Myocardial Revascularization through Cardiac Venous System.

MYOCARDIAL REVASCULARIZATION THROUGH THE CARDIAC VENOUS SYSTEM MARIA ERMELINDA ANTUNES SOARES RODRIGUES MUNZ Tese de doutoramento em Ciências Veterinárias 2012 MARIA ERMELINDA ANTUNES SOARES RODRIGUES MUNZ MYOCARDIAL REVASCULARIZATION THROUGH THE CARDIAC VENOUS SYSTEM Tese de Candidatura ao grau de Doutor em Ciências Veterinárias submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador – Prof. Doutor Artur Manuel Perez Neves Águas Categoria – Professor Catedrático Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto Co-orientador – Prof. Doutor Carlos Alberto da Silva Lopes Categoria – Professor Catedrático Jubilado Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto Co-orientador – Dr. Mário Jorge Gonçalves Santos Matos Amorim Categoria – Docente Voluntário Afiliação – Faculdade de Medicina da Universidade do Porto This work was supported by grants from FCT (Fundação para a Ciência e a Tecnologia) to UMIB (Unit for Multidisciplinary Biomedical Research), project reference PEstOE/SAU/UI0215/2011-12. vi vii "A pessoa isolada é uma abstracção teórica que não tem sentido, porque cada um é uma consequência dos que se cruzam no trajecto de vida e compartilham o circunstancialismo existencial.” Nuno Grande viii ix Acknowledgments In this section (which I have heard is the most read!), I will mainly use my mother language, as most of the contributors are Portuguese. I apologize to my English-speaking friends… Este trabalho e respetiva dissertação são um tributo, uma homenagem ao Prof. Doutor Nuno Grande (meu mestre de sempre e para sempre) e à Dra. Ana Maria Grande. Ao Prof. Nuno, gostaria de lha ter oferecido em vida, mas infelizmente já não foi possível… As palavras nesta altura de pouco valem e nunca serão suficientes. Neste momento, serve-me de consolo que tentei sempre transmitir-lhe todo o meu respeito, a minha admiração e o prazer de gozar da sua companhia, da sua amizade e dos seus ensinamentos. Um homem como nunca conheci, com qualidades ímpares nas mais diversas vertentes: como professor, como cientista, como amigo, e como pai e marido. Como professor, o Prof. Nuno possuía uma capacidade incrível de captar a atenção de todos nas suas aulas, nas suas apresentações, recorrendo às suas interessantes (e muitas vezes engraçadas) vivências e descobertas pessoais para manter a audiência cativada. Como cientista, com uma versatilidade e genialidade impressionantes, era capaz de debater e envolver-se nos mais variados temas. Este trabalho de investigação advém das suas experiências pioneiras nos anos sessenta… Como amigo, de uma bondade extrema para com todos e sempre disponível para uma palavra meiga. Como pai e marido,… um exemplo que tentarei sempre seguir, sem dúvida! Em segundo lugar, devo agradecer ao meu ex-chefe e orientador, Prof. Doutor Artur Águas. Reconhecendo o trajeto já traçado nas minhas experiências prévias (estudo anatómico da circulação coronária no suíno) e a minha aptidão pela área cirúrgica, foi o Prof. Águas quem me propôs a ideia deste trabalho tão interessante e empolgante. Agradeço-lhe ter aceitado o ónus da orientação e me ter proporcionado sempre as condições necessárias para que esta investigação seguisse em frente. Em seguida, devo agradecer a duas pessoas importantíssimas: o Dr. Mário Jorge Amorim e o Prof. Doutor Adelino Leite-Moreira. Sem o Jorge e as suas mãos mágicas, esta investigação nunca teria começado. Sem o Adelino e o seu apoio científico, esta tese nunca teria terminado. Obrigada, Jorge, espero manter sempre a tua amizade! Ao Adelino, devo dizer que, aparte o exemplo do Prof. Nuno, o admiro como um profissional xvi ABSTRACT Ischemic coronary disease is considered a major cause for mortality in developed countries and accounts for 7 million worldwide deaths per year. Most deaths are fast and current treatments tend to be more urgent and aggressive. At present, coronary artery bypass grafting and percutaneous transluminal coronary angioplasty are the routine standard procedures to obtain myocardial revascularization in patients with severe ischemic coronary disease. Bypass grafting is a demanding surgical technique, whereas angioplasty does not involve anesthesia, is a much simpler method but of more limited long-term success. In fact, most angioplasty-treated vessels suffer restenosis, while bypass grafting has a lower incidence of reoperations. Each of these methods presents other important limitations, which are described in detail further on. As a major drawback, both bypass grafting and angioplasty overcome focal narrowings of proximal coronary arteries but are of little benefit for patients with diffuse multivessel coronary disease. These are the so-called “no-option” patients, which consist of 12 to 15% of the total population of candidates for myocardial revascularization. Alternative surgical options for salvaging ischemic myocardium have been somewhat disregarded. Our research proposal aimed at revisiting, testing and optimizing cardiac venous arterialization as a surgical alternative to treat ischemic patients. This reperfusion technique consists on the use of cardiac veins to deliver arterial blood from a pulmonary vein or systemic artery to infarcted myocardial areas. Our goal was to evaluate the benefit of cardiac venous arterialization in reducing acute myocardial infarct size and its effects on cardiac performance. For this purpose, cardiac data from two groups of pigs were compared. In the control group, myocardial infarction was induced through simple surgical ligation of a branch from the left coronary artery. In the experimental group, besides coronary artery occlusion, cardiac veins were arterialized. Over 5 days, several diagnostic procedures were used in each group to characterize and measure the extent of myocardial infarct, namely ECG, echocardiography, cardiac biomarkers and histopathology. Initially, to attain the most significant differences between the two groups and high survival rates in the control group, an ideal myocardial infarction model was investigated. This surgical model should present a reproducible and standardized myocardial lesion, associating the largest possible infarct extent to the lowest morbidity and mortality. The recognition of the most proper coronary occlusion site was achieved by separating pigs in groups subjected to permanent occlusions in different locations of either the left circumflex xvii artery or the left anterior descending artery. The results demonstrated that proximal occlusions lead to high mortality rates, while distal occlusions induce rather small myocardial infarction areas. The optimal occlusion site was produced at the mid-point of the left anterior descending artery. This swine model was considered to be easily reproducible and consistent, with precise location of the site of occlusion, low mortality and surgical complication rates, and minimization of pain, suffering, and distress to the animals involved. Our experiments on cardiac venous arterialization during acute myocardial infarction also suffered several changes for melioration of the model, both in the surgical technique and in the diagnostic protocols used. In the final stage, the left anterior descending vein was arterialized through anastomosis with the left internal mammary artery. Both the left anterior descending artery and vein were ligated, to produce ischemia and avoid backflow into the coronary sinus, respectively. Our results were remarkable. There was clear evidence of myocardial perfusion, with reduction of over 50% on infarct size (especially on the amount of necrotic tissue) and full protection of cardiac performance. Nevertheless, cardiac venous arterialization on our pig model was not the full success we expected. Cardiac damage was still present: small endocardial infarcts were observed and concentration levels of cardiac biomarkers were as high as the ones from the control group. We speculated on several possible reasons for this event. Our major belief was that myocardial reperfusion through retrograde flow might be insufficient due the frequency and morphology of competent valves within the porcine cardiac venous system. Being remarkably close to the human in vascular arrangement and heart topography, the pig is an excellent model for experimental cardiac surgery. In earlier studies, we have characterized the arterial cardiac system of the pig heart and compared it to the human coronary anatomy. For our study on the venous cardiac system of the pig, we have used the same vascular injection techniques and basic dissection, which are adequate for detailed anatomical characterizations. The analysis of the cardiac venous anatomy included the distribution pattern and course of cardiac veins, and a new insight on frequency, location, morphology and efficiency of venous valves. A comparison with the human pattern was performed, through examination of two human hearts and available human literature. Our results have shown that, although venous drainage was deemed quite similar between the two species, cardiac venous valves presented pertinent differences. In the analyzed porcine hearts, these anatomical barriers were found more frequently and showed less variability in shape than in humans. Additionally, they were xviii also considered more competent in pigs, with complete luminal coverage in all examined valves. These results indicate that efficacy of retrograde flow should be compromised by cardiac venous valves when pigs are used as experimental models. In humans, however, retrograde flow might be more successful, as cardiac venous valves are fewer and less competent. Ideally, techniques for overcoming these anatomical obstacles should be developed/planned for better efficacy of cardiac venous arterialization as an alternative to restore myocardial perfusion. xix xx xxi RESUMO A cardiopatia isquémica constitui uma elevada causa de morte em países desenvolvidos, com cerca de 7 milhões de mortes anuais a nível mundial. A maioria das mortes são rápidas e os tratamentos atuais tendem a ser mais urgentes e agressivos. Os procedimentos mais comuns para revascularização miocárdica de pacientes com sinais severos de doença coronária isquémica são atualmente a cirurgia de revascularização coronária (comummente designado por “bypass”) e a angioplastia coronária. O “bypass” é uma técnica cirúrgica exigente, ao invés da angioplastia, que não requer anestesia e é um método bastante mais simples, mas com taxas de sucesso inferiores a longo prazo. De facto, a maioria das artérias coronárias tratadas por angioplastia sofrem re-estenoses, enquanto o “bypass” tem uma baixa incidência de reoperações. Cada um destes métodos apresenta limitações importantes, que são descritas em pormenor adiante. Tanto o “bypass” como a angioplastia são tratamentos adequados em caso de estenoses focais e proximais das artérias coronárias, mas apresentam a mesma desvantagem importante: os pacientes com doença coronária multivascular difusa são considerados pacientes “sem opção”. Estes pacientes constituem 12 a 15% do total da população de candidatos à revascularização miocárdica. As opções cirúrgicas alternativas para reperfusão do miocárdio têm sido um pouco negligenciadas. Propusemo-nos revisitar, testar e otimizar a arterialização de veias cardíacas como opção cirúrgica para o tratamento de pacientes com doença isquémica do coração. Esta técnica de reperfusão baseia-se na utilização de veias cardíacas como via de transporte de sangue arterial desde uma veia pulmonar ou artéria sistémica até áreas isquémicas do miocárdio. O nosso objetivo consistiu em avaliar a capacidade das veias cardíacas arterializadas em reduzir a dimensão de enfartes agudos do miocárdio e medir os seus benefícios na performance cardíaca. Para esse efeito, foram comparados dados cardíacos de dois grupos de suínos. No grupo controlo, foi induzido enfarte miocárdico por laqueação cirúrgica de um dos ramos principais da artéria coronária esquerda. No grupo experimental, para além desta oclusão coronária, as veias cardíacas foram arterializadas. Durante 5 dias, foram utilizados vários métodos de diagnóstico em ambos os grupos para caracterização e medição da extensão do enfarte, nomeadamente ECG, ecocardiografia, biomarcadores cardíacos e histopatologia. No início do projeto, foi criado um modelo ideal de enfarte miocárdico que demonstrasse diferenças significativas em relação ao grupo experimental e com elevadas taxas de sobrevivência. Este modelo cirúrgico deveria apresentar uma lesão miocárdica xxii reprodutível e padronizada, associando a maior extensão de enfarte à menor mortalidade e morbilidade possíveis. De forma a reconhecer o local mais apropriado para oclusão coronária, foram criados vários grupos de suínos sujeitos a laqueação permanente a diferentes níveis da artéria circunflexa esquerda ou da artéria descendente anterior. Os resultados demonstraram que as oclusões proximais resultam em elevadas taxas de mortalidade, enquanto as oclusões distais induzem lesões miocárdicas relativamente pequenas. O ponto de oclusão ideal foi conseguido no ponto médio da artéria descendente anterior. Este modelo suíno foi considerado facilmente reprodutível e consistente, com localização precisa do ponto de oclusão, baixas taxas de mortalidade e de complicações cirúrgicas, com minimização da dor, sofrimento e stress pelos animais envolvidos. As nossas experiências em arterialização venosa cardíaca em caso de enfartes miocárdicos agudos sofreu alterações para melhoramento do modelo, tanto na técnica cirúrgica como nos protocolos de diagnóstico utilizados. Na fase final, a artéria descendente anterior foi arterializada através de uma anastomose com a artéria mamária interna. A artéria e veia descendente anterior foram laqueadas, de forma a produzir isquémia e evitar o refluxo de sangue para o seio coronário, respetivamente. Os resultados obtidos foram notáveis. A perfusão do miocárdio foi claramente evidente, com redução superior a 50% na dimensão do enfarte (especialmente na quantidade de tecido necrosado), e possibilitou a proteção total da performance cardíaca. Apesar de tudo, a arterialização venosa cardíaca no nosso modelo suíno não alcançou o sucesso esperado. O dano cardíaco ainda era visível, com presença de pequenos enfartes endocárdicos e as concentrações dos biomarcadores cardíacos a atingirem valores tão elevados quanto os do grupo controlo. Especulámos sobre as possíveis razões para estas ocorrências. A nossa principal convicção residia numa insuficiente reperfusão do miocárdio por fluxo retrógrado em resultado da frequência e morfologia de válvulas competentes no sistema cardíaco venoso dos suínos. O suíno é considerado um modelo excelente para experimentação cardíaca cirúrgica, devido às extraordinárias semelhanças aos humanos, em termos de arquitetura vascular e topografia cardíaca. Em estudos anteriores, fizemos a caracterização detalhada do sistema cardíaco arterial em corações de suíno e estabelecemos comparações com a anatomia vascular cardíaca do Homem. Para o estudo do sistema venoso cardíaco em suínos, utilizámos as mesmas técnicas de injeção vascular e disseção básica, métodos adequados em caracterizações anatómicas detalhadas. A análise da anatomia venosa cardíaca incluiu o estudo da distribuição e trajeto das veias cardíacas e uma nova perspetiva sobre a frequência, localização, morfologia e eficiência das válvulas venosas. Foram estabelecidas comparações ao sistema venoso cardíaco xxiii humano através da avaliação de dois corações humanos e literatura disponível. Os nossos resultados demonstraram que, apesar da drenagem venosa ser considerada semelhante entre as duas espécies, foram observadas diferenças pertinentes ao nível das válvulas venosas cardíacas. Nos corações de suíno analisados, estas barreiras anatómicas foram observadas com maior frequência e evidenciavam menor variabilidade na forma, comparativamente à espécie humana. Adicionalmente, foram consideradas mais competentes no caso dos suínos, com cobertura total do lúmen por todas as válvulas examinadas. Estes resultados apontam para uma menor eficácia do fluxo retrógrado (devido a comprometimento por parte das válvulas venosas cardíacas) quando se utilizam suínos como modelos experimentais. Em humanos, no entanto, o fluxo retrógrado poderá ser mais bem-sucedido tendo em conta que as válvulas venosas cardíacas são menos numerosas e menos competentes. Idealmente, deveriam ser criadas/planeadas técnicas para ultrapassar estes obstáculos anatómicos de forma a melhorar a eficácia da arterialização de veias cardíacas como uma alternativa para restauração da perfusão miocárdica. xxiv xxv INDEX Chapter 1 - Introduction _________________________________________________ 1 1. Ischemic