Anatomia, Histologia, Embryologia
ORIGINAL ARTICLE
The Pulmonary Veins of the Pig as an Anatomical Model for the Development of a New Treatment for Atrial Fibrillation T. Vandecasteele*, K. Vandevelde, M. Doom, E. Van Mulken, P. Simoens and P. Cornillie Address of authors: Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
*Correspondence: Tel.: +3292647713; fax: +3292647790; e-mail: [email protected] With 11 figures and 1 table Received November 2013; accepted for publication November 2013 doi: 10.1111/ahe.12099
Summary The layout of the porcine atriopulmonary junction and immediately adjacent structures was investigated by gross anatomical and vascular corrosion casting studies to meet the need for more in-depth anatomical insights when using the pig as an animal model in the development of innovative approaches for surgical cardiac ablation in man. The veins from the right cranial and middle lung lobes drain through a common ostium in the left atrium, whereas a second ostium receives the blood returning from all other lung lobes, although limited variation to this pattern was observed. Surrounding anatomical structures that are most vulnerable to ablation damage as reported in man are located at a safer distance from the pulmonary veins in pigs, yet a certain locations, comparable risks are to be considered. Additionally, it was histologically confirmed that myocardial sleeves extend to over a centimetre in the wall of the pulmonary veins.
Introduction Atrial fibrillation is the most commonly diagnosed cardiac arrhythmia responsible for substantial mortality and morbidity in man. The incidence increases with age and can be associated with several internal diseases or genetic disorders (Brugada et al., 1997; Kannel et al., 1998). Focal triggering sites of cardiac arrhythmia may be resided in various anatomical structures such as the crista terminalis, the ostium of the coronary sinus, the interatrial septum and the atrial free wall (Haissaguerre et al., 1996, 1998; Chen et al., 1999; Hsieh et al., 1999; Pappone et al., 2000; Cappato et al., 2003; Gerstenfeld et al., 2003; Nanthakumar et al., 2004) as well as in myocardial sleeves located in the pulmonary veins. The latter structures are myocardial fibres spreading out from the left atrium into the wall of the pulmonary veins and are believed to be one of the most important origin of ectopic electric pulses (Nathan and Gloobe, 1970; Haissaguerre et al., 1998; Chen et al., 1999; Saito et al., 2000; Roux et al., 2004). These myocardial fibres act as an ectopic pacemaker transmitting electrical pulses which interfere with the pulses originating from the sinoatrial node, disturbing the normal heart rhythm and inducing atrial fibrillation © 2013 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 1–12
in humans (Haissaguerre et al., 2000; Saito et al., 2000; Tagawa et al., 2001). The sleeves exhibit different arrangement patterns in humans and in various mammals. Patients suffering from atrial fibrillation are often not treated when their quality of life is not affected, or they receive only pharmacological treatment when a surgical intervention is not considered appropriate. When a pharmacological approach and electric cardioversion are unsuccessful and return to sinus rhythm is thought to be necessary, ablation procedures can be applied by creating local transmural lesions at the left atrial ostia draining the pulmonary veins to eliminate the influence of ectopic myocardial sleeve pacemakers (Haissaguerre et al., 1998; Haissaguerre et al., 2000 Chen et al., 1999; Pappone et al., 2000). Those procedures still have some disadvantages because they require difficult and prolonged interventions and are therefore more prone to failure, which may lead to conduction recurrence and major complications (Haissaguerre et al., 1996; Robbins et al., 1998; Pappone et al., 1999; Mohr et al., 2002) including phrenic nerve injuries (Lee et al., 2004; Bunch et al., 2005; Okumura et al., 2008, 2009; Ahsan et al., 2010; Andrie et al., 2012), atrio-oesophageal fistulae (Gillinov et al., 2001; Sonmez et al., 2003; Pappone et al., 2004) and
1
The Pulmonary Veins of the Pig
pulmonary vein stenosis (Robbins et al., 1998; Chen et al., 1999; Arentz et al., 2003). To perform research into possible treatments for atrial fibrillation in humans, a suitable animal model is needed. The porcine model has multiple times been proven to be a good animal model for humans because of comparable body size and numerous anatomical, immunological, biochemical, physiological and genetic similarities between pigs and humans (Gregg et al., 1980; Shulman et al., 1988; Delange et al., 1992; Bermejo et al., 1993; Rowan et al., 1994; Jones et al., 1999; Paterson et al., 2002; Sommerer et al., 2004; Perry et al., 2005; Barker et al., 2006; Brunet et al., 2006; Ibrahim et al., 2006; Glenny et al., 2007; Hotchkiss et al., 2007; Wang et al., 2007; Rogers et al., 2008; Khatri et al., 2010). The porcine heart, of which the microanatomy is representative for the mammalian heart, has an average weight of approximately 300 g, which is similar to the average weight of the human heart (approximately 300 g in men and 250 g in women) (Barone, 1997). The development of an appropriate animal model for ablation therapy studies requires in-depth data on the fine anatomical and histological architecture of the pulmonary veins and the myocardial sleeves and on the consequences of a heating process at the level of this myocardial tissue in pigs. This essential information is indispensable when approaching the ostia of the pulmonary veins from the left atrium to perform an intraluminal ablation at this level, but is lacking in current literature. Therefore, in the present study, the microanatomy of the porcine pulmonary veins was investigated to document the atriopulmonary junction in swine. This includes the number of pulmonary vein ostia, the different pulmonary veins draining into their specific ostia, the branching pattern of the various pulmonary veins and its variability, and the position of the pulmonary veins in relation to their surrounding structures such as the trachea, the oesophagus and the pulmonary arteries. These parameters, together with previous data provided by Vollmerhaus et al. (1999), who described the relation between the pulmonary arteries and the veins, between the pulmonary arteries and the trachea, and between the pulmonary veins and the trachea, are essential for the use of the porcine animal model, enabling an intraluminal ablation procedure of the pulmonary veins in swine.
