cell protein was extracted in the non-deformed protein lysate. Non-specific proteins were removed by adding protein A/G-beads . An antibody targeting mTOR was incubated at room temperature to bind the antibody to the target antigen. The antigen-antibody complex was incubated overnight at 4°C and then, boiled for 5 mins. SDS-polyacrylamide gel electrophoresis was performed by adding the sample buffer on the protein .For immunofluorescence staining, lung tissue sections were blocked in 2% BSA for 30 min and incubated overnight at 4°C with alpha-smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA). After extensive washes with PBS for 30 min, lung tissue was incubated with Alexa 488 anti-rabbit secondary or Alexa 594 anti-mouse secondary antibodies at a 1:500 dilution for 1 h at room temperature. Nuclei were stained with DAPI .p < 0.05 was considered as statistically significant.Data were presented as means ± standard error (SE) and analyzed by GraphPad Prism software. Statistical differences among multiple experimental groups were tested by one-way analysis of variance (ANOVA) and two-way ANOVA. While rapamycin is well-established as an mTORC1 inhibitor, its effects on mTORC2 has not been well studied. Thus, we initially investigated the effects of longer rapamycin exposure on mTORC2 in hPASMCs. As shown in Rapamycin works through a gain-of-function mechanism in which it binds to the intracellular protein FKBP12 to generate a drug-receptor complex that binds to and inhibits mTORC1 . To deteRictorSM-/-)  from smooth muscle-specific Rictor knockout mice (rSM-/-) . Base onrSM-/-) . In paraSince hPASMCs proliferation and migration drives obliterative pulmonary arterial vascular remodeling in PAH, we investigated the impact of rapamycin and imatinib on each process. We first detected impact of rapamycin and imatinib on cell viability. Result showed that both rapamycin and imatinib alone significantly inhibited hPASMCs viability. Importantly, the inhibitory effects of rapamycin combined with imatinib was more significant than that of either drug alone . Both drWe evaluated combination therapy with rapamycin and imatinib in MCT and Hypoxia/Sugen rat models of PAH. Rats develop PH in both MCT and Hypoxia/Sugen treated , 6A, as We next evaluated the effects of combination therapy on the extensive vascular remodeling and muscularized vessels in both rat models. For the MCT rodent model , H&E stain vitro data, rapamycin inhibited the activity of both, mTORC1 and mTORC2. Imatinib alone inhibited the phosphorylation of PDGFR. Rapamycin combined with imatinib inhibited mTORC1, mTORC2 and PDGFR signaling mediators.We evaluated the effects of combination therapy on mTOR and PDGFR signaling pathways in isolated PAs from rat lungs. As shown in in vitro and in vivo. While mTORC1 is a known target, our data suggest the PDGFR activation is due, in part, to additional inhibition of mTORC2 by rapamycin. Devising a strategy to address both seemingly related pathological cascades, the current study found greater attenuation of PH in two pre-clinical rat models when rapamycin and imatinib were combined compared to either drug alone.Both rapamycin and imatinib have been tested in clinical trials in patients with PAH . While tThe PI3K/AKT/mTOR signaling pathway mediates PAH pathogenesis via its canonical role in cell survival and proliferation . RapamycBeyond reductions in p-S473AKT with longer exposures, rapamycin also inhibited p70S6k phosphorylation at 6 h, with a trend toward activation of p-S473AKT. A compensatory response by mTORC2 could explain this discrepant finding. Specifically, mTOR is a common component of the two complexes, and both mTORC1 and mTORC2 compete for mTOR binding . With shRictorSM-/- mice that demonstrated increased expression of PDGFRs with aging (Bolstering findings from our previous studies in th aging , we founth aging . PDGF prth aging . PDGF anth aging . The curIn summary, in contrast to mTORC1 inhibition with shorter duration, prolonged rapamycin treatment results in the inhibition of both mTORC1 and mTORC2. As illustrated in