heart disease ________________________________________________ 2 1.1. Epidemiology and relevance ________________________________________ 3 1.2. Pathophysiology __________________________________________________ 5 1.2.1. Causes _______________________________________________________ 5 1.2.2. Consequences _________________________________________________ 6 1.2.2.1. At the cellular level __________________________________________ 6 1.2.2.2. At the tissue level __________________________________________ 10 1.2.2.3. At the organ level __________________________________________ 13 1.3. Therapeutic strategies ____________________________________________ 14 1.3.1. Revascularization techniques ____________________________________ 15 1.3.1.1. Disadvantages/risks/limitations ________________________________ 17 1.3.1.2. Alternatives _______________________________________________ 19 1.3.1.3. Myocardial Revascularization through the Cardiac Venous System ___ 21 Chapter 2 – Published manuscripts _______________________________________ 24 2.1. Surgical porcine myocardial infarction model through permanent coronary occlusion __________________________________________________________ 26 2.1.1. Abstract _____________________________________________________ 26 2.1.2. Introduction __________________________________________________ 26 2.1.3. Materials and Methods__________________________________________ 28 2.1.3.1. Anesthetic protocol _________________________________________ 28 2.1.3.2. Surgical procedures ________________________________________ 29 2.1.3.3. Diagnostic tests ____________________________________________ 31 2.1.3.4. Statistics _________________________________________________ 33 2.1.4. Results ______________________________________________________ 33 2.1.5. Discussion ___________________________________________________ 38 2.2. Cardiac venous arterialization in acute myocardial infarction: how great is the benefit? ____________________________________________________________ 44 2.2.1. Abstract _____________________________________________________ 44 2.2.2. Introduction __________________________________________________ 45 2.2.3. Materials and Methods__________________________________________ 46 xxxii Chapter 1 - Introduction Myocardial Revascularization through the Cardiac Venous System Introduction 2 1. Ischemic heart disease Ischemia is a reduction (or loss) of blood supply in a tissue due to blocked arterial flow or reduced venous drainage (Myers RK, 2007; Mitchell RN, 2003). When the organ affected is the heart, the consequences are devastating, as vital organs such as kidney, liver and lungs are eventually stricken (Burns DK, 2003). Ischemic heart diseases (IHD) are usually caused by changes in atherosclerotic plaques at the coronary arteries (Burns DK, 2003; Van Vleet JF, 2007; Myers RK, 2007; Schoen FJ, 2010). Hence, IHD is also Myocardial Revascularization through the Cardiac Venous System Introduction 3 named coronary heart disease or coronary artery disease (Burns DK, 2003; Myers RK, 2007; Schoen FJ, 2010). IHD include a group of related syndromes, such as angina pectoris (chest pain), myocardial infarction (MI), as well as sudden death and congestive heart failure of ischemic origin (Burns DK, 2003; Schoen FJ, 2010; Woolf, 1992). Although the ischemic cause underlies them all, these syndromes have different pathogenic mechanisms and clinical pictures (Mc Gee JO'D, 1992). Angina pectoris may be stable or unstable. It is caused by reversible, transient ischemia and only induces intermittent chest pain (Burns DK, 2003). MI, also called “heart attack”, is a more severe form, with local ischemia producing an area of myocardial necrosis. It is usually caused by complete thrombotic occlusion of a coronary artery (Burns DK, 2003; Schoen FJ, 2010). Sudden cardiac death results from a lethal rapid arrhythmia usually caused by marked coronary occlusion (more than 75%) (Burns DK, 2003; Schoen FJ, 2010; Mc Gee JO'D, 1992). Less frequently (in only 10 to 20% of cases), however, these fatal arrythmias may result from other nonatherosclerotic conditions (Van Vleet JF, 2007). Congestive heart failure of ischemic aetiology (CHF), also referred as ischemic cardiomyopathy, is usually a consequence of chronic and progressive myocardial dysfunction secondary to ischemia (Burns DK, 2003). CHF may also derive from non-atherosclerotic pathologies, such as chronic work overload, and may even occur in acute conditions, such as fluid overload, acute valvular dysfunction or a substantial MI (Myers RK, 2007). 1.1. Epidemiology and relevance Cardiovascular disease is a global health problem. Eighty per cent of deaths from cardiovascular diseases occur in developed countries, mainly due to the outstanding aging of these modern societies (Schoen FJ, 2010; Galiñanes, 2005). In 2010, with the increased life span and the decrease in both mortality and birth rates, 11% of the world population was older than 60 years old. Within the high income group of countries*, however, this percentage had already reached 21% and it is predicted that, for Portugal itself, this number will rise close to 30% by 2050 (World Health Organization, 2012; Machado, 2007). Life expectancy has increased due to biological changes, such as cleanliness of drinking water, improvements in hygiene and nutrition, and progress in quality of health care (including vaccination) and medication itself. Moreover, the quality of life has improved through enhanced psychosocial aspects, such as economic security, and family Myocardial Revascularization through the Cardiac Venous System Introduction 4 and social stability (Petrov, 2007). According to the WHO (World Health Organization), life expectancy at birth has globally increased from 64 (in 1990) to 68 years (in 2009). Considering only the high income group of countries, life expectancy rose from 68 to 78 years during this period (World Health Organization, 2012). Thus, as a consequence of the elderly living healthier and longer, it is understandable that most deaths occur from chronic diseases, such as cardiovascular disease, chronic obstructive lung disease, cancers, diabetes or dementia (World Health Organization, 2012). In fact, in a broad study from the WHO in 2008, it was estimated that, in high income countries, 71% of the total deaths would pertain to people older than 70 years old (World Health Organization, 2011). Another important demographic issue is the amount of population living in urban areas. According to the WHO (2012), in the high income countries, the percentage of population living in urban areas was as high as 77%. These people are subject to higher stress, traffic pollution, smoking and obesity (due to a poorer nutrition and reduced physical activities), which are known for being common risk factors for chronic cardiovascular diseases (World Health Organization, 2012; Hoffmann B, et al., 2009; Brook, 2007; Petrov, 2007; Burns DK, 2003; Mitchell RN, 2010; Maxie MG, 2007; Schoen FJ, 2010). Approximately one third of the world population dies from cardiovascular diseases, largely from IHD and stroke (Thygesen K, 2007; World Health Organization, 2011; Burns DK, 2003; Schoen FJ, 2010). They remain the leading causes of hospitalization and death in high and middle income groups (World Health Organization, 2011; Apple FS, 2006; Schoen FJ, 2010). In the study performed by WHO in 2008, the deaths caused by IHD were estimated to reach 15.6% in high income countries, while stroke and other cerebrovascular diseases were responsible for 8.7% of deaths. As for the middle income countries, numbers were also very high: 13.7% and 12.8%, respectively (World Health Organization, 2011). In the group of diseases included in IHD, MI is a major manifestation (Thygesen K, 2007). Approximately one third of patients who suffer an MI will eventually die (Mitchell RN, 2003; Burns DK, 2003). From these fatalities, 50% do not reach the hospitals alive (Burns DK, 2003; Mc Gee JO'D, 1992). The risk of MI increases throughout life. Within the range of 40 and 50 years old, men are 4 to 5 times more likely to develop MI than women (Burns DK, 2003; Machado, 2007). However, the risk is the same in both sexes after the 80s (Burns DK, 2003). Myocardial Revascularization through the Cardiac Venous System Introduction 5 1.2. Pathophysiology 1.2.1. Causes As mentioned above, IHD is mainly (90%) caused by atherosclerotic plaques (Burns DK, 2003; Apple FS, 2006; Schoen FJ, 2010). These plaques (atheromas) are intimal lesions protruding into the arterial lumen, generating arterial stenosis, and have a tendency for disruption and thrombus formation (Mitchell RN, 2010; Apple FS, 2006). They are usually composed of a soft, necrotic, lipid core covered by a fibrous cap (Mitchell RN, 2010; Maxie MG, 2007) (Fig.1). The core contains cholesterol and cholesterol esters, dead cell debris, foam cells (lipid laden macrophages and smooth muscle cells), and eventually organized thrombus and plasma proteins (Mitchell RN, 2010; Ross, 1992). Macrophages, T cells and smooth muscle cells involve this central core, and, more superficially, a fibrous cap is visible, composed of smooth muscle cells and dense collagen (Mitchell RN, 2010; Ross, 1992). Figure 1 – The major components of a well-developed intimal atheromatous plaque overlying an intact media. © 2010 Kumar V et al, Robbins and Cotran Pathologic Basis of Disease, (Fig. 11-6, p. 496). Used with permission. The most recent theory about the formation of atherosclerotic plaques states that, after an initial endothelial damage, the lesion progresses with accumulation of lipoproteins and monocyte and platelet adhesion, which is followed by migration of monocytes and smooth muscle cells into the intima (and activation of macrophages), and finally smooth muscle proliferation and accumulation of lipids and collagen (Mitchell RN, 2010; Apple FS, 2006; Maxie MG, 2007). These plaques are continuously growing due to on-going cell death, remodeling, and organization of thrombus, and may reach more than 1.5 cm in Myocardial Revascularization through the Cardiac Venous System Introduction 6 diameter (Mitchell RN, 2010). Thus, as they progress towards the arterial lumen, they may cause critical stenosis in these vessels and compromise distal blood flow. Moreover, the atherosclerotic plaques might also suffer abrupt alterations, such as erosion, ulceration, hemorrhage, fissuring or rupture (Burns DK, 2003; Mitchell RN, 2010; Schoen FJ, 2010; Ross, 1992). Besides expanding the total size of the plaque, these changes activate platelet aggregation with consequent induction of thrombosis, which may partially or completely occlude the arterial lumen (Schoen FJ, 2010; Burns DK, 2003; Mitchell RN, 2010). These sudden changes also activate inflammation, an important factor for atherosclerosis formation and remodeling (Schoen FJ, 2010). Additionally, as the plaque ruptures, the release of small atheromatous debris may cause embolization at distal branches of coronary arteries (Burns DK, 2003; Mitchell RN, 2010). Another complication of atherosclerotic plaques is aneurysmal dilation as the arterial wall gets progressively destroyed (Mitchell RN, 2010; Maxie MG, 2007). Other possible causes for IHD (or factors which may worsen the deleterious effects of atherosclerotic plaques) may include coronary artery vasospasms, lowered systemic blood pressure, emboli originating from vegetations in cardiac valves, systemic hypertension and pre-existing cardiac changes such as hypertrophy (Burns DK, 2003; Apple FS, 2006; Schoen FJ, 2010). Atherosclerotic plaques are most frequently found at the abdominal aorta, but the coronary arteries are the second most affected vessels (Mitchell RN, 2010). The frequency of occlusion on the different coronary artery branches is variable. The most commonly hit is the left anterior descending artery, reaching 40 to 50% of MI cases. As for the right coronary artery, the occlusion occurs in 30 to 40% of the cases, while occlusion at the left circumflex artery only happens in 15 to 20% of the cases (Burns DK, 2003). 1.2.2. Consequences 1.2.2.1. At the cellular level The ischemic injury to cells can be reversible or irreversible, depending on the severity and duration of the pathologic stimuli. In case of incomplete or brief blood flow occlusion, cells may undergo reversible changes. However, myocytes are very susceptible to ischemic injury because they depend almost exclusively on oxidative phosphorylation (Schoen FJ, 2010; Woolf, 1992). Therefore, with complete or prolonged occlusion (such as in MI), cells are irreversibly injured, cannot recover and will die (Mitchell RN, 2003; Myers RK, 2007) (Fig.2). Myocardial Revascularization through the Cardiac Venous System Introduction 7 Figure 2 – The relationship between normal, adapted, reversibly injured, and dead myocardial cells. The cellular adaptation is myocardial hypertrophy (lower left), caused by increased blood flow requiring greater mechanical effort by myocardial cells. This adaptation leads to thickening of the left ventricular wall to over 2 cm (normal, 1-1.5cm). In reversibly injured myocardium (illustrated schematically, right) there are generally only functional effects, without any readily apparent gross or even microscopic changes. In the specimen showing necrosis, a form of cell death (lower right), the light area in the postolateral left ventricle represents an acute myocardial infarction caused by reduced blood flow (ischemia). All three transverse sections of the heart have been stained with triphenyltetrazolium chloride, an enzyme substract that colors viable myocardium magenta. Failure to stain is due to enzyme loss following cell death. © 2010 Kumar V et al, Robbins and Cotran Pathologic Basis of Disease, (Fig. 1-2, p. 6). Used with permission. When irreversible injury is present, although gross or even microscopical changes are not evident, functional changes are already present (Mitchell RN, 2003). This occurs because cardiac cell activity relies on the integrity of all cellular systems. Thus, cells will stop contracting after 1 or 2 minutes of ischemic stimulus, although they will only die 20 to 30 minutes later (Mitchell RN, 2003; Kumar V, 2010; Maxie MG, 2007; Apple FS, 2006). Moreover, changes in the appearance of the cell will only be visible histologically after 4 to 12 hours (Burns DK, 2003; Mitchell RN, 2003; Myers RK, 2007; Kumar V, 2010; Maxie MG, 2007). Gross morphological changes will be evident even later (usually 18 to 24 hours after initial insult) (Mitchell RN, 2003; Burns DK, 2003; Myers RK, 2007; Maxie MG, 2007) (Fig.3). Myocardial Revascularization through the Cardiac Venous System Introduction 8 Figure 3 - Sequential development of biochemical and morphologic changes in cell injury. Cells may become rapidly non-functional after the onset of injury, although they are still viable, with potentially reversible damage; a longer duration of injury may eventually lead to irreversible injury and cell death. Note that irreversible biochemical alterations may cause cell death, and typically this precedes ultrastructural, light microscopic, and grossly visible morphologic changes. © 2010 Kumar V et al, Robbins and Cotran Pathologic Basis of Disease, (Fig. 1-7, p. 12). Used with permission. A cell is considered irreversibly injured when mitochondrial and membrane structure and function cannot be restored (Mitchell RN, 2003; Myers RK, 2007; Maxie MG, 2007). At this point, necrosis takes place, a sequence of morphological cellular events following death (Mitchell RN, 2003). In fact, the age of a MI is possible to be determined as these sequential morphological changes are somehow predictable through time (Fig. 4): - during the first 4 to 12 hours, coagulative necrosis develops and becomes microscopically evident (Kumar V, 2010). Coagulative necrosis results from enzymatic digestion of the cell and denaturation of proteins (Mitchell RN, 2003). After enzymatic degradation of organelles, the cytoplasm becomes vacuolated and calcification may ultimately occur (Kumar V, 2010). The nucleus may undergo condensation (pyknosis), fragmentation (karyorrhexis) or dissolution (karyolysis) (Burns DK, 2003; Mitchell RN, 2003). The cell membranes lose integrity, so leakage of cellular contents occurs and elicits inflammation of surrounding tissues (Kumar V, 2010). Cells appear swollen and hypereosinophilic, mainly due to the denaturation of intracytoplasmic proteins (Mitchell RN, 2003; Myers RK, 2007; Van Vleet JF, 2007; Kumar V, 2010). Interstitial edema is also evident at this initial stage and some degree of hemorrhage may be present (Apple FS, 2006; Burns DK, 2003). Myocardial Revascularization through the Cardiac Venous System Introduction 15 benefit from urgent invasive revascularization (Stanger O, 2006; Galiñanes, 2005; May SA, 2009; Apple FS, 2006). Patients with smaller myocardial damage often suffer repetitive episodes of MI, leading to increased morbidity and mortality over time (Apple FS, 2006). Thus, management of MI suggested by most guidelines is now aggressive and invasively oriented (Apple FS, 2006). Also prevention through control of risk factors is considered essential and is included in these guidelines (Apple FS, 2006; Schoen FJ, 2010). Prevention is used before the patients experience MI (primary prevention) or to avoid reinfarctions (secondary prevention) (Schoen FJ, 2010). Given the pathophysiology of atherosclerotic plaques, the inhibition of platelet aggregation and inflammation are important steps in treating MI (Apple FS, 2006; Loop, 1998). Currently, the routine pharmacological and surgical therapies for patients with acute MI include: aspirin and heparin (to prevent further thrombosis); oxygen (to minimize ischemia); nitrates (for vasodilation and reversion of vasospasm); beta-blockers (to diminish cardiac oxygen demand and lower the risk for arrhythmias); ACE inhibitors (to lessen ventricular dilation) and revascularization maneuvers (to recover blood supply to injured area) (Schoen FJ, 2010; Apple FS, 2006). 