Materials and Methods Anatomical dissection The anatomical examination was performed on cardiopulmonary sets of healthy pigs of 20–40 kg used for other studies and of pigs of approximately 100 kg obtained from
2
T. Vandecasteele et al.
the slaughterhouse (Table 1). The fresh cardiopulmonary sets were incised from the left ventricle into the left atrium to get an overview of the draining area of the pulmonary veins at the roof of the left atrium. Subsequently, the ostia and the pulmonary veins were incised longitudinally to provide an overview of the more distally located branches and to determine both the branching pattern of the pulmonary veins and their drainage area. Silicone casting Before casting the pulmonary veins with silicone, the left atrioventricular orifice was occluded by placing a clamp at the level of the coronary groove. Thereafter, both components of the silicone (base and catalyst, ratio 1:1) were combined and coloured by adding the desired dye to indicate different structures. All pulmonary veins were casted with blue silicone [ZA22, 22 ShA silicone (type 1); Zhermack, Badia Polesine, Italy], unless indicated otherwise, whereas the pulmonary arteries and bronchi were, respectively, casted with red and white silicone [HT33, 30 ShA silicone (type 2); Zhermack]. Subsequently, the silicone was injected into the left atrium, and the pulmonary veins through a blunt needle inserted through the left auricular wall. Three cardiopulmonary sets (from two pigs of approximately 100 kg and one piglet of 20 kg) were casted with silicone (250 and 75 ml of both components for the large and smaller pigs, respectively) to visualize the pulmonary veins. The pulmonary trunk was transversely disconnected from the right ventricle by placing a clamp near the arterial cone to be able to cast the pulmonary arteries. Subsequently, silicone was injected through a blunt needle placed in the pulmonary trunk near the applied clamp. The trachea was casted by injection of silicone through a blunt needle placed in the trachea approximately 5 cm
Table 1. Number of cardiopulmonary sets used in the different techniques and for the various silicone casts Number of cardiopulmonary sets used Anatomical dissection Silicone casting of the pulmonary veins Silicone casting of the pulmonary veins, pulmonary arteries and trachea Silicone casting of the pulmonary veins and arteries, trachea, oesophagus and aorta Silicone casting of complete hearts Silicone casting of the pulmonary veins in relation to the phrenic nerves Total number of used cardiopulmonary sets
57 3 2 1 2 2 67
© 2013 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 1–12
T. Vandecasteele et al.
cranial to the origin of the tracheal bronchus, after the trachea was transversely clamped just cranial to the injection spot. To visualize the position of the oesophagus in relation to the pulmonary veins and the other adjacent structures, a plastic tube of 20 cm was inserted in the oesophagus. Cardiopulmonary sets of pigs of 35 kg were casted to demonstrate the pulmonary arteries (100 ml of both components), the pulmonary veins (100 ml of both components) and the trachea and bronchi (150 ml of both components). A silicone cast of a cardiopulmonary set of a pig of 30 kg was made to visualize the pulmonary veins (100 ml of both components), pulmonary arteries (100 ml of both components), trachea and bronchi (150 ml of both components) and the oesophagus and aorta. The cardiopulmonary sets of pigs of 30 kg were casted in situ with silicone (100 ml of both components) to visualize the ostia of the pulmonary veins in relation to the phrenic nerves after opening the thorax bilaterally. The position of both phrenic nerves was determined by removing the pericardium and the walls of the left auricle and the pulmonary veins surrounding the hardened silicone. To localize the position of both ostia on the heart base and in relation to the other arterial and venous structures, cardiopulmonary sets of pigs of approximately 20 kg, suspended at the apex of the heart, were completely casted by pouring red silicone (type 2, 100 ml of both components) into the right ventricle and blue silicone (type 2, 100 ml of both components) into the left ventricle after clamping the aorta and the cranial and caudal vena cava. After hardening overnight at room temperature, the casted cardiopulmonary sets were macerated in 25% potassium hydroxide during approximately 5 days and then rinsed in running tap water for 24 h. Histology To get an idea of the presence and the relative length of the myocardial sleeves, a reference point was defined by applying India ink on the luminal side of the wall of fresh pulmonary vein tissue at the level of the atrial-pulmonary junction of a pig of 100 kg, after which the myocardial sleeve length was measured on 5-lm-thick H&E-stained histological sections of 4% formalin-fixed (12 h) paraffinembedded pulmonary vein samples. The distance was measured between the India ink spot and the most distal point of the myocardial sleeve on the histological sections. Immunohistochemistry The myocardial sleeve of the pulmonary vein draining the right middle lung lobe was stained immunohistochemically with the polyclonal myosin marker MYBPC3 (K-16: © 2013 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 1–12