1.3.1. Revascularization techniques Reperfusion is considered the most effective mean to salvage ischemic myocardium, limit infarct size and improve function, through rapid restoration of blood flow (Schoen FJ, 2010). Ideally, reperfusion should be initiated within 20 min of initial ischemic insult, so that necrosis is completely prevented. After that, reperfusion will not be fully successful, but nonetheless important and beneficial in rescuing viable cells if within the first 6 hours post-ischemic insult (Schoen FJ, 2010). Reperfusion changes the appearance of a MI. The presence of a patchy nontransmural infarction is common after reperfusion as necrosis is stopped before it reaches the full thickness of the myocardium (Burns DK, 2003). Small infarcts also heal faster than larger ones (Burns DK, 2003). Coronary interventions for reperfusion may include thrombolysis, percutaneous transluminal coronary angioplasty (PTCA) with or without stent placement, or coronary artery bypass graft (CABG) surgery (Schoen FJ, 2010). Thrombolysis (streptokinase or tPA) is used for dissolution of the lesion that caused the MI, while angioplasty and stenting aim to mechanically change it. Percutaneous coronary interventions (PCI) include PTCA and PTCA with stenting. PTCA consists on the dilation of focal stenosis through insertion and inflation of intravascular balloon-tipped catheters. In 90% of PTCAs, endovascular Myocardial Revascularization through the Cardiac Venous System Introduction 16 stents (expandable tubes of metallic mesh) are additionally implanted to reinforce arterial lumen dilation, by supporting the new stretched open position of the artery (Mitchell RN, 2010). CABG is a surgical technique which simply bypasses the lesion itself, providing blood distally to the occluded vessel, either by use of autologous or synthetic vascular grafts (Schoen FJ, 2010; Mitchell RN, 2010). For proximal vascular narrowings in all presented scenarios, such as severe coronary disease, diabetic patients, or multi-vessel disease, CABG is the elective treatment and has become the “gold standard” since 1969 (Galiñanes, 2005; May SA, 2009). In these patients, it has been shown to improve survival compared to medical therapy (Stanger O, 2006; Galiñanes, 2005; May SA, 2009). This technique is performed through median sternotomy and allows direct visualization of all areas of the heart, hence the possibility of treating multi-vessel disease (Ochi M, 2003). Several studies report a better surgical outcome with off-pump CABG (OPCAB) than by use of cardiopulmonary bypass (on-pump CABG). It has been demonstrated that oxidative stress, inflammatory response, aortic dissection and postoperative bleeding were relatively higher when extracorporeal circulation, cardioplegia and aortic manipulation were used. The consequences included pulmonary and renal insufficiency, and neurological complications due to reduced cerebral perfusion and development of cerebral micro-emboli (Galiñanes, 2005; Scarborough JE, 2003; Ochi M, 2003; Stanger O, 2006; May SA, 2009). With new technologic advances, these problems were overcome with surgeries being performed on the beating heart (OPCABG) and the perioperative mortality is as low as 1 to 2% (Galiñanes, 2005; Pennington, 2006; Scarborough JE, 2003; May SA, 2009). However, the option for onor off-pump CABG is still debatable considering the type of patients, clinical conditions and surgeon’s experience (Galiñanes, 2005; Pennington, 2006; Ochi M, 2003; Stanger O, 2006; May SA, 2009). An alternative to reduce invasive procedures is PTCA (Galiñanes, 2005). PTCA was introduced later than CABG and has the advantage that no anesthesia is required and hospital stay is short (Galiñanes, 2005). PCI was usually reserved for high-risk surgical patients or those with single-vessel disease (Stanger O, 2006; Galiñanes, 2005). With the development of newer stents coated with certain medicines (drug-eluting stents) for PTCA, restenosis was reduced (10-15%) and may nowadays also be applied to patients with left main and three-vessel coronary artery disease (Galiñanes, 2005; Loop, 1998; May SA, 2009; Mitchell RN, 2010; Jensen J, 2006). Myocardial Revascularization through the Cardiac Venous System Introduction 17 1.3.1.1. Disadvantages/risks/limitations As a disadvantage of reperfusion techniques in general, hemorrhages are common as a result of leakage from injured microvascular ischemic vessels (Burns DK, 2003; Schoen FJ, 2010). Contraction bands, due to hypercontraction of myofibrils, are also prominent as a consequence of the rapid influx of calcium into the cells through the damaged plasma membranes (Burns DK, 2003; Schoen FJ, 2010). Reperfusion also involves risks, namely reperfusion-injury, “no-reflow” phenomenon, “stunned myocardium” and reperfusion arrhythmias. When ischemic myocardium is reperfused, cell injury may be reversible and cells may recover completely. However, under certain circumstances, this blood reflow may induce new injury processes, causing death to cells that might otherwise recover (Mitchell RN, 2003; Myers RK, 2007; Kumar V, 2010). This phenomenon is referred as ischemia-reperfusion injury or simply reperfusioninjury (Myers RK, 2007; Schoen FJ, 2010). This mechanism is still unclear, but may occur due to overload in calcium influx, inflammatory cells and free radicals, and activation of the complement system (which cause loss of cell integrity) (Mitchell RN, 2003; Kumar V, 2010; Schoen FJ, 2010). These excessive free radicals and calcium also trigger undue arrhythmias during reperfusion. In the “no-reflow” phenomenon, the haemorrhages and endothelial swelling resulting from reperfusion in pre-injured ischemic microvascular walls lead to capillary occlusion and therefore limit blood supply to ischemic areas (Schoen FJ, 2010). After reperfusion, the affected area might also remain in a state of reversible cardiac failure with post-ischemic contractile dysfunction. This so-called “stunned myocardium” will only recover after several days (Schoen FJ, 2010). The different reperfusion techniques also present limitations. Thrombolytic agents, for instance, will remove the thrombus, but not modify the original cause which is the underlying atherosclerotic plaque (Schoen FJ, 2010). These agents are also contraindicated when there is a risk of cranial hemorrhages. All three most common approaches for treatment of coronary diseases (PTCA, stenting and CABG) cause endothelial injury to arterial walls, which, as mentioned before, is considered the initial cause for atheroma formation (Mitchell RN, 2010; Jensen J, 2006). Limitations of PCI PTCA causes arterial wall stretching and rupture of plaques, in order to restore blood flow, and adjacent arterial walls suffer hemorrhages due to dissection (Mitchell RN, 2010). As short-term risks, abrupt occlusion may re-occur due to elastic recoil or negative Myocardial Revascularization through the Cardiac Venous System Introduction 18 remodeling (vessel contracture) when this dissection is too extensive, or due to luminal thrombosis (Mitchell RN, 2010). As long-term risks, 30 to 50% of patients suffer from proliferative restenosis, a result from intimal thickening similar to atherosclerotic disease (Mitchell RN, 2010; Loop, 1998; May SA, 2009). The introduction of stents enabled a reduction of this percentage and the procedure is less time-consuming than PTCA alone (Galiñanes, 2005). As disadvantages, stenting also causes direct trauma with plaque and arterial erosion and potential acute thrombosis (Schoen FJ, 2010; Mitchell RN, 2010; May SA, 2009). Proliferative restenosis may also occur but is now less common due to the development of new-generation coated stents which considerably reduce proliferative intimal thickening (Mitchell RN, 2010; May SA, 2009; Jensen J, 2006). The preference of PCI to CABG is very strongly debated. Although PCI procedures are perceived to be associated with smaller immediate risks and are therefore more popular, some studies show similar survival rates after PCI or CABG, with cardiac events and need for repeat revascularization being also higher with PCI (Stanger O, 2006; May SA, 2009; Serruys PW, 2012). In most left-main disease patients, bifurcating or multivessel disease and heavy calcification are usually present, which are challenging conditions for performing PCI. In these cases, CABG is a more appropriate option (May SA, 2009). CABG is also preferable to PCI in chronic total occlusions and angulated lesions (May SA, 2009). Limitations of CABG The endothelial insult after CABG derives from abnormal mechanical forces caused by insertion of vascular grafts into the arterial circulation (Mitchell RN, 2010; Schoen FJ, 2010). Generally speaking, early graft failure in CABG is expressed as a poor “run-off”, usually due to disproportionate anastomotic lumen diameters or distal arterial obstructions, and less commonly due to technical problems (Allwork, 1992; Loop, 1998). The reasons for long-term failure (3 months to 7 years) include thrombosis and fibrous intimal hyperplasia, particularly of veins grafts (Allwork, 1992). Short to medium-term benefits with CABG are superior to long-term advantages (Galiñanes, 2005). In fact, reoperation rates increase with time, rising up to 30% 15 years after surgery (Loop, 1998). According to the material used for CABG, also different limitations apply. Synthetic grafts are usually successful in large high-flow arteries, but will fail if in arteries thinner than 8mm in diameter (such as coronary bypasses) due to thrombosis and intimal hyperplasia at the anastomotic site (Mitchell RN, 2010; Stanger O, 2006). For replacement or bypass of these smaller vessels, reverse autologous saphenous veins or internal mammary arteries (IMAs) are frequently used. IMAs are the gold standard conduit Myocardial Revascularization through the Cardiac Venous System Introduction 19 for CABG. The use of saphenous venous grafts presents several disadvantages. First, their long-term patency is much shorter than that of arterial grafts (Mitchell RN, 2010; Allwork, 1992; Stanger O, 2006; Galiñanes, 2005; Loop, 1998; May SA, 2009; González Santos JM, 2005). Indeed, occlusion as a result of thrombosis and intimal thickening, and vein atherosclerosis are common consequences with venous grafting (Mitchell RN, 2010; Stanger O, 2006; Loop, 1998; González Santos JM, 2005). Second, manipulation of the aorta is usually performed for proximal implantation of the venous graft (González Santos JM, 2005). Third, saphenous veins may present abnormalities, such as varicous dilatations, limiting their use (Allwork, 1992; Stanger O, 2006). And fourth, wound morbidity from vein harvesting causes patient discomfort and complaints, with increased in-hospital stays and treatment costs (Stanger O, 2006). Despite all these disadvantages, most patients undergoing CABG need three bypass grafts, and venous grafts have to be frequently used. In these patients, the left IMA is used for revascularizing the LAD and venous grafts are applied in the remaining diseased coronary branches (Galiñanes, 2005; Stanger O, 2006). The use of both left and right IMAs or other arterial conduits as an alternative or a complement (such as the radial, the splenic and the right gastroepiploic arteries) appears to be more beneficial than the use of venous grafts, but the procedures are technically more demanding, with increase in operative time (González Santos JM, 2005). Additionally, arterial graft lengths are sometimes insufficient and vasospasms are frequent, so careful patient selection must be performed (Stanger O, 2006; May SA, 2009; González Santos JM, 2005). When both IMAs are used, the bleeding resulting from their dissection is a considerable risk in old or obese patients, with general increase in length for the procedure and morbidity (Allwork, 1992; González Santos JM, 2005). Also sternal blood supply is compromised, with higher risk for wound dehiscence and mediastinitis (González Santos JM, 2005). Hence, all-arterial grafting remains a controversial issue between surgeons (Galiñanes, 2005; Loop, 1998; Stanger O, 2006; May SA, 2009; González Santos JM, 2005). 1.3.1.2. Alternatives The future surgical treatment for IHD is envisioned as minimally invasive techniques (ideally with three-dimensional optics and robotic control) accomplishing complete revascularization, with routine use of total-arterial grafting (Loop, 1998; Pennington, 2006; Stanger O, 2006; May SA, 2009; González Santos JM, 2005). In order to reduce morbidity, costs and length of hospital stays, surgical incisions may be reduced with Myocardial Revascularization through the Cardiac Venous System Introduction 20 MIDCABG (minimally invasive direct coronary artery bypass grafting), through parasternal approaches or minithoracothomies (Pennington, 2006; Stanger O, 2006). However, besides technically difficult and expensive, MIDCABG and TECAB (totally endoscopic coronary artery bypass) techniques cannot be applied in multivessel disease as the access to selected vessels is limited (Stanger O, 2006). The combination of MIDCABG and PTCA is possible and known as “hybrid revascularization” (Matsumoto Y, 2001). However, the frequency of coronary restenosis is still high and it is unfeasible in patients with multi-vessel or diffuse coronary disease, whose incidence is growing (Stanger O, 2006; Matsumoto Y, 2001). In these cases, endarterectomy is an option, which consists on the excision of occluding material, including intima and most of the media, from the coronary artery. However, this procedure is associated with increased risk of perioperative infarction, namely incomplete removal of the atheromatous core, dissection of arterial wall and creation of occlusive flaps (Stanger O, 2006). Although highly argued, selective use of this technique may justify these risks and improve mortality and morbidity of patients with diffuse disease, but only in vessel diameters of at least 2mm (Stanger O, 2006). Other interesting approaches, such as transmyocardial laser revascularization and regenerative therapies (using stem-cells or genes) are also under investigation and have been used in clinical trials mainly as adjunctive therapies in CABGs. However, besides the risks involved, these techniques require optimization and larger-scale studies need to be performed for clear demonstration of efficacy and safety (Galiñanes, 2005; Genovese J, 2007; Kanashiro-Takeuchi RM, 2011; Sansone F, 2011). Considering all the advantages and risks from all these procedures, the selected method for performing myocardial revascularization has to take into consideration the number of affected vessels, the clinical status of the patient (renal dysfunction, diabetes, etc.) and the surgeon’s skills (Loop, 1998). Patients presenting for revascularization are nowadays considerably older and present comorbidities, such as severe pulmonary disease, dementia, left ventricular dysfunction, diabetes, acute coronary syndromes, high stroke risk, gastrointestinal bleeding, contraindications to specific medication, etc (Stanger O, 2006; May SA, 2009; González Santos JM, 2005). Regardless of treatment approach, diabetic patients, for instance, are a challenging group in which complete revascularization is usually harder to attain and the need for repeat revascularization and the risk for cardiac events and death are higher (Loop, 1998; May SA, 2009). CABG after PTCA and CABG re-operations are commonplace in ischemic heart disease, and alternative surgical procedures have been somewhat disregarded (Loop, 1998). A major pitfall of both PCI and CABG is that they cannot be applied to patients with Myocardial Revascularization through the Cardiac Venous System Introduction 21 widespread lesions in the coronary vascular tree, which consist of 12 to 15% of the revascularization candidates and whose incidence is growing (Galiñanes, 2005; Stanger O, 2006). CABG can only overcome arterial stenosis located at the beginning of the major vessels and PTCA is only feasible in vessels greater than 3mm in diameter (Galiñanes, 2005). Additionally, and regardless of the revascularization technique used, the underlying atherosclerotic disease process continues to progress (May SA, 2009). Is there any alternative surgical treatment to propose to those patients? 1.3.1.3. Myocardial Revascularization through the Cardiac Venous System For patients in whom previous CABG or redos have failed, or whose arteries are too small for anastomosis or in those with diffuse disease, cardiac venous arterialization may be viewed as a pertinent alternative route to an otherwise inaccessible ischemic myocardium. Cardiac venous arterialization allows arterial blood coming from a pulmonary vein or a systemic artery to reach the myocardium through the use of cardiac veins. Since the first attempt by Beck in the 1940s, cardiac venous arterialization has been investigated by several research teams worldwide. Retroperfusion through the coronary sinus or arterialization of selected cardiac veins have been performed in experimental animal models (and even in a few human patients) mainly during the 1970s. The common goal was to provide an alternate surgical mean to revascularize and rescue ischemic myocardial lesions. Despite the obsolete diagnostic methods used, several scientists have provided good evidence that, in the acute phase of MI, these techniques are able to perfuse the myocardium, maintain metabolism, limit infarct size and maintain left ventricular function (Bhayana JN, 1974; Gardner RS, 1974; Benedict JS, 1975; Hochberg, 1977; Rhodes GR, 1978; Marco JD, 1977; Hochberg MS, 1979; Park SB, 1975). After the 1970s, research on cardiac venous arterialization was much reduced and only scarce literature can be found, probably as a result of the success of the two newly developed techniques, namely PTCA and CABG (Krombach GA, 2005; Stanger O, 2006; May SA, 2009; O'Byrne GT, 1991; Resetar ME, 2007; Møller CH, 2008; Harig F, 2011; Oesterle SN, 2003; Raake P, 2005). The most remarkable reports published after the 1970s concern the development of catheter devices to arterialize veins, thereby avoiding surgical invasive approaches (Oesterle SN, 2003; Raake P, 2005). Oesterle SN et al. (2003) created a percutaneous insitu coronary venous arterialization (PICVA), connecting and stenting the proximal Myocardial Revascularization through the Cardiac Venous System Introduction 22 coronary artery to the adjacent cardiac vein. Then, a blocking device was implanted proximally in the vein to avoid drainage to the coronary sinus. This group was not very successful on its first trials, mainly due to variations in vascular anatomy (vessel crossovers and distance), presence of atherosclerotic plaques in the proximal segment of the artery and technical problems (perforation of veins and undersized migrating vein blockers). Raake P et al. (2005) tested the efficacy of a percutaneous ventricle-tocoronary-vein bypass, by introducing a stent between the left ventricle and the left interventricular vein. The results were not satisfactory, because there was no valve in the stent, so the blood could backflow into the ventricle and thus the pressure in the venous system was not high enough to reach the capillaries. 1.3.1.3.1. Our model and its validation The herein experimental investigation has revisited cardiac venous arterialization. In order to test the potential therapeutic benefits of an arteriovenous bypass in rescuing infarcted myocardial areas, we have performed surgical experiments in two groups of pigs. In the first group (control), pigs were submitted to focal infarction by surgical ligation of one main branch of the left coronary artery. In the second group of pigs (experimental), pigs were submitted to the same procedure but this was preceded by surgical creation of an arteriovenous shunt between the left internal mammary artery and the left anterior descending vein. By opening the shunt, blood flowed retrogradely through the cardiac venous system promoting myocardial revascularization. To avoid blood flow into the coronary sinus, the vein was ligated proximally to the established shunt. We studied the effects of cardiac venous arterialization during the acute phase of myocardial infarction, thus the animals have been sacrificed on the 5th day. Several parameters were evaluated before and after the surgical procedure in order to characterize and measure the extent of the myocardial infarction in the two groups of pigs. These diagnostic exams included ECGs, echocardiographies, cardiac biomarkers and histopathologies. In our understanding, new information on myocardial revascularization through cardiac venous arterialization will be helpful in several clinical situations: a) it is ideal for patients with distal and diffuse coronary disease (ex. diabetic patients), since blood can flow reversely through the extensive capillary network, b) the surgical procedure is as technically simple as the classical CABG, c) within the surgical options, it enables a fast myocardial revascularization with arterial blood, d) only one anastomosis is necessary, Myocardial Revascularization through the Cardiac Venous System Introduction 23 and not multiple grafts in the LAD territory as sometimes seen in CABG, and e) time required for the surgical procedure is comparable to a simple CABG. We expect this technique to reduce mortality and morbidity of patients going through coronary artery surgery, at least for those considered inoperable by other revascularization methods, which represent 12-15% of the total candidates (Chowdhry MF, 2005). 24 Chapter 2 – Published manuscripts Myocardial Revascularization through the Cardiac Venous System Published manuscripts 31 or left lateral saphenous vein was dissected and cannulated with a 16-gauge, 20-cm central venous catheter (Arrow International, Reading, PA) for postsurgical collection of blood samples and administration of medication. 2.1.3.3. Diagnostic tests 2.1.3.3.1. Cardiac biochemical markers A blood sample was collected from the jugular vein of each anesthetized pig for baseline analysis of cardiac biomarkers. In order to capture the true up-slopes and peak values of cardiac biomarkers (and thus more accurately estimate the infarct size), serial blood samples (approximately 3mL each) were collected every 2 h during the first 10 h after surgery, at 24 h after surgery, and then once daily until euthanasia (Gibbons RJ, 2004). Blood samples were centrifuged at 3100 × g for 5 min within 30 min of sampling, and the serum was stored in 2 microfuge tubes at −4 °C. The analysis included assessment of troponin I, myoglobin, total creatinekinase, isoenzyme MB of creatine kinase (measured by mass and activity assays), and lactate dehydrogenase. Although several biomarkers were used, analysis focused on the combination of 3: myoglobin (helpful in early detection of MI), cardiac troponin I (a marker that takes longer to rise but is highly specific) and MMB, as mass assay of creatine kinase (considered as effective as cardiac troponins in estimating infarct size) (Panteghini, 2002; Thygesen K, 2007). Calculations of areas under the curve (AUC) are presented to complement data provided by peak values. Myoglobin, cardiac troponin I, and MMB were determined by enzyme immunoassays (Dimension, Dade Behring, Newark, DE) which were performed according to the manufacturer’s instructions. 2.1.3.3.2. Electrocardiography and echocardiography At baseline, with the pigs under general anesthesia and in right lateral recumbency, 12-lead electrocardiography and 2D and M-mode echocardiography were performed. By using a multichannel electrocardiography analyzer (Cardisuny, Fukuda ME, Tokyo, Japan), precordial leads were included to precisely determine the ventricular wall segment affected. Transthoracic echocardiography (Vivid 3 Pro digital ultrasonic scanner, GE Medical Systems, Ontario, Canada) allowed assessment of myocardial echogenicity, cardiac chamber size, wall thicknesses and amplitudes of motion, and valve morphology and motion. Wall and cavity dimensions allowed for calculation of indices of cardiac motion (shortening fraction and ejection fraction). For these measurements, short-axis views were obtained at the level of the chordae tendineae, by using the right parasternal Myocardial Revascularization through the Cardiac Venous System Published manuscripts 32 window. To avoid intraobserver variability, 3 different M-mode images were obtained for each pig, and the mean of these 3 measurements was calculated. To detect the maximal value of ST deviation, 12-lead electrocardiography was repeated 1 h after coronary occlusion (with pigs still under anesthesia), by using the same technical approach (Smith GT, 1979). On day 4 after surgery, pigs again were sedated and anesthetized for reevaluation of cardiac performance and electrical activity. Echocardiography and 12-lead electrocardiography as performed at baseline were repeated by using the same technical approach. To estimate left ventricular performance, the recommendations from ASE Committee for echocardiographic measurements of left ventricular dimensions, volumes, and wall thicknesses were followed (Lang RM, 2005). To avoid operator bias, both baseline and postocclusion results from electrocardiography and echocardiography were analyzed by independent observers who were blinded to groups and the focus of the investigation. 2.1.3.3.3. Histopathologic studies After euthanasia of pigs on day 5 after surgery, hearts were excised immediately, cleaned, and washed in saline solution for macroscopic and histologic evaluation. In all cases, the lengths to the occlusion site in the proximal and distal coronary artery segments were measured. By cannulating the proximal segment of the coronary artery, saline solution (NaCl 0.9%) was injected to confirm complete occlusion. Hearts were stored in 10% buffered formaldehyde (Panreac Quimica SAU, Barcelona, Spain) for 24h. Each heart then was filled and embedded in agar–agar suspension (Merck KGaA, Darmstadt, Germany) at 4%, which was allowed to harden by incubating for 4h at 4°C. The hearts were sliced circumferentially parallel to the atrioventricular sulcus from the apex to the base. Each slice was 10 mm thick. The basal surface of each slice was photographed with a digital camera (FE360, Olympus, Tokyo, Japan). From the basal surfaces of the cardiac transverse slices, circumferential transmural 2to 3-mm thick samples were cut (including septum and both ventricles). These tissue samples were processed routinely for histologic analysis and stained with hematoxylin and eosin and Masson trichrome. Masson trichrome provided a clear contrast of colors between viable myocardium (bright red to pink), rim of infarct with inflammatory tissue (gray), and infarction (purple to blue) (Ouyang J, 2009). We used Image J software (NIH, Bethesda, MD) to measure these 3 regions in each trichrome-stained sample by tracing the corresponding tissue boundaries. Infarct size of both ventricles was expressed Myocardial Revascularization through the Cardiac Venous System Published manuscripts 33 as the percentage of affected myocardial area (necrosis + inflammatory tissue) in all myocardial areas analyzed, with infarct area % = infarct area × 100 / total myocardial area. As for electrocardiographic and echocardiographic studies, an independent blinded investigator calculated mean infarct size through histologic studies. 2.1.3.4. Statistics Statistical analysis was performed using the PASW package, version 18.00 (SPSS, Chicago, IL). Data are presented as mean ± SE (where appropriate) and were considered to differ significantly if the P value was less than 0.05. 2.1.4. Results The results presented are from 15 pigs; 2 pigs (both from the LADm group) were excluded from the study. One pig developed malignant hyperthermia during surgery (before coronary occlusion) and died. Sternotomy revealed that the other pig had pleural effusion and adherences from underlying disease; we therefore rejected this animal from analysis. Macroscopically, the effusion was serous and transparent, and culture demonstrated that it was aseptic. At echocardiography and necropsy, this pig showed an abnormal dimension of the right ventricle and thickness of its walls. In the LCXp group, only 1 pig of 3 survived. This pig had very high peak values and AUC of cardiac biomarkers (Table 1). In addition, echocardiography reflected severe ventricular dysfunction 4 d after surgery (Table 2): ejection fraction fell from 60.9% to 36.8% (a 40% decrease) and the thickness of the posterior wall fell from 12.7 to 5.6 mm (a 56% decrease); the septal wall was nearly unaffected. Macroscopic analysis revealed a transmural infarct located at the inferiolateral myocardial region, affecting mostly the posterior wall. Calculated infarct size was 17.91%. Neither of the 2 pigs in the LADp group survived. Therefore, no cardiac data could be obtained from diagnostic tests. In the LADd group (n = 2), both pigs survived. Some discrepancies were observed in peak values and AUC of the various analyzed cardiac biomarkers (Table 1). At 4 d after ligation, the ejection fraction fell from 69.9% to 65.2% (a 7% decrease); changes in the thickness of the septal and posterior walls were negligible. Infarct sizes were 9.95% and 10.09%. Myocardial Revascularization through the Cardiac Venous System Published manuscripts 34 Occlusion site Troponin I Myoglobin MMB Peak (ng/ml) AUC Peak (ng/ml) AUC Peak (ng/ml) AUC LCXp N=1 109 7.964 2.341 81.342 165 10.620 LADd (mean±SE) N=2 41 ±17 2.637 ±227 696 ±301 18.079 ±2973 16 ±3 587 ±150 LADm (mean±SE) N=6 43 ±10 2.647 ±310 668 ±115 10.374 ±1.186 15 ±4 360 ±102 Table 1. Peak values and areas under the curve (AUC; mean ± SE) of troponin I, myoglobin, and mass assay of creatine kinase of surviving pigs with coronary occlusions. Results from LADp are not shown, because only baseline values were obtained. Occlusion site EF (%) IVSs (mm) LVDs (mm) PWs (mm) 0h 96h 0h 96h 0h 96h 0h 96h LCXp N=1 60.9 36.8 12.4 13.7 28.1 40.3 12.7 5.6 LADd (mean±SE) N=2 69.9 ±2.7 65.2 ±0.6 16.2 ±0.8 14.5 ±1.5 20.8 ±1.4 32.4 ±3.2 13.9 ±1.4 13.9 ±2.8 LADm (mean±SE) N=7 65.5 ±4.1 53.4 ±3.9 14.9 ±1.1 10.3 ±1.2 24.6 ±2.1 33.8 ±2.4 13.7 ±1.2 16.0 ±1.2 Table 2. Variation (mean ± SE) of ejection fraction, interventricular septal thickness at end-systole, left ventricular diameter at end-systole, and posterior wall thickness at end-systole of surviving pigs with coronary occlusions. Results from LADp are not shown, because only baseline values were obtained. At baseline, all 8 pigs in the LADm group had normal values of biochemical markers, ECG tracings, and echocardiographic data. In this group, 7 of the 8 pigs survived. As shown in Figures 2 through 4, levels of cardiac biomarkers rose during the first 10h after occlusion, followed by a gradual decrease throughout the remaining 4d. The curves of cardiac biomarkers were somewhat distinct in the 6 pigs analyzed (2 animals were excluded from analysis). In all 8 pigs of the LADm group, electrocardiographic recordings obtained 1h after coronary occlusion confirmed MI as represented by STsegment deviations (elevations or depressions; Figure 5). These ST changes resolved completely by day 4 after surgery. Inversions of T waves occurred also. On average, heart rate at baseline was 85.1 ± 3.0 bpm, at 1h after occlusion was 86.7 ± 10.3 bpm, and on day 4 was 105.0 ± 19.0 bpm. Myocardial Revascularization through the Cardiac Venous System Published manuscripts 35 Figure 2. Average variation of troponin I from baseline values to the fifth day, in LADm occlusions. Two pigs are not included: pig 8 did not survive MI and pig 7 was submitted to numerous cardioversion maneuvers and showed extraordinary increase of troponin I. Figure 3. Average variation of myoglobin from baseline to day 5 after LADm occlusion. Two pigs are not included: pig 8 did not survive MI, and pig 7 underwent numerous cardioversion maneuvers and showed extraordinary increase of myoglobin. Myocardial Revascularization through the Cardiac Venous System Published manuscripts 36 Figure 4. Average variation of MMB (mass assay of creatine kinase) from baseline to day 5 after LADm occlusion. Two pigs are not included: pig 8 did not survive MI, and pig 7 underwent numerous cardioversion maneuvers and showed extraordinary increase of myoglobin. Figure 5. Derivation II on ECG recording of pig 6 at 1h after LADm occlusion, showing marked STsegment depression. Echocardiography in all 7 LADm pigs that survived until analysis revealed a decrease in systolic function from baseline to 4d after occlusion, with remarkable wallmotion abnormalities. Akinesis and dyskinesis were predominant at the septal wall (Figure 6). In addition, LADm pigs showed an 18% reduction (P < 0.01) in ejection fraction compared with baseline (from 65.5% to 53.4%), which was accompanied by enlargement (P < 0.01) of the left ventricular diameter from 24.6 to 33.8 mm (a 37% increase); Table 2). The thickness of the septal wall fell (P < 0.01) from 14.9 to 10.3 mm (a 31% decrease), and the thickness of the posterior wall tended (P = 0.09) to increase. Myocardial Revascularization through the Cardiac Venous System Published manuscripts 37 Figure 6. M-mode echocardiography of pig 6 on day 4 after LADm occlusion. IVSd, thickness of interventricular septum at end-diastole; LVIDd, left ventricular internal diameter at end-diastole; LVPWd, left ventricular posterior wall at end-diastole; IVSs, thickness of interventricular septum at end-systole; LVIDs, left ventricular internal diameter at end-systole; LVPWs, left ventricular posterior wall at end-systole; HR, heart rate; EF, ejection fraction; %FS, percentage of fractional shortening. Histopathologic studies after necropsy confirmed complete coronary vessel occlusion in all LADm pigs. All hearts showed some degree of pericarditis, with layers of fibrin covering the epicardium, especially over ischemic areas. Macroscopic analysis of transverse cardiac sections revealed transmural MI located at the anteroseptal myocardial region. The infarcted area was lighter in color than was normal tissue, shrunken, thin, and firm (Figure 7 A). No abnormal changes were detected in any other organ evaluated (lung, liver, and kidney). Histologic total mean infarct size was 14.9% ± 0.8%, with coagulative necrosis (6.8% ± 0.8%) in approximately equal proportion to granulation tissue (8.1% ± 0.6%). All infarcts were transmural in both ventricles, but the left ventricle was always more severely damaged than was the right (11.9% ± 1.0% compared with 3.0% ± 0.3%). Myocardial Revascularization through the Cardiac Venous System Published manuscripts 38 Figure 7. (A) Transverse cardiac slice at the level of the papillary muscles of pig 6 after LADm. A transmural infarct is visible at the left part of interventricular septum and anterior wall of the left ventricle. Dark arrow shows the location of the occluded LAD. (B) Histologic section stained with Masson trichrome, with clear distinction of necrotic tissue (purple), surrounding granulation tissue (gray), and viable myocardium (bright pink). Magnification, 1.0× (A); 3.0× (B). 2.1.5. Discussion In our search for the ideal surgical model of MI, we were confronted with 2 main issues: achievement of a standard insult of MI along with minimal morbidity and mortality. Surgical models are associated with high rates of complications, resulting in high mortality (Dib N, 2006; Krombach GA, 2005; Mitsos S, 2009). We believe mortality rates depend mostly on the occlusion site, duration of occlusion, and the anesthetic strategies and precautions. Two of the 3 pigs in the LCXp group died from ventricular arrhythmias refractory to cardioversion: one during surgery and the other at 21 h after surgery. The remaining pig in this group survived likely due to an individual variation: its LAD was more developed than was typical and thus supplied a wider myocardial region. However, this pig showed distress throughout postsurgical period. In fact, the diagnostic tests confirmed and justified its clinical instability: extremely elevated peaks and AUC of analyzed cardiac biomarkers (Table 1), substantial ST-segment elevation, severe decreases in the motion and thickness of the posterior wall (compensated by increase of the septal wall), along with remarkable enlargement of left ventricular lumen and reduction in ejection fraction (Table 2). The calculated infarct size for this pig was 17.91%, the highest value in our study (Table 3). Myocardial Revascularization through the Cardiac Venous System Published manuscripts 39 Table 3. Ventricular fibrillation (VF) rate, mortality rate, and calculated mean infarct size of pigs with coronary occlusions at different locations. Due to severe left ventricular dysfunction and intractable ventricular arrhythmias, both pigs of the LADp group died shortly after coronary ligation, thereby preventing subsequent diagnostic tests. We therefore abandoned LADp experiments to proceed with more distal occlusions. Only 1 of the 8 LADm pigs died before final evaluation. In the pig that died, the LAD supplied a larger cardiac region than was typical. This pig developed several arrhythmias after coronary occlusion, all of which were reversed successfully. Nevertheless, the pig died 2 h after surgery. One other pig in the LADm group presented with ventricular fibrillation and survived only after several cardioversion maneuvers, including defibrillation. The mortality rate and diagnostic data from the LADm group typically fell between the extremes of those pigs with proximal occlusion (LCXp and LADp) and those with distal occlusion (LADd; Tables 1 through 3). Most changes in cardiac values in LADd pigs were not significant. Contrary to our expectation, the peak values and AUC of myoglobin and MMB of LADd pigs were higher than the ones from the LADm group. This difference is explained by the fact that, due to technical difficulties with the placement of a central venous catheter, blood samples had to be collected from the jugular vein (inducing muscular damage) in 1 LADd pig (Panteghini, 2002; Thygesen K, 2007). Although mortality was 0% in the LADd group, infarct sizes were considered to be too small (9.95% and 10.09%) to be useful, and these experiments were abandoned. As we confirmed for proximal occlusions (LCXp and LADp), if the infarcted myocardial region is too large, pigs develop ventricular fibrillation and die (Peukert D, 2009; Suzuki Y, 2008). Therefore, during the acute phase of MI, these models do not provide enough information from diagnostic tests to correlate with, for instance, experimental groups in which new treatments for coronary heart disease are tried. In Occlusion site VF rate (%) Mortality rate (%) Mean infarct size (%) LCXp N=3 67 67 17.9 LADp N=2 100 100 - LADd N=2 0 0 10.0±0.1 LADm N=8 25 12.5 14.9±0.8 Myocardial Revascularization through the Cardiac Venous System Published manuscripts 40 contrast, if the infarcted area is too small (as in LADd), diagnostic tests will detect only mild changes which may not be significantly different from those obtained from treatment groups. Pigs that underwent LADm surgery seem to offer the optimal balance between low mortality (12.5%) and sufficient infarct size (14.9%). Echocardiographic changes correlated well with histopathologic results, with a reduction of 18% in ejection fraction. The increase in heart rate corroborated left ventricular dysfunction and insufficiency. Ventricular fibrillation occurred in 2 pigs (25%) of the LADm group and resulted only in one death. Intragroup variability in most cardiac data was small, so reproducibility of the swine LADm model is feasible. Our results are in accordance to other studies performed in domestic swine (Table 4). Analysis of Table 4 reveals several important points. First, closed-chest porcine models are associated with high mortality rates (Peukert D, 2009), similar to or even higher than those of open-chest models. Second, independent of method, mortality rates of LCX occlusions (even in its distal part) are higher than those for LADm occlusions. Finally, mortality rates (as regards infarct size) in LADp occlusions are higher than those of LADm occlusions. Table 4. Ventricular fibrillation (VF) rate, mortality rate and calculated mean infarct size in previous porcine models. Occlusion site Method used VF rate (%) Mortality rate (%) Mean infarct size (%) Reference Open-chest models LADp Surgical ligation — >70 — (White FC, 1986) LADm Surgical ligation 34.6 11.5 ≈15.8 (Brooks H, 1975) LADm Surgical ligation 33 19.4 — (Cinca J, 1995) Closed-chest models LADp Balloon inflation 90 66.7 18.3 (Suzuki Y, 2008) LADm Balloon inflation 18.4 16.3 13 (Suzuki Y, 2008) LADm Embolization coils — 13.6 14.8 (Dib N, 2006) LADm Balloon inflation 59.1 31.8 21.5 (Krombach GA, 2005) LCXd Balloon inflation 76.2 52.4 — (Reffelmann T, 2004) LCXd Tungsten spirals 50 27.8 — (Peukert D, 2009) Myocardial Revascularization through the Cardiac Venous System Published manuscripts 47 atraumatic clamp (Geister T03, Tuttlingen, Germany) used at the LIMA was then released, and observation of blood flow from the LIMA to the LADV confirmed the integrity of the anastomosis. Figure 1. Schematic illustration of cardiac vessels, occlusion sites and arteriovenous anastomosis. GCV: great cardiac vein; LAD: left anterior descending coronary artery; LADV: left anterior descending coronary vein; LCx: left circumflex artery; LIMA: left internal mammary artery. Exactly as for the control group, an intravenous injection of 5,000 IU heparin was administered prior to LAD occlusion. This was followed by daily intravenous administration of acetylsalicylic acid until the fifth day after surgery. Chest closure and placement of chest tubes was performed as for the control group. The surviving animals were killed on the fifth day. For comparison of cardiac performance and the affected myocardial area between the two groups, the same experimental conditions were used for both groups, and quantification of cardiac biomarkers, ECGs, echocardiography and detailed histological studies were performed blindly by independent investigators. A brief description of the diagnostic methods is stated here; more details are found in another report (Munz MR, 2011). Myocardial Revascularization through the Cardiac Venous System Published manuscripts 48 Cardiac biomarkers included troponin I, myoglobin, total creatine kinase, isoenzyme MB of creatine kinase (activity and mass assays) and lactate dehydrogenase. Serial blood samples were collected via a central venous catheter prior to surgery, every 2 h during the first 10 h after surgery and then every 24 h until the pigs were killed. Results are presented as the area under the curve (AUC) and peak values of each biomarker. Electrocardiograms were performed prior to surgery and then 1 h and 4 days after surgery, whereas echocardiograms were done before surgery and 4 days later. Pigs were always under general anaesthesia during these examinations. For ECGs, pre-cordial leads were included in order to determine the ventricular wall segment affected more precisely. For comparison of results, the sum of absolute ST segment deviation (elevation or depression) was calculated for all leads that measured 60 ms after the J point relative to the TP segment. With echocardiography, the ventricular wall contraction was visually evaluated, and measurements of left ventricular lumen and wall thicknesses allowed calculation of the ejection fraction, for ascertainment of cardiac performance. Full design and data collection for both ECGs (method GUSTO-I) and echocardiography are described elsewhere (Munz MR, 2011; Hathaway WR, 1998). Histological studies were performed after the pigs were killed on the fifth day. Hearts were collected and fixed in 10% formaldehyde. For confirmation of complete arterial occlusion, the LAD was cannulated and, through injection of saline solution, visual inspection for leaks at the ligature site and absence of filling of distal arterial branches was performed. Likewise, the LIMA was cannulated for injection of saline solution to evaluate patency of the arteriovenous grafts and filling of the distal venous tributaries. The hearts were then sectioned in transverse slices (1cm wide) from the apex to the heart base. From each of these slices, the full basal surface (including septum and both ventricles) was divided into small blocks, which were processed, paraffinized and stained with haematoxylin and eosin and Masson’s trichrome. These stainings allowed recognition of infarcted and healthy myocardial areas, which were measured using specific software (ImageJ ® ) to determine with precision the percentage of infarcted myocardial area in each pig. To enhance the macroscopic difference of the extent of infarcts in the two groups, and for photographic purposes only, three hearts were selected (two from control pigs and one of the experimental group) and stained with 2% 2,3,5-triphenyltetrazolium chloride. The apical surfaces of their transverse slices (where infarcts were visually identified) were incubated in 2,3,5-triphenyltetrazolium chloride at 37ºC for 20 min. With this stain, necrotic myocardium appears pale and viable myocardium stains dark red (Krombach GA, 2005). Myocardial Revascularization through the Cardiac Venous System Published manuscripts 49 Statistics Statistical analysis was performed using the PASW package, version 18.00. All data are presented as means ± SEM. For all the variables included in this study, a Kolmogorov-Smirnov one-sample-goodness-of-fit test was used to confirm normality. Student’s t test for independent samples was used when comparing means. 2.2.4. Results Four animals from each group (control and experimental) died either during or shortly after surgery, owing to technical problems or the MI itself; therefore, we present here the results from nine animals in each group. 2.2.4.1. Electrocardiography Control group. One hour after coronary occlusion, all ECGs presented ST segment deflections, as well as variable changes in heart rate and polarity of the T wave. The average sum of ST segment shift was 7.8±1.9 mm. The ST segment changes disappeared completely by the fourth day postsurgery in six pigs. All pigs presented positive T waves at baseline, which shifted 1 h after coronary occlusion in three animals. Four days later, five pigs presented a negative T wave. The average heart rate was 85.2±3.1 beats/min at baseline and 86.8±8.9 beats/min 1 h after surgery. On the fourth day, it had increased to 115.1±15.4 beats/min. Experimental group. ST deflections were present in all pigs 1 h after surgery. The average sum of ST segment shift was 3.8±1.1 mm. On the fourth day, these ST segment changes were totally resolved in five animals. The T waves were positive in all pigs at baseline. One hour after surgery and at the fourth day, all ECGs except one displayed negative T waves. Heart rate was unchanged from basal values to 1 h postsurgery (80.0±3.3 to 79.1±4.3 beats/min, respectively), but it increased until the fourth day (122.4±8.5 beats/min). 