The Pulmonary Veins of the Pig
sc-50115; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for the detection of the myosin-binding protein C (the cardiac type), which is reactive with porcine tissue and is present in the myofibrils of cardiac and other striated muscle tissue (Winegrad, 1999). The slides were immunostained using the Dako automated Autostainer Plus. No antigen retrieval was carried out. The sections were first incubated for 5 min with 3% hydrogen peroxide and 30 min with rabbit serum. After primary antibody incubation for 60 min, secondary antibody (rabbit/anti-goat, biotinylated, polyclonal, 1:500; Dako, Glostrup, Denmark) was applied for 30 min followed by streptavidin and horseradish peroxidase (streptavidin-HRP, 1:1500, Dako) for 30 min. Visualization was done with DAB (Dako) for 5 min. To visualize the damage caused by heating myocardial tissue, mimicking ablation, immunohistochemical staining was performed, because this is almost impossible to demonstrate on general histology performed immediately after the procedure (own observations). Three-cm-thick samples of fresh myocardial tissue were unilaterally heated for 3 min on a hot plate on one side until the heated side was discoloured, subsequently fixated in formalin (4%) for 12 h and then embedded in paraffin. The histological sections were stained with the MYBPC3 marker (1:100). The same protocol was followed as described above, except for the application of a 1:500 streptavidin dilution as tertiary reagent.
Results Pulmonary veins The branching pattern In all hearts examined, all pulmonary veins drained into the left atrium through two distinct ostia. An ostium is being defined as the common draining orifice of a number of pulmonary veins into the left atrium, while the common terminal part of the pertaining veins is referred to as the antrum of the respective ostium. The antrum therefore collects the venous blood of a set of pulmonary veins and discharges into the left atrium through the ostium. One principal branching pattern (type I) was observed in 34 of 57 cases (Figs 1,3 and 7). In this type, the pulmonary veins from the right caudal and left caudal lung lobes (resp. V4 and V5) debouch together in a venous antrum which drains through ostium I. At their point of convergence, or slightly more peripheral in one of either veins, the orifice of the vein from the accessory lobe (V3) of the right lung can be located (highly variable pattern). The orifice of the pulmonary veins from the left cranial
3
The Pulmonary Veins of the Pig
lobe (V7) is situated in the left wall of the antrum leading to ostium I. By dissection, it was seen that myocardial tissue extends into V7. The pulmonary veins draining the right cranial and right middle lung lobes (resp. V1 and V2) debouch together in a venous antrum which drains into the left atrium through ostium II. This general branching pattern of the pulmonary veins presented some variations (Fig. 2). In a single case, V2 debouched together with V4 into the common venous antrum, together with V5, leading to ostium I (Fig. 1 type VI). In one other case, V1 and V2 drained separately into the left atrium at the level of ostium II (Fig. 1 type VII). The orifice of V3 was most variable as shown in Fig. 1 (types II (1 of 57 cases), III (14 of 57 cases), IV (3 of 57 cases) and V (2 of 57 cases). The draining pattern of the left caudal lung lobe showed some variation as well. The different parts of the left caudal lung lobe can incidentally be drained not through a common vein but instead directly through two separate pulmonary veins
T. Vandecasteele et al.
(V5a and V5b) which opened together with V4, V6, V7 into the venous antrum leading to ostium I (type VIII, 1 of 57 cases). The principal branching pattern is illustrated in Fig. 3 (silicon cast of the pulmonary veins). Topography of the pulmonary ostia The lungs of the pig can be defined on the basis of the different lung lobes. The right lung is divided into a cranial, an intermediate and a caudal lung lobe. The left lung consists of a cranial and a caudal lung lobe. The position of the different pulmonary veins draining the corresponding lung lobes is visualized in Fig. 3. The position of both pulmonary vein ostia in relation to their surrounding structures is visualized in Figs 4–7. Both ostia (I and II) are located ventral to the pulmonary arteries. From a dorsal view of the heart, the right pulmonary artery separates antrum I from antrum II (Fig. 4b). The larger pulmonary ostium, referred to as
Fig. 1. Left image: schematic drawing of the branching pattern of the pulmonary veins (cranial view). Middle image: equivalent intra-atrial view of the left atrium. Right image: overview of the branching pattern of the pulmonary veins and the corresponding lung lobes (ventrocaudal view). For abbreviations, see Fig. 7.