2.2.4.2. Echocardiography Control group. The echocardiography performed 4 days after surgery revealed a severe impairment in contractility of the interventricular septum (example in Fig. 2A and B). The ejection fraction decreased by 21% (P<0.001), with a reduction of 31% (P <0.004) in the thickness of the interventricular septum at end systole and evidence of enlargement Myocardial Revascularization through the Cardiac Venous System Published manuscripts 50 (28%) of the left ventricular lumen (P <0.001; Table 1). The thickness of the posterior wall did not show significant differences (P =0.207; Table 1). Experimental group. After surgery, the ejection fraction was similar to baseline (Table 1; example in Fig. 2C and D). Regarding the measurements of the left ventricular wall and lumen, there were also no changes, as can be seen in Table 1. Figure 2. M-Mode echocardiography in one pig from the control group, at baseline (A) and on the fourth day (B) and in one pig from the experimental group, at baseline (C) and on the fourth day (D). EF: ejection fraction; HR: heart rate; IVSd: thickness of interventricular septum at end diastole; IVSs: thickness of interventricular septum at end systole; LVIDd: left ventricular internal diameter at end diastole; LVIDs: left ventricular internal diameter at end systole; LVPWd: left ventricular posterior wall at end diastole; LVPWs: left ventricular posterior wall at end systole; %FS: percentage of fractional shortening. Table 1. Echocardiographic results at baseline and on the fourth day in each group. EF: ejection fraction; IVSs: thickness of interventricular septum at end systole; LVDs: left ventricular internal diameter at end systole. LVPWs: left ventricular posterior wall at end systole. **P<0.01, ***P<0.001 vs. 0h. Group EF (%) IVSs (mm) LVIDs (mm) LVPWs (mm) 0h 96h 0h 96h 0h 96h 0h 96h Control (mean±SEM; n=9) 59.8 ±2.6 47.2 ±2.4*** 12.2 ±0.7 8.4 ±0.7** 29.1 ±1.6 37.2 ±1.7*** 12.5 ±0.7 14.2 ±1.1 Experimental (mean±SEM; n=9) 56.0 ±3.3 53.9 ±3.4 10.3 ±0.7 10.8 ±1.2 31.5 ±2.4 34.2 ±2.8 12.0 ±0.8 13.5 ±0.6 Myocardial Revascularization through the Cardiac Venous System Published manuscripts 51 2.2.4.3. Histological studies Control group. At post-mortem examination, complete occlusion of the LAD at its mid-point was confirmed in all pigs. Histological results were fairly similar among the different animals. The total measured area of MI was 13.7±1.0%. Within this area, necrotic tissue occupied 5.5±1.0% while the surrounding granulation tissue occupied 8.2±0.5%. Infarcts were transmural in both ventricles, with the main area of lesion being located at the interventricular septum (Fig. 3A). The total infarcted lesion in the left ventricle was more extensive than the one in the right ventricle (14.8±1.2 versus 10.8±1.9%; P=0.048). Granulation tissue was in identical proportion in both ventricles (7.9±0.8% in the left ventricle and 9.3±1.7% in the right ventricle; P=0.47); however, the left ventricle had a much wider necrotic area compared with the necrotic area in the right ventricle (6.8±1.3% and 1.5±0.5% respectively; P=0.03). Experimental group. Post-mortem examination confirmed that total occlusion of the LAD at its mid-point was achieved in all animals, as well as a patent arteriovenous anastomosis. The measured MI lesion in this group was 6.8±1.2%. Within this area, necrotic tissue occupied 1.6±0.5% while the surrounding granulation tissue occupied 5.2±0.7%. Infarcts were transmural in only three hearts. The other six hearts displayed subendocardial or scattered areas of lesion (Fig. 3B). The left ventricle was more affected than the right ventricle (7.7±0.9% vs. 3.9±0.3%; P=0.0026). The granulation tissue was present in identical proportions in both ventricles (5.6±0.6% in the left ventricle and 3.9±1.3% in the right ventricle; P=0.28). In the left ventricle, the area of necrotic tissue was only 2.1±0.7%, while the right ventricle did not present any necrotic areas. Myocardial Revascularization through the Cardiac Venous System Published manuscripts 52 Figure 3. Transverse cardiac slices from one pig in the control group (A) and one pig in the experimental group (B), stained with triphenyltetrazolium chloride. x1.0 magnification. (A) From the total of seven cardiac slices, the five shown here presented lesions; the infarct was transmural in both ventricles and mainly located at the left part of the interventricular septum and anterior wall of both ventricles. (B) From a total of eight cardiac slices, only the three shown here were affected; the area of lesion was hardly recognized, but mainly located at the subendocardial part of the interventricular septum. 2.2.4.4. Cardiac biochemical markers In both groups, the concentrations of cardiac biomarkers increased rapidly within the first 10 h and then slowly decreased until the fifth day. Differences in the curve shape, peak values and AUC were observed between individuals within each group. The AUC and peak values of troponin I, myoglobin and mass assay of creatine kinase (MMB) are shown in Table 2. A B Myocardial Revascularization through the Cardiac Venous System Published manuscripts 53 Table 2. Average peak values and AUC of cardiac biomarkers in each group. AUC - area under the curve; cTnI – concentration of troponin I; MMB - mass assay of creatine kinase; Myo – myoglobin. 2.2.5. Discussion 2.2.5.1. Our optimized model Retroperfusion through the coronary sinus or arterialization of selected cardiac veins has been performed in experimental animal models (and even in a few human patients), mainly during the 1970s. Despite the obsolete diagnostic methods used, several scientists have provided good evidence that, in the acute phase of MI, these techniques are able to perfuse the myocardium, maintain metabolism, limit infarct size and maintain left ventricular function (Bhayana JN, 1974; Gardner RS, 1974; Benedict JS, 1975; Hochberg, 1977; Rhodes GR, 1978; Marco JD, 1977; Hochberg MS, 1979; Park SB, 1975). After the 1970s, research on cardiac venous arterialization was much reduced and only scarce literature can be found, probably as a result of the success of the two newly developed techniques, namely angioplasty and surgical bypass (Krombach GA, 2005; O'Byrne GT, 1991; Resetar ME, 2007; Møller CH, 2008; Harig F, 2011; Oesterle SN, 2003; Raake P, 2005). The most remarkable reports published after the 1970s concern the development of catheter devices to arterialize veins, thereby avoiding surgical invasive approaches (Oesterle SN, 2003; Raake P, 2005). In one of these studies, the catheter was introduced from the proximal LAD to the LADV (Oesterle SN, 2003). However, these trials were not very successful, mainly due to variations in vascular anatomy and technical problems. In the second study, the LADV was arterialized directly from the left ventricle, but the lack of a valve resulted in backflow into the ventricle and low venous pressure (Raake P, 2005). Our research aimed to optimize selective cardiac venous arterialization and measure its benefit. Our strategy is quite different from those reported in previous studies, as follows. Group cTnI Myo MMB Peak (ng/ml) AUC Peak (ng/ml) AUC Peak (ng/ml) AUC Control (mean±SE) N=9 47 ±7 2.989 ±344 703 ±95 10.441 ±874 19 ±5 537 ±190 Experimental (mean±SE) N=9 46 ±8 2.406 ±194 586 ±109 9.915 ±1.788 18 ±6 472 ±114 Myocardial Revascularization through the Cardiac Venous System Published manuscripts 54 (i) We used the pig as animal model, instead of dogs and sheep which were used in former investigations (Hochberg MS, 1979; Resetar ME, 2007). Pigs are a more appropriate experimental model because their arterial and venous cardiac architecture resembles that of humans and they display a relative lack of natural collateral circulation (Munz MR, 2011; Gardner RS, 1974). (ii) We have compared our results with a control group of animals subjected to LAD ligation only. Most previous studies, however, did not establish these comparisons and thus could not really evaluate the benefit of cardiac venous arterialization. Hochberg et al. (1979), Møller et al. (2008) and Harig F et al. (2011) were three of the few groups who performed these comparisons. However, the LAD was occluded more proximally in these studies, which resulted in early death of all control animals, thus impeding the collection and comparison of cardiac data during the following days. (iii) We have applied several more accurate diagnostic examinations to evaluate the benefits of cardiac venous arterialization. Thus, unlike previous studies, quantitative analysis of left ventricular function and precise evaluation of the reduction in infarct size was made possible (Bhayana JN, 1974; Gardner RS, 1974; Benedict JS, 1975; Hochberg, 1977; Rhodes GR, 1978; Marco JD, 1977; Hochberg MS, 1979; Park SB, 1975). (iv) Our team employed up-to-date anaesthetics agents and surgical equipment. Also, as recommended for anastomotic surgeries, heparin and antiplatelet therapy were administered to prevent clotting from interfering with blood flow through the arterialized cardiac veins. (v) In the experimental group, the LIMA-LADV anastomosis was opened immediately after LAD ligation, so the area at risk was never exposed to ischemia. The following two main reasons justified this procedure. (a) Despite the standardized timings of ischemia, the individual LAD distribution creates variable amounts of necrotic tissue before venous arterialization is active. Necrotic tissue is irreversibly recovered and will not be recovered, even if within the area perfused by the arterialized LADV. It would be impossible to perceive whether necrosis was due to the induced ischemia or to inefficiency of the arterialized LADV. (b) Also, the induced ischemia would increase the number of deaths in the experimental group. Would they be caused by a wider LAD arborization, by an abnormally long exposure to ischemia, by an extended surgical procedure and/or increased recovery periods, or simply by the limited revascularization capacity from the arterialized LADV? All these variables would overshadow the real benefit of venous arterialization. In our study, by not inducing ischemia, quantification of the exact perfused/non-perfused myocardium by the arterialized LADV was made clear. Myocardial Revascularization through the Cardiac Venous System Published manuscripts 55 The information provided by our research is limited to the acute phase of MI, and the animals lacked any atherosclerotic disease; therefore, further investigations should focus on the patency of grafts in long-term studies and in atherosclerotic animal models for translation into the ischemic disease in humans. Chowdhry et al. (2005) have experienced poor results with the patency of arteriovenous grafts, although all four patients were free of angina 2 years later. Three reasons might explain this failure of graft patency, as follows: (i) the saphenous vein was used as a conduit between the aorta and the middle cardiac vein, so two anastomoses and a longer circuit were used; ii) in each patient, two additional arterial grafts were performed at major coronary arteries, which could have led to a higher capillary pressure; and iii) collateral circulation might have developed during those 2 years. 2.2.5.2. Analysis of results In our experimental group, three deaths were caused most probably by anatomical variations (thin LIMA, bifurcated LADV and overdeveloped LAD) and one was due to technical problems (after extubation, the position of the epiglottis was changed during transportation and delivery to the animal’s quarters, leading to respiratory arrest and death). Regarding the control group, three deaths were due to the induced MI, while the other one seemed to be a result of technical problems (the pig was not stable enough to be moved to the recovery room). In the experimental group, there was still a clear ST segment deflection 1 h after surgery, indicating the presence of MI. Nevertheless, these changes were less pronounced than in the control group, as revealed by the sum of absolute ST segment shift (50% lower in the experimental group; P=0.038), one of the strongest ECG predictors of mortality (Hathaway WR, 1998). On the fourth day, complete resolution of these ST deflections was recognized in about half of the pigs in each group. Also, in both groups the ECGs presented T wave shifts and tachycardia. Although not as specific as ST segment deflections, these two events are also common in MI (Savonitto S, 1999). After surgery, echocardiography in the experimental group showed that left ventricular wall thicknesses and contractility remained unchanged. The control group, on the contrary, suffered a clear decline in ejection fraction, with thinning of the interventricular septum and enlargement of the left ventricular lumen. In the control group, the tendency for posterior wall thickening seemed to occur in order to compensate for the dysfunction associated with the interventricular septum. Myocardial Revascularization through the Cardiac Venous System Published manuscripts 56 Peak values and AUCs of all cardiac biomarkers analysed did not differ between the two groups (P>0.2). There may be two reasons for this event, as follows: (i) several studies have demonstrated that reperfusion to ischemic tissues may exacerbate or accelerate injury, although the mechanisms are still not fully understood (Mitchell RN, 2003); and (ii) we hypothesize that the impaired venous drainage (LADV was ligated) might have been another possible cause (Savonitto S, 1999). A study by Fredericks S et al. (2001) suggests that troponin I in pigs is a suitable biomarker for myocardial damage, owing to its cardiac specificity. In fact, in their retroperfused group, Harig F et al. (2011) reported a troponin I curve that reached its peak at 4 h and then declined to basal values. They also documented a much lower troponin I concentration in their retroperfused group compared with all other groups at 1 h after surgery. We did not observe any differences between our two groups during the first hour and peak values were reached at the same times. We believe this phenomenon requires further investigation. In our study, because troponin I and MMB are considered to be the most reliable biomarkers for their cardiac specificity and myoglobin can detect MI as early as the first 12 h, only the results for these biomarkers are presented in Table 2. The percentage of histological lesion in the experimental group was 50% lower than in the control group (with P<0.001), with a large differences (70%) in the amount of necrotic tissue (5.5±1.0% vs. 1.6±0.5%; P<0.001). There was also nearly 40% less granulation tissue in the experimental group (8.2±0.5 vs. 5.2±0.7%; P=0.003). In both groups, the area of the lesion was mainly located at the interventricular septum. In conclusion, all our cardiac data evidenced a severe MI in the control group, with high concentrations of cardiac biomarkers, changes in electrical activity reflecting a high value in the sum of ST segment deviations and a strong impairment in contractility of the left ventricle, especially from the interventricular septum. A high percentage of infarcted myocardial lesion (~14%) was identified in those animals. In the experimental group also, MI was identified by the rise of cardiac biomarkers, changes in ECGs and by histological studies, but no dysfunction was revealed by echocardiography on the fourth day. Moreover, the changes detected in ECGs were of lesser amplitude than those of the control group and, most importantly, the percentage of histological lesion was much lower than in the control group. Curiously, cardiac biomarkers remained as high as in the control group and did not correlate with the results from the other diagnostic tests. This phenomenon would require further investigation. Myocardial Revascularization through the Cardiac Venous System Published manuscripts 63 Figure 1. Corrosion cast of arterial (red) and venous (blue) cardiac systems. The arrows indicate postero-lateral and posterior left ventricular stumps which were not filled by methacrylate. Figure 2. Corrosion cast of arterial (red) and venous (blue) cardiac systems: left lateral view (image on the left) and dorsal view (image on the right). Smaller veins, such as apical and septal veins, seem more numerous because methacrylate easily flows through them owing to lack of venous valves. Myocardial Revascularization through the Cardiac Venous System Published manuscripts 64 2.3.4.2. Dissection of cardiac veins Venous valves were present at the openings to the coronary sinus and its tributaries, in all porcine and human hearts analyzed. They were more numerous in pig hearts (7, 7, 8, 10 and 10 per heart) than in the human hearts (6 and 7). Location In both species, the Vieussens valves were always present, as openings to the coronary sinus. All other observed valves were located at (or very close to) the venous ostia (Figs. 3 and 4). Besides the Vieussens valve, all venous valves were located at the openings from second-order tributaries to the great cardiac vein (anterior, anterolateral, lateral, posterolateral and posterior veins) or large second-order tributaries to the anterior and posterior IV veins, in both porcine and human hearts. The ostia of smaller tributaries, such as second-order tributaries located close to the heart apex for instance, did not include venous valves. Also no venous valves were found in third-order tributaries, in any species. Type The morphology of venous valves displayed in pig hearts was consistently flat (Fig. 3). No sinuses were identified. In human hearts, venous valves were very variable in form (Fig. 4). In fact, three types were found: single, semilunar cusp (n=7); endothelial fold (n=3); and double pouched (n=1). At the Vieussens valves, fenestrations and fine strands were also observed (n=2). In both cusped and pouched valves, variable sized intramural collecting chambers or sinuses were identified. Dimension In all porcine venous valves, complete coverage of the venous ostia was observed. In humans, ostial coverage was very variable, from nearly 0% (with small threads of endothelial folds) to close to 100% (in double pouched valves). Through qualitative assessment, we may firmly state that overall ostial coverage was considerably smaller in the analyzed human hearts. Myocardial Revascularization through the Cardiac Venous System Published manuscripts 65 Figure 3. The standard flat configuration of cardiac venous valves found in porcine hearts (x4 magnification). 