Fig. 2. Different branching patterns of the pulmonary veins (cranial view). Type I: 32 of 57 cases (V7 single) and 2 of 57 cases (V7 double); types II, III, IV and VIII: respectively 1, 14, 3 and 1 cases of 57 cases (V7 single); types VI and VII: both 1 of 57 cases (V7 double); type V: 2 of 57 cases (V7 single or double). For abbreviations, see Fig. 7.
4
© 2013 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 1–12
T. Vandecasteele et al.
The Pulmonary Veins of the Pig
Fig. 3. Cranial view of a silicon cast of the porcine pulmonary veins (left image) and schematic drawing of the pulmonary veins (right image). For abbreviations, see Fig. 7.
(a)
(c)
(b)
(d)
(e)
Fig. 4. Different views on silicone casts of the porcine heart and the major vessels (a, b, c, d, e) and schematic representation (b). (a) Right lateral cranio-dorsal view. (b) Dorsal view. (c) Right lateral view. (d) Right caudo-lateral view. (e) Caudal view. (1) Right pulmonary artery; (1′) left pulmonary artery; (2) pulmonary veins draining into ostium I; (3) pulmonary veins draining into ostium II; (4) cranial vena cava; (5) aorta; (6) right auricle; (7) left auricle; (8) caudal vena cava; (9) pulmonary trunk; (10) left azygos vein.
ostium I, is adjacent to and separated from the left auricle by the left azygos vein (Fig. 4b and e), while the caudal vena cava is located caudally and on its right side (Figs 4c and d). Ostium II is located more cranially towards the septal side of the left atrium (Fig. 4a), just above the oval fossa, at the level of the intervenous tubercle (Fig. 4b). © 2013 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 1–12
The venous ostium I is located ventral to the left pulmonary artery and more distally, the antrum slightly deflects to the right side of the left pulmonary artery (Figs 5 and 6). The bifurcation of this antrum into V4 and V5 is situated ventral to the left principal bronchus. V6 is situated ventral to the left pulmonary artery, cranial to the bronchus of the left caudal lung lobe and caudal to the bronchi of the cranial
5
The Pulmonary Veins of the Pig
and caudal parts of the left cranial lung lobe. V7, on the other hand, is located cranial to the latter bronchi (Fig. 7). The venous ostium II is situated ventral to the origin of the pulmonary artery of the cranial right lung lobe (A1) (Figs 5–7). Its corresponding venous antrum is located between this artery (A1) and the more caudal
Fig. 5. (left image) Ventral view of a silicon cast of the lungs showing the pulmonary veins (blue), pulmonary trunk (red) and trachea (white).
Fig. 6. (right image) Ventral view of a silicone cast of the lungs showing the pulmonary veins (blue), pulmonary trunk (red) and bronchi (white).
T. Vandecasteele et al.
pulmonary artery irrigating the right middle (A2), accessory (A3) and caudal (A4) lung lobes (Figs 5–7). After giving off the bronchus trachealis to the right cranial lung lobe, the trachea bifurcates into the primary bronchi at the level of the heart base, dorsal to the left atrium and to the right side of the median plane. The trachea is located dorsal to and on the right side of the pulmonary trunk. Both pulmonary veins ostia are located at the left side of the trachea, ostium II being situated closer to the trachea as compared to ostium I. Similar to the relative position of the trachea, the bronchi are also located dorsal to the pulmonary arteries, except in three places specified below (Fig. 7). 1. A branch of the right primary bronchus bends ventrally and crosses the right pulmonary artery ventrally, just cranial to the separation between the pulmonary arteries of the right accessory (A3) and middle (A2) lung lobes. Subsequently, the right pulmonary artery continues into A4. 2. The left primary bronchus divides into two large branches. The cranial branch enters the cranial part of the left cranial lobe, while the caudal branch ramifies in the caudal part of the left cranial lung lobe and the left caudal lung lobe. The caudal branch of the left bronchus is located ventral and between the pulmonary arteries, which irrigate the caudal part of the left cranial (A6) lung lobe and the left caudal (A5) lung lobe. This caudal bronchial branch is ventrally accompanied by V6. 3. The cranial branch of the left bronchus crosses the left pulmonary artery ventrally, just cranial to the origin
Fig. 7. Ventral view of the lung showing the pulmonary veins (blue), pulmonary trunk (red) and trachea (white) with abbreviations of the pulmonary veins, arteries and bronchi.