2.3.5. Discussion In our analyzed porcine casts, we observed variability in the number and distribution of left ventricular tributaries draining to the coronary sinus. This feature is also described in humans (Singh JP, 2005; Ho SY, 2004; Gerber TC, 2001; Abbara S, 2005; M, 1987; Gensini GG, 1965; Loukas M, 2009). However, according to our results and the reviewed literature on human cardiac venous anatomy, we can state that the general location, course and distribution of these cardiac veins are similar in both species (Singh JP, 2005; Ho SY, 2004; Gerber TC, 2001; Abbara S, 2005; Anderson SE, 2009; Bales, 2004; Crick SJ, 1998; Gensini GG, 1965; Loukas M, 2009). Loukas M et al. (2009), for example, also described duplicated anterior IV veins. For vascular casting, our first attempts to inject the whole cardiac venous system through cannulation of the coronary sinus failed. In fact, the venous valves at the ostia of the cardiac veins precluded the access of resin from the coronary sinus to the veins. Thus, cannulation of the coronary sinus was performed only after injection of the great cardiac vein and the posterior IV vein. However, even after taking care of transposing the Vieussens valve and some ostial valves of major left ventricular tributaries (second-order veins), our venous casts were incomplete. In fact, independently of the pressure used for methacrylate injection or the concentration of methacrylate solution, other ostial valves Myocardial Revascularization through the Cardiac Venous System Published manuscripts 66 impeded its progression through some large tributaries, as observed in Fig. 1. This event was less frequent in smaller left ventricular tributaries, where cardiac venous valves are absent, and the resin was able to perfuse these veins (Fig. 2). Alternative methods other than vascular casting have been used for studying the distribution of cardiac veins. Coronary retrograde venography, for example, has been performed since 1950 (Loukas M, 2009). Nevertheless, this technique is also not ideal: it is challenging (due to the tortuosity and pattern of veins) and not able to identify smaller tributaries (Loukas M, 2009). In human hearts, Singh et al. (2005) also mention that retrograde venography is limited by vigorous backwash of the injected contrast (due to the presence of venous valves), which impairs the detailed definition of the cardiac venous system. By use of retrograde venography, both Gerber TC et al. (2001) and Abbara S et al. (2005) report a 5% failure on the identification of the 3 major cardiac veins (great, posterior and anterior IV cardiac veins). Furthermore, over 50% of the smallest and more distal veins could also not be identified. These authors have tried different techniques to optimize venous imaging, by use of electron beam computed tomography and 16multidetector row computerized tomography, but both were considered suboptimal for visualization of smaller tributaries (Gerber TC, 2001; Abbara S, 2005). Kini S et al. (2007) did not get better results using a high resolution 64-multidetector row computerized tomography. Echocardiography, on the other hand, is an imaging method unable to display lateral branches and catheters create visual obstructions (Loukas M, 2009). Thus, although injection/corrosion may not fill larger tributaries due to the presence of venous valves, no other imaging method has been found to be ideal for studies on cardiac venous anatomy. In the present study, we have found differences in number, type and dimension of venous valves between porcine and human hearts. We now compare our results to available and pertinent literature on cardiac venous valves. Frequency According to former anatomical studies, the frequency of Vieussens valves is inconstant in humans, as observed in Table I. Venous valves within all major left ventricular veins (anterior and posterior IV veins, posterolateral and posterior veins) have been characterized and quantified by Anderson SE et al. (2008) through use of endoscopy. These authors have found venous valves only in 89% of the human hearts analyzed. Their results showed a prevalence of venous valves in human hearts of around 5,5 per male heart and 2,3 per female heart. Myocardial Revascularization through the Cardiac Venous System Published manuscripts 67 Although our sample size on human hearts was small due to difficulties in obtaining whole fresh human hearts, the values in our study were higher (6 and 7 venous valves per heart). This difference could be explained by the impediment of the fiberscope used by Anderson et al. (2008) in accessing tortuous or thinner veins. In agreement with our study, these authors also conclude that smaller tributaries possess fewer valves. In accordance to our study, Pan-Chih et al. (1994) also documented a higher number of venous valves in pig hearts than in adult human hearts. Type The variability in shape of venous valves found in our examined human hearts is also documented in several other studies, as can be observed in Table I (Ho SY, 2004; Karaca M, 2005; Zawadzki M, 2004; Cendrowska-Pinkosz M, 2004; Loukas M, 2009). Zawadzki M et al. (2004) have observed the number of leaflets on the Vieussens valve and described them as flat or concave. They found single, double and one case of triple leaflet valves. Most leaflets were concave. These authors believe that the concave shape of leaflets is more likely to impede the advancement of catheters into the great cardiac vein. As for venous valves in second-order ventricular veins, Ho SY et al. (2004) describe them as “flimsy endothelial ridges” present in venous ostia. Loukas M et al. (2009) also describe human venous valves within left ventricular tributaries. Their findings are similar to ours. They identified two types of venous valves: unifoliate (mostly) and bifoliate, but considered them both incompetent. Table I. Frequency and number of leaflets in Vieussens valves found in human hearts. Authors Present Single leaflet Double leaflet Triple leaflet Karaca et al. (2005) 75% - - - Cendrowska-Pinkosz M et al. (2004) 92% - - - Zawadzki et al. (2004) 78% 36% 40% 2% Duda and Grzybiak (2000) 65.1% 34.6% 29.9% 0.6% von Lüdinghausen M (1987) 87% 62% 25% <1% Silver and Rowley (1988) 76% 20% 56% 0 Myocardial Revascularization through the Cardiac Venous System Published manuscripts 68 Dimension Some studies looked into the dimension of valves and their degree of coverage of ostia rather than categorizing their type, as they consider this to be of worse prognostic for catheterization of the cardiac veins (Karaca M, 2005; Duda B, 2000). In accordance to our study, different authors also document high variability on the dimension of human venous valves, ranging from 0 to 100% coverage of ostia (Karaca M, 2005; Anderson SE, 2007; Hill AJ, 2003; Loukas M, 2009). Conclusions The overall anatomical arrangement of the cardiac venous system can be considered similar in both human and porcine hearts, although it is very variable in both species. However, several differences were encountered between the two species concerning the prevalence, type and dimension of cardiac venous valves. These differences were found experimentally on analyzed hearts, but were confirmed in the reviewed literature. The porcine hearts displayed a higher prevalence of venous valves, presented no variability in shape, location or dimension, and covered completely the venous ostia. The human venous valves, on the other hand, were fewer, much more variable in shape and were mostly considered incompetent due to their shape and small dimension. Facing these results, we may state that the domestic pig might underestimate the benefits of retrograde flow, due to the presence of more frequent and competent venous valves. If pigs are to be used in these studies, then we can only presume that a better outcome should be expected in human trials. Myocardial Revascularization through the Cardiac Venous System Published manuscripts 70 Chapter 3 - Discussion Myocardial Revascularization through the Cardiac Venous System Discussion 79 3.2.2. Research progress Throughout the research, the surgical technique has been modified and the analytical diagnostic methods improved in order to optimize cardiac venous arterialization and to evaluate its putative benefits. The project underwent 5 different phases, which are described below. In each phase, the anastomosis of pulmonary vein/systemic artery to the cardiac venous system was opened right after ligation of LCX/LAD. This allowed the exact quantification of perfused/non perfused myocardium by the arterialized cardiac veins. 3.2.2.1. Phase I In the beginning, we have followed the technique proposed by Grande NR et al. (1970), by anastomosing one left pulmonary vein to the great cardiac vein, and performing a proximal ligation of LCX in two pigs. Also the great cardiac vein was ligated close to its opening into the coronary sinus. Due to the distance between the two vessels, a Gore-tex connector was used for the anastomosis. This model was not successful. One animal died 8 h after the surgery. The other pig survived, despite the extreme high levels of cardiac biomarkers at 12-24 h after surgery and the evidence of contractility impairment of the free ventricular wall on echocardiographies. At necropsy of both pigs, the anastomosis was not patent and the affected lung lobe was congested, as well as the entire cardiac venous system. Significant transmural infarcts were macroscopically visible at the lateral wall of the left ventricle and, from histological studies, the infarct size measured was as high as 18% in the surviving pig. Facing these unsuccessful results, we have postulated that the low venous pressure from the pulmonary vein was the most probable cause, as also proposed by other authors (Oh BH, 1992). Furthermore, Gore-tex is a potential thrombogenic material as compared to autologous vessels. The technique was revised and we stepped into phase II. Myocardial Revascularization through the Cardiac Venous System Discussion 80 3.2.2.2. Phase II In order to solve both above-mentioned experimental problems, our surgeon suggested a direct anastomosis of a systemic artery to the great cardiac vein. The pressure would be higher and heterologous material would not be used. Thus, in phase II, the left internal mammary artery (LIMA; so-called left internal thoracic artery in pigs) was grafted to the great cardiac vein and the LCX was ligated proximally. The results were more promising with this technique: ST segment elevations were absent, and concentrations of cardiac biomarkers, changes in echocardiographic data and infarct size were lower than in the control group. Nevertheless, mortality in the control group was too high, as demonstrated in our previous work (Munz MR, 2011). Thus, to reach statistical significance, massive number of animal deaths would be required. 3.2.2.3. Phase III Having in mind the higher percentage of MIs in the LAD (named interventricular paraconal artery, in pigs), our surgeon suggested the ligation of the LAD rather than the LCX. Moreover, we agreed that the ligation of the LAD vein (LADV) instead of the great cardiac vein would restrict the circulation of blood to a smaller venous myocardial territory, with expected improvements on the outcome of revascularization. Thereby, in this phase, the pigs were submitted to a LIMA-LADV anastomosis, with proximal ligation of the LAD and adjacent LADV. The effects of such occlusions were violent and devastating, with only one survival from a total of 5 animals. The surviving pig presented evident ST segment elevation and inversion of T waves on ECGs, and very high levels on concentrations of cardiac biomarkers post-operatively. Echocardiographies on the 4 th day presented clear akinesia of the interventricular septum, with reduction of its thickness and of the calculated ejection fraction. Infarct size measured by histological studies was nearly 25%! The deaths of the remaining 4 pigs were happening so quickly that no cardiac data from diagnostic tests was obtained after the surgery. Hence, no comparison to the control group was possible. In addition, also all animals in the control group died from massive MIs, as shown in our previous study (Munz MR, 2011). In fact, the territory supplied by the LAD is superior to the one from the LCX, so this outcome was predictable. We only had mortality rate as a comparison factor between the 2 groups, and this rate was still too high for the Myocardial Revascularization through the Cardiac Venous System Discussion 81 experimental group. We considered that continuing with these experiments would only raise the number of deaths and ethical issues would arise, so we abandoned this phase. 3.2.2.4. Phase IV The surgical protocol was amended once again. In this phase, we agreed to ligate the distal third of the LAD in order to drastically reduce infarct size and, thus, avoid high mortality rates. As foreseen, all pigs survived in this phase. In the experimental group, the slight ECG changes were stabilized by day 4, peak values of cardiac biomarkers were rather low and no qualitative (observable contractility) or quantitative changes (ejection fraction or wall thicknesses) were observed at echocardiographic examinations. The infarct size was quite small, with values under 10% (around 3, 8 and 9%). However, because the infarct size was relatively small also at the control group (10% in both pigs), results from diagnostic exams were quite subtle and not significantly different from the experimental group (Munz MR, 2011). Additionally, some discrepancies in cardiac data (such as elevation of troponin I over unchanged concentrations of other cardiac biomarkers, for instance) were also occurring, probably due to variability in individual arterial distribution or in pharmacological reactions. Before exponentially increasing the number of animals to attain significant differences between the two groups, we have decided to increase the infarct size, expecting to show clearer evidences on the benefit of cardiac venous arterialization. 3.2.2.5. Phase V In this final phase, our efforts were finally rewarded. Pigs underwent ligation of the LAD at its middle point and adjacent LADV was anastomosed to the LIMA. As in previous phases, LADV was ligated proximally to this arteriovenous graft to avoid blood backflow towards the great cardiac vein and into the coronary sinus. These pigs seemed to offer the optimal balance between low mortality and sufficiently large infarct size, i.e., the infarcted area was wide enough to reveal significant differences between the experimental and control groups. In our experimental group, three deaths were caused most probably by anatomical variations (thin LIMA, bifurcated LADV and overdeveloped LAD) and one was due to Myocardial Revascularization through the Cardiac Venous System Discussion 82 technical problems (after extubation, the position of the epiglottis was changed during transportation and delivery to the animal’s quarters, leading to respiratory arrest and death). Regarding the control group, three deaths were due to the induced MI, while the other one seemed to be a result of technical problems (the pig was not stable enough to be moved to the recovery room). In the experimental group, there was still a clear ST segment deflection one hour after surgery, indicating the presence of MI. Nevertheless, these changes were less pronounced than in the control group, as revealed by the sum of absolute ST segment shift (50% lower in the experimental group; P=0.038), one of the strongest ECG predictors of mortality (Hathaway WR, 1998). On the fourth day, complete resolution of these ST deflections was recognized in about half of the pigs in each group. Also, in both groups the ECGs presented T wave shifts and tachycardia. Although not as specific as ST segment deflections, these two events are also common in MI (Savonitto S, 1999). After surgery, echocardiography in the experimental group showed that left ventricular wall thicknesses and contractility remained unchanged. The control group, on the contrary, suffered a clear decline in ejection fraction, with thinning of the interventricular septum and enlargement of the left ventricular lumen. In the control group, the tendency for posterior wall thickening seemed to occur in order to compensate for the dysfunction associated with the interventricular septum. Peak values and AUCs (areas under the curve) of all cardiac biomarkers analysed did not differ between the two groups (P>0.2). We hypothesized two reasons for this surprising event, as follows: (i) several studies have demonstrated that reperfusion to ischemic tissues may exacerbate or accelerate injury, although the mechanisms are still not fully understood (Mitchell RN, 2003); and (ii) we hipothesize that the impaired venous drainage (LADV was ligated) might have been another possible cause (Savonitto S, 1999). A study by Fredericks S et al. (2001) suggests that troponin I in pigs is a suitable biomarker for myocardial damage, owing to its cardiac specificity. In fact, in their retroperfused group, Harig F et al. (2011) reported a troponin I curve that reached its peak at 4 h and then declined to basal values. They also documented a much lower troponin I concentration in their retroperfused group compared with all other groups at 1 h after surgery. There were no more comparisons after one hour. We did not observe any differences between our two groups during the first hour, and peak values were reached at the same times. We believe this phenomenon requires further investigation. The percentage of histological lesion in the experimental group was 50% lower than in the control group (with P <0.001), with a large difference (70%) in the amount of necrotic tissue (5.5±1.0% vs. 1.6±0.5%; P<0.001). There was also nearly 40% less Myocardial Revascularization through the Cardiac Venous System Discussion 83 granulation tissue in the experimental group (8.2±0.5 vs. 5.2±0.7%; P=0.003). In both groups, the area of the lesion was mainly located at the interventricular septum. In conclusion, all our cardiac data evidenced a severe MI in the control group, with high concentrations of cardiac biomarkers, changes in electrical activity reflecting a high value in the sum of ST segment deviations and a strong impairment in contractility of the left ventricle, especially of the interventricular septum. A high percentage of infarcted myocardial lesion (~14%) was identified in those animals. In the experimental group also, MI was identified by the rise of cardiac biomarkers, changes in ECGs and by histological studies, but no dysfunction was revealed by echocardiography on the fourth day. Moreover, the changes detected in ECGs were of lesser amplitude than those of the control group and, most importantly, the percentage of histological lesion was much lower than in the control group. Curiously, cardiac biomarkers remained as high as in the control group and did not correlate with the results from the other diagnostic tests. This phenomenon would require further investigation. 