6
© 2013 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 1–12
T. Vandecasteele et al.
of the pulmonary artery (A7) irrigating the cranial part of the cranial left lung lobe. The position of the oesophagus in relation to the surrounding structures is demonstrated by the silicone cast presented in Fig. 8a and b. In the caudal part of the neck, the oesophagus is situated slightly to the left side of the trachea. More caudally, it is situated completely dorsal to the trachea in the mediastinum until the level of the tracheal bifurcation. At this point, the oesophagus is located to the right side of the aorta. Both pulmonary veins ostia are separated from the oesophagus by the pulmonary trunk and the trachea. To the right of the venous ostium II, the most adjacent structure in ventral direction is the sinus venarum cavarum. While dissecting in dorsal direction, there can be passed between A1 and the pulmonary artery leading to A2, A3 and A4. Following this path, the trachea perfectly shields the oesophagus, which lies on the dorsal side of the trachea. To the right of the venous ostium I the caudal vena cava, while to the left side, the left azygos vein is reached. In dorsal direction, on the other hand, the passage is partially shielded by the left pulmonary artery on the right side, while the aorta and oesophagus are located more dorsally and the left primary bronchus is encountered more caudally. This indicates that intravascular ablation in both venous ostia in the pig is not likely to induce atriooesophageal fistulae, although ostium II may constitute more risks compared with ostium I (Figs 5, 6, 8a and b). The position of the phrenic nerves in relation to the pericardium at the level of the left and right auricle is shown in Fig. 9. Both nerves lie dorsal to the auricles and the level of the ostia. The left phrenic nerve crosses ostium I, which might include a potential risk of causing phrenic nerve injuries during intra- and extra-ostial ablation procedures at this level (Fig. 9a–d). The right phrenic nerve lies lateral to the cranial and caudal vena cava (Fig. 9e and f). The right phrenic nerve is not lying adjacent to the pulmonary ostia, but the nerve crosses ostium I, ostium II, V1 and V2 ventrally and the right atrium dorsally, which might compromise an extra-ostial ablation procedure (Fig. 9g and h).
Fig. 8. Overview of the casted porcine lungs. (a) Ventral view of the casted porcine lungs. (b) Dorsal view of the casted porcine lungs.
© 2013 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 1–12
The Pulmonary Veins of the Pig
Myocardial sleeve Myocardial sleeves were localized in both antra. The myocardial sleeve in the wall of the venous antrum draining through ostium II is visualized in Fig. 10. The measurement indicates a relative length of the myocardial sleeve of approximately 13 mm at this sampling spot. The immunohistochemical staining of myocardial sleeve tissue clearly distinguishes the cardiac muscle tissue from the surrounding smooth muscle tissue and connective tissue (Fig. 11a). The staining of the ex-vivo-heated myocardial tissue (referring to ablation procedures) with the myosin marker demonstrated the disappearance of the myosin fibres caused by the high temperatures at the heated side of the sample (Fig. 11b). In contrast, the non-heated tissue of the sample was still clearly stained, indicating that the myosin fibres were still present (Fig. 11c). Discussion A complication incidence after ablation procedures for atrial fibrillation of 4.5% has been reported by Anderson (2012) in 16309 patients studied between 2003 and 2006. Catheter manipulations can cause several complications, of which the majority are not directly caused by the delivery of radiofrequency energy (Angkeow and Calkins, 2001). Apart from the complications, such procedures can fail and cause conduction recurrence. Conduction recurrence may occur when the targeted pulmonary veins are not completely isolated, when the induced lesions are not completely transmural or when the pathological conduction is caused by stimuli which originate from ectopic foci located outside the pulmonary veins (Haissaguerre et al., 1996, 1998; Chen et al., 1999; Hsieh et al., 1999; Pappone et al., 2000; Cappato et al., 2003; Gerstenfeld et al., 2003; Nanthakumar et al., 2004). An easily executable, fast ablation procedure with a high success rate, which can be repeated with low impact on the patient, if necessary, would be a big leap forward in terms of quality of the surgical technique and applicability in various
(a)
(b)
7
The Pulmonary Veins of the Pig
T. Vandecasteele et al.
(a)
(b)
(d)
(c)
(f) (e)
(g)
Fig. 10. Histologic section of the myocardial sleeve of a left pulmonary vein (HE staining).
patients. The continuous search for new ablation techniques requires appropriate animal models, in which the left atrium-pulmonary vein junction is documented in detail. In this investigation, we described all important characteristics of the pulmonary veins and their ostia, including the topography and the branching pattern of the pulmonary veins, the histological structure of the myocardial sleeves and their destruction as visualized by immunochemistry after heating.
8
(h)
Fig. 9. Overview of the topography of the phrenic nerves in relation to the pulmonary veins. (a and b) Left view of the topography of the left phrenic nerve (indicated in yellow) before (left image) and after (right image) fenestration of the pericardium. (c and d) Left view of the topography of the left phrenic nerve (indicated in yellow) in relation to casted pulmonary veins with ostium I. (e, f, g and h) Right view of the topography of the right phrenic nerve (indicated in yellow) before (e) and after (f–h) fenestration of the pericardium in relation to the casted pulmonary veins (g and h).