3.3. Diagnostic exams used In the past, several diagnostic procedures have been used in different studies to evaluate the benefits of cardiac venous arterialization. Nevertheless, most of them could not accurately quantify myocardial perfusion (like ECG, angiography, pressure and blood flow measurements) or the size of MI (macroscopic histopathological studies) (Bhayana JN, 1974; Gardner RS, 1974; Chiu CJ, 1974; Benedict JS, 1975; Hochberg, 1977; Rhodes GR, 1978; Hochberg MS, 1979; Marco JD, 1977; Park SB, 1975). Besides, most did not evaluate ventricular function. In our research, we have used up-to-date diagnostic methods to evaluate cardiac function and infarct size in each animal, including cardiac biochemical markers, ECG, echo imaging and pathological studies. In fact, the serial measurements of cellular damage, the evaluation of cardiac electrical activity, of left ventricular performance and the measurement of histological infarct size, should allow for a precise quantification of infarct size. In phase V, the estimation of infarct size was considered to be reliable, because intragroup variation was negligible in all diagnostic tests. Besides providing important data on cardiac morphology and function, these diagnostic tools used are simple, readily available, noninvasive, and applicable in routine clinical practice. Throughout the research, each diagnostic exam was refined in different ways, as follows. Myocardial Revascularization through the Cardiac Venous System Discussion 84 3.3.1. Cardiac biomarkers The porcine species has few available superficial veins. Initially, in our research, blood collections for analysis on cardiac biomarkers were all performed at the external jugular vein. However, this procedure was arduous and blood collections had to be performed frequently, so we decided to use central venous catheters. After several unsuccessful attempts to introduce and use these catheters in an auricular vein, we have used a lateral saphenous vein, with excellent results. As an additional advantage, animal restraint would be unnecessary by use of this latter vein, as mentioned before. During research, we have also increased the number of blood samples during the first 24 hours in order to capture the true up-slopes and peak values of cardiac biomarkers (and thus more accurately estimate the infarct size) (Gibbons RJ, 2004). Serial blood samples (approximately 3 mL each) were collected prior to surgery, every 2 hours during the first 10 hours after surgery, at 24 hours after surgery, and then once daily until euthanasia, by use of the central venous catheter. Blood samples were centrifuged at 3100 × g for 5 min within 30 min of sampling, and the serum was stored in 2 microfuge tubes at −4 °C. The analysis included assessment of troponin I, myoglobin, total creatine kinase, isoenzyme MB of creatine kinase (measured by mass and activity assays), and lactate dehydrogenase. Yet, analysis focused on the combination of 3: myoglobin (helpful in early detection of MI), cardiac troponin I (a marker that takes longer to rise but is highly specific) and MMB, as mass assay of creatine kinase (considered as effective as cardiac troponins in estimating infarct size) (Panteghini, 2002; Thygesen K, 2007). Results were presented as AUC and peak values of each biomarker. Myoglobin, cardiac troponin I and MMB were determined by enzyme immunoassays which were performed according to the manufacturer’s instructions. 3.3.2. Electrocardiographic evaluation For electrocardiographic studies in animals, it is a common veterinary practice to include only leads I, II, III, AvF, AvL and AvR. In this research, however, by using a multichannel electrocardiography analyzer, precordial leads were included to determine the ventricular wall segment affected more precisely. Design and data collection for ECGs was according to method GUSTO-I (Hathaway WR, 1998). Myocardial Revascularization through the Cardiac Venous System Discussion 85 At baseline, with the pigs under general anesthesia and in right lateral recumbency, 12-lead electrocardiographic study was performed. To detect the maximal value of ST deviation, electrocardiography was repeated 1 h after coronary occlusion (with pigs still under anesthesia), by using the same technical approach (Smith GT, 1979). For comparison of results, the sum of absolute ST segment deviation (elevation or depression) was calculated for all leads that measured 60 ms after the J point relative to the TP segment. To repeat ECGs on the fourth day after surgery, we have initially sedated the animals and avoided a second anesthesia. However, pigs had variable reactions to the sedatives and some would require second and third boluses. Therefore, to have fair comparisons to the values at baseline and 1 h after surgery, we agreed that the conditions should be kept the same, so we decided to entubate and keep the animals under general anesthesia on the fourth day as well. To avoid operator bias, both baseline and postocclusion results from ECGs were analyzed by independent observers who were blinded to groups and the focus of the investigation. 3.3.3. Echocardiographic evaluation At baseline, 2D and M-mode echocardiographic evaluations were performed with the pigs under general anesthesia and in right lateral recumbency, Transthoracic echocardiography (Vivid 3 Pro digital ultrasonic scanner, GE Medical Systems, Ontario, Canada) allowed assessment of myocardial echogenicity, cardiac chamber size, wall thicknesses and amplitudes of motion, and valve morphology and motion. Wall and cavity dimensions allowed the calculation of indices of cardiac motion (shortening fraction and ejection fraction). Hence, qualitative and quantitative assessment of left ventricular function was made possible. For these measurements, short-axis views were obtained at the level of the chordae tendineae, by using the right parasternal window. To avoid intraobserver variability, 3 different M-mode images were obtained for each pig, and the mean of these 3 measurements was calculated. To estimate left ventricular performance, the recommendations from ASE Committee for echocardiographic measurements of left ventricular dimensions, volumes, and wall thicknesses were followed (Lang RM, 2005). On day 4 after surgery, pigs were again sedated and anesthetized for reevaluation of cardiac performance. Echocardiography was repeated as performed at baseline by using the same technical approach. As for ECGs, echocardiographic results at baseline and post-occlusion were analysed by independent observers. Myocardial Revascularization through the Cardiac Venous System Discussion 86 In phase V, we have also used planimetric measurements (modified Simpson’s rule) through apical 4-chamber or 2-chamber views, but unfortunately the narrow acoustic windows precluded a reasonable amount of observations. We could only analyze 4 animals in the control group and 6 from the experimental group. The mean values for ejection fraction are concordant with the data provided through the Teichholz formula: a strong decline in ejection fraction was observed at the control group (from 52.4% to 37.4%), while the experimental group remained unchanged (from 51.0% to 47.0%). However, the small sample size in the control group prevented the observed reduction to reach statistical significance (p=0.2). 3.3.4. Histopathological studies After euthanasia of pigs on day 5 after surgery, hearts were excised immediately, cleaned, and washed in saline solution for macroscopic and histologic evaluation. For confirmation of complete arterial occlusion, the LCX/LAD was cannulated and, through injection of saline solution, visual inspection for leaks at the ligature site and absence of filling of distal arterial branches was performed. Likewise, the LIMA was cannulated for injection of saline solution to evaluate patency of arteriovenous grafts and filling of the distal venous tributaries. Hearts were stored in 10% buffered formaldehyde for 24 h. Each heart was then filled and embedded in agar–agar suspension at 4%, which was allowed to harden by incubating for 4 h at 4 °C. The hearts were sliced circumferentially parallel to the atrioventricular sulcus from the apex to the base. Each slice was 10 mm thick. A special device was designed and built for this purpose. The basal surface of each slice was photographed with a digital camera. From each of these slices, the full basal surface (including septum and both ventricles) was divided into small, transmural, 2to 3-mm thick blocks. These serial blocks were processed, paraffinized and stained with hematoxylin-eosin and Masson trichrome. Optimization of timings and stains was necessary for successful preservation and coloration of tissues. Masson trichrome provided a clear contrast of colours between viable myocardium (bright red to pink), rim of infarct with inflammatory tissue (gray), and infarction (purple to blue) (Ouyang J, 2009). We used Image J software (NIH, Bethesda, MD) to measure these 3 regions in each trichrome-stained sample by tracing the corresponding tissue boundaries. Thus, the percentage of infarcted myocardial area in each pig was determined with precision. Infarct size of both ventricles was expressed as Myocardial Revascularization through the Cardiac Venous System Discussion 87 the percentage of affected myocardial area (necrosis + inflammatory tissue) in all myocardial areas analyzed, with infarct area % = infarct area × 100 / total myocardial area. Once again, an independent blinded investigator calculated mean infarct size through histological studies. To enhance the macroscopic difference of the extent of infarcts in the two groups, and for photographic purposes only, some hearts were selected (from both control and experimental group) and stained with 2% 2,3,5-triphenyltetrazolium chloride. The apical surfaces of their transverse slices (where infarcts were visually identified) were incubated in triphenyltetrazolium chloride at 37ºC for 20 min. With this stain, necrotic myocardium appears pale and viable myocardium stains dark red (Krombach GA, 2005). Methylene blue colorations were also experimented but the outcome was worse. 3.4. Understanding the incomplete success of cardiac venous arterialization 3.4.1. Variations in the distribution of epicardial veins, and frequency and morphology of competent valves may affect myocardial revascularization Cardiac venous arterialization was expected to be a major success, facing the evidence that veins are spreading through similar myocardial extensions as arteries and that they do not develop atherosclerosis (Chiu CJ, 1974). Actually, Kassab GS et al. (2008) documented a larger surface area of venules compared to the arteriolar surface, owing to the higher number of venules in general (two venules per arteriole). Nevertheless, our results from cardiac venous arterialization were not 100% satisfactory. Although ventricular function was completely recovered, small endocardial infarcts were still present within the area at risk and cardiac biomarkers (such as troponin I) have still reached high concentration levels. During surgery, we have visually perceived that a few epicardial tributaries (diagonal veins) were not becoming filled with blood flowing retrogradely. We suspected that this “failure” for complete myocardial revascularization in the domestic pig could be explained by the presence of venous valves as anatomical obstacles. Indeed, these valves have been held responsible for complicating several cardiovascular procedures using retrograde flow through the cardiac venous system Myocardial Revascularization through the Cardiac Venous System Discussion 88 (namely drug delivery, retrograde cardioplegia administration and cardiac resynchronization therapy), by hindering the entrance or advancement of catheters, pacing leads or guide wires (Anderson SE, 2007; Shinbane JS, 2004). Most cardiovascular experimental studies have used the pig as animal model, due to its close similarities to humans. However, comparative studies have usually only addressed the arterial coronary system (Rodrigues M, 2005; White FC, 1986; Sahni D, 2008; Fozzard, 1975). We found no available literature describing the porcine cardiac venous anatomy in detail. In a recent study, we have sought for establishing comparisons between human and pig cardiac venous systems. Through basic anatomical techniques, such as dissection and corrosion casting, we have studied the architectural distribution of the porcine cardiac veins, focusing on the major left ventricular veins and also reviewed the anatomical barriers which may impair retrograde flow through cardiac veins. For this latter purpose, we investigated and compared the prevalence and type of venous valves in porcine and human hearts. Literature available on porcine cardiac venous valves is extremely rare. As for humans, the best known are the Thebesian and Vieussens valves, which are described by many authors (Ho SY, 2004; Karaca M, 2005; Zawadzki M, 2004; Shinbane JS, 2004; Hill AJ, 2003; Hellerstein HK, 1951; Loukas M, 2009). The Thebesian valve refers to the opening of the coronary sinus to the right atrium, whereas the Vieussens valve is the ostial valve of the great cardiac vein (Karaca M, 2005; Zawadzki M, 2004; Anderson SE, 2007; Loukas M, 2009). Information about valves in second-order ventricular tributaries (epicardial tributaries draining to the anterior and posterior interventricular veins and great cardiac vein), though, is rarely found in human anatomic literature. In this study, we have categorized and compared cardiac venous valves in both species, through anatomical dissections in hearts and revision of available literature. The results from this study have reaffirmed the resemblance on the overall anatomical arrangement of the cardiac venous system in human and porcine hearts, even though individual variability is observed within each species. However, several differences were encountered concerning the prevalence, type and dimension of cardiac venous valves in pigs and humans. These differences were found experimentally on analyzed hearts, but were confirmed in the reviewed literature. The porcine hearts displayed a higher prevalence of venous valves, presented no variability in shape, location or dimension, and covered completely the venous ostia. The human venous valves, on the other hand, were fewer, much more variable in shape and were mostly considered incompetent due to their shape and small dimension. 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