Because of the adverse effects that may occur after an ablation procedure in man, this article has focused mainly on the risk for atrio-oesophageal fistulae and phrenic nerve injuries, because these are two of the most important complications. This emphasizes the need of having information about such possible risks in pigs. While the human oesophagus covers the posterior side of the left atrium (Ho et al., 2012), the porcine oesophagus passes dorsal to the base of the heart, hereby separated from the pulmonary veins ostia by the pulmonary trunk and the trachea. While Barone (1997) already discussed in detail the topography of the porcine oesophagus and trachea, the present paper elaborates on their positions relative to the ostia of the pulmonary veins. In 40% of human cases, the distance between the oesophagus and the endocardium is
ORIGINAL ARTICLE
The Pulmonary Veins of the Pig as an Anatomical Model for the Development of a New Treatment for Atrial Fibrillation T. Vandecasteele*, K. Vandevelde, M. Doom, E. Van Mulken, P. Simoens and P. Cornillie Address of authors: Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
*Correspondence: Tel.: +3292647713; fax: +3292647790; e-mail: [email protected] With 11 figures and 1 table Received November 2013; accepted for publication November 2013 doi: 10.1111/ahe.12099
Summary The layout of the porcine atriopulmonary junction and immediately adjacent structures was investigated by gross anatomical and vascular corrosion casting studies to meet the need for more in-depth anatomical insights when using the pig as an animal model in the development of innovative approaches for surgical cardiac ablation in man. The veins from the right cranial and middle lung lobes drain through a common ostium in the left atrium, whereas a second ostium receives the blood returning from all other lung lobes, although limited variation to this pattern was observed. Surrounding anatomical structures that are most vulnerable to ablation damage as reported in man are located at a safer distance from the pulmonary veins in pigs, yet a certain locations, comparable risks are to be considered. Additionally, it was histologically confirmed that myocardial sleeves extend to over a centimetre in the wall of the pulmonary veins.
Introduction Atrial fibrillation is the most commonly diagnosed cardiac arrhythmia responsible for substantial mortality and morbidity in man. The incidence increases with age and can be associated with several internal diseases or genetic disorders (Brugada et al., 1997; Kannel et al., 1998). Focal triggering sites of cardiac arrhythmia may be resided in various anatomical structures such as the crista terminalis, the ostium of the coronary sinus, the interatrial septum and the atrial free wall (Haissaguerre et al., 1996, 1998; Chen et al., 1999; Hsieh et al., 1999; Pappone et al., 2000; Cappato et al., 2003; Gerstenfeld et al., 2003; Nanthakumar et al., 2004) as well as in myocardial sleeves located in the pulmonary veins. The latter structures are myocardial fibres spreading out from the left atrium into the wall of the pulmonary veins and are believed to be one of the most important origin of ectopic electric pulses (Nathan and Gloobe, 1970; Haissaguerre et al., 1998; Chen et al., 1999; Saito et al., 2000; Roux et al., 2004). These myocardial fibres act as an ectopic pacemaker transmitting electrical pulses which interfere with the pulses originating from the sinoatrial node, disturbing the normal heart rhythm and inducing atrial fibrillation © 2013 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 1–12
in humans (Haissaguerre et al., 2000; Saito et al., 2000; Tagawa et al., 2001). The sleeves exhibit different arrangement patterns in humans and in various mammals. Patients suffering from atrial fibrillation are often not treated when their quality of life is not affected, or they receive only pharmacological treatment when a surgical intervention is not considered appropriate. When a pharmacological approach and electric cardioversion are unsuccessful and return to sinus rhythm is thought to be necessary, ablation procedures can be applied by creating local transmural lesions at the left atrial ostia draining the pulmonary veins to eliminate the influence of ectopic myocardial sleeve pacemakers (Haissaguerre et al., 1998; Haissaguerre et al., 2000 Chen et al., 1999; Pappone et al., 2000). Those procedures still have some disadvantages because they require difficult and prolonged interventions and are therefore more prone to failure, which may lead to conduction recurrence and major complications (Haissaguerre et al., 1996; Robbins et al., 1998; Pappone et al., 1999; Mohr et al., 2002) including phrenic nerve injuries (Lee et al., 2004; Bunch et al., 2005; Okumura et al., 2008, 2009; Ahsan et al., 2010; Andrie et al., 2012), atrio-oesophageal fistulae (Gillinov et al., 2001; Sonmez et al., 2003; Pappone et al., 2004) and
1
The Pulmonary Veins of the Pig
pulmonary vein stenosis (Robbins et al., 1998; Chen et al., 1999; Arentz et al., 2003). To perform research into possible treatments for atrial fibrillation in humans, a suitable animal model is needed. The porcine model has multiple times been proven to be a good animal model for humans because of comparable body size and numerous anatomical, immunological, biochemical, physiological and genetic similarities between pigs and humans (Gregg et al., 1980; Shulman et al., 1988; Delange et al., 1992; Bermejo et al., 1993; Rowan et al., 1994; Jones et al., 1999; Paterson et al., 2002; Sommerer et al., 2004; Perry et al., 2005; Barker et al., 2006; Brunet et al., 2006; Ibrahim et al., 2006; Glenny et al., 2007; Hotchkiss et al., 2007; Wang et al., 2007; Rogers et al., 2008; Khatri et al., 2010). The porcine heart, of which the microanatomy is representative for the mammalian heart, has an average weight of approximately 300 g, which is similar to the average weight of the human heart (approximately 300 g in men and 250 g in women) (Barone, 1997). The development of an appropriate animal model for ablation therapy studies requires in-depth data on the fine anatomical and histological architecture of the pulmonary veins and the myocardial sleeves and on the consequences of a heating process at the level of this myocardial tissue in pigs. This essential information is indispensable when approaching the ostia of the pulmonary veins from the left atrium to perform an intraluminal ablation at this level, but is lacking in current literature. Therefore, in the present study, the microanatomy of the porcine pulmonary veins was investigated to document the atriopulmonary junction in swine. This includes the number of pulmonary vein ostia, the different pulmonary veins draining into their specific ostia, the branching pattern of the various pulmonary veins and its variability, and the position of the pulmonary veins in relation to their surrounding structures such as the trachea, the oesophagus and the pulmonary arteries. These parameters, together with previous data provided by Vollmerhaus et al. (1999), who described the relation between the pulmonary arteries and the veins, between the pulmonary arteries and the trachea, and between the pulmonary veins and the trachea, are essential for the use of the porcine animal model, enabling an intraluminal ablation procedure of the pulmonary veins in swine.
Materials and Methods Anatomical dissection The anatomical examination was performed on cardiopulmonary sets of healthy pigs of 20–40 kg used for other studies and of pigs of approximately 100 kg obtained from
2
T. Vandecasteele et al.
the slaughterhouse (Table 1). The fresh cardiopulmonary sets were incised from the left ventricle into the left atrium to get an overview of the draining area of the pulmonary veins at the roof of the left atrium. Subsequently, the ostia and the pulmonary veins were incised longitudinally to provide an overview of the more distally located branches and to determine both the branching pattern of the pulmonary veins and their drainage area. Silicone casting Before casting the pulmonary veins with silicone, the left atrioventricular orifice was occluded by placing a clamp at the level of the coronary groove. Thereafter, both components of the silicone (base and catalyst, ratio 1:1) were combined and coloured by adding the desired dye to indicate different structures. All pulmonary veins were casted with blue silicone [ZA22, 22 ShA silicone (type 1); Zhermack, Badia Polesine, Italy], unless indicated otherwise, whereas the pulmonary arteries and bronchi were, respectively, casted with red and white silicone [HT33, 30 ShA silicone (type 2); Zhermack]. Subsequently, the silicone was injected into the left atrium, and the pulmonary veins through a blunt needle inserted through the left auricular wall. Three cardiopulmonary sets (from two pigs of approximately 100 kg and one piglet of 20 kg) were casted with silicone (250 and 75 ml of both components for the large and smaller pigs, respectively) to visualize the pulmonary veins. The pulmonary trunk was transversely disconnected from the right ventricle by placing a clamp near the arterial cone to be able to cast the pulmonary arteries. Subsequently, silicone was injected through a blunt needle placed in the pulmonary trunk near the applied clamp. The trachea was casted by injection of silicone through a blunt needle placed in the trachea approximately 5 cm
Table 1. Number of cardiopulmonary sets used in the different techniques and for the various silicone casts Number of cardiopulmonary sets used Anatomical dissection Silicone casting of the pulmonary veins Silicone casting of the pulmonary veins, pulmonary arteries and trachea Silicone casting of the pulmonary veins and arteries, trachea, oesophagus and aorta Silicone casting of complete hearts Silicone casting of the pulmonary veins in relation to the phrenic nerves Total number of used cardiopulmonary sets
57 3 2 1 2 2 67
© 2013 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 1–12
T. Vandecasteele et al.
cranial to the origin of the tracheal bronchus, after the trachea was transversely clamped just cranial to the injection spot. To visualize the position of the oesophagus in relation to the pulmonary veins and the other adjacent structures, a plastic tube of 20 cm was inserted in the oesophagus. Cardiopulmonary sets of pigs of 35 kg were casted to demonstrate the pulmonary arteries (100 ml of both components), the pulmonary veins (100 ml of both components) and the trachea and bronchi (150 ml of both components). A silicone cast of a cardiopulmonary set of a pig of 30 kg was made to visualize the pulmonary veins (100 ml of both components), pulmonary arteries (100 ml of both components), trachea and bronchi (150 ml of both components) and the oesophagus and aorta. The cardiopulmonary sets of pigs of 30 kg were casted in situ with silicone (100 ml of both components) to visualize the ostia of the pulmonary veins in relation to the phrenic nerves after opening the thorax bilaterally. The position of both phrenic nerves was determined by removing the pericardium and the walls of the left auricle and the pulmonary veins surrounding the hardened silicone. To localize the position of both ostia on the heart base and in relation to the other arterial and venous structures, cardiopulmonary sets of pigs of approximately 20 kg, suspended at the apex of the heart, were completely casted by pouring red silicone (type 2, 100 ml of both components) into the right ventricle and blue silicone (type 2, 100 ml of both components) into the left ventricle after clamping the aorta and the cranial and caudal vena cava. After hardening overnight at room temperature, the casted cardiopulmonary sets were macerated in 25% potassium hydroxide during approximately 5 days and then rinsed in running tap water for 24 h. Histology To get an idea of the presence and the relative length of the myocardial sleeves, a reference point was defined by applying India ink on the luminal side of the wall of fresh pulmonary vein tissue at the level of the atrial-pulmonary junction of a pig of 100 kg, after which the myocardial sleeve length was measured on 5-lm-thick H&E-stained histological sections of 4% formalin-fixed (12 h) paraffinembedded pulmonary vein samples. The distance was measured between the India ink spot and the most distal point of the myocardial sleeve on the histological sections. Immunohistochemistry The myocardial sleeve of the pulmonary vein draining the right middle lung lobe was stained immunohistochemically with the polyclonal myosin marker MYBPC3 (K-16: © 2013 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 1–12
The Pulmonary Veins of the Pig
sc-50115; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for the detection of the myosin-binding protein C (the cardiac type), which is reactive with porcine tissue and is present in the myofibrils of cardiac and other striated muscle tissue (Winegrad, 1999). The slides were immunostained using the Dako automated Autostainer Plus. No antigen retrieval was carried out. The sections were first incubated for 5 min with 3% hydrogen peroxide and 30 min with rabbit serum. After primary antibody incubation for 60 min, secondary antibody (rabbit/anti-goat, biotinylated, polyclonal, 1:500; Dako, Glostrup, Denmark) was applied for 30 min followed by streptavidin and horseradish peroxidase (streptavidin-HRP, 1:1500, Dako) for 30 min. Visualization was done with DAB (Dako) for 5 min. To visualize the damage caused by heating myocardial tissue, mimicking ablation, immunohistochemical staining was performed, because this is almost impossible to demonstrate on general histology performed immediately after the procedure (own observations). Three-cm-thick samples of fresh myocardial tissue were unilaterally heated for 3 min on a hot plate on one side until the heated side was discoloured, subsequently fixated in formalin (4%) for 12 h and then embedded in paraffin. The histological sections were stained with the MYBPC3 marker (1:100). The same protocol was followed as described above, except for the application of a 1:500 streptavidin dilution as tertiary reagent.
Results Pulmonary veins The branching pattern In all hearts examined, all pulmonary veins drained into the left atrium through two distinct ostia. An ostium is being defined as the common draining orifice of a number of pulmonary veins into the left atrium, while the common terminal part of the pertaining veins is referred to as the antrum of the respective ostium. The antrum therefore collects the venous blood of a set of pulmonary veins and discharges into the left atrium through the ostium. One principal branching pattern (type I) was observed in 34 of 57 cases (Figs 1,3 and 7). In this type, the pulmonary veins from the right caudal and left caudal lung lobes (resp. V4 and V5) debouch together in a venous antrum which drains through ostium I. At their point of convergence, or slightly more peripheral in one of either veins, the orifice of the vein from the accessory lobe (V3) of the right lung can be located (highly variable pattern). The orifice of the pulmonary veins from the left cranial
3
The Pulmonary Veins of the Pig
lobe (V7) is situated in the left wall of the antrum leading to ostium I. By dissection, it was seen that myocardial tissue extends into V7. The pulmonary veins draining the right cranial and right middle lung lobes (resp. V1 and V2) debouch together in a venous antrum which drains into the left atrium through ostium II. This general branching pattern of the pulmonary veins presented some variations (Fig. 2). In a single case, V2 debouched together with V4 into the common venous antrum, together with V5, leading to ostium I (Fig. 1 type VI). In one other case, V1 and V2 drained separately into the left atrium at the level of ostium II (Fig. 1 type VII). The orifice of V3 was most variable as shown in Fig. 1 (types II (1 of 57 cases), III (14 of 57 cases), IV (3 of 57 cases) and V (2 of 57 cases). The draining pattern of the left caudal lung lobe showed some variation as well. The different parts of the left caudal lung lobe can incidentally be drained not through a common vein but instead directly through two separate pulmonary veins
T. Vandecasteele et al.
(V5a and V5b) which opened together with V4, V6, V7 into the venous antrum leading to ostium I (type VIII, 1 of 57 cases). The principal branching pattern is illustrated in Fig. 3 (silicon cast of the pulmonary veins). Topography of the pulmonary ostia The lungs of the pig can be defined on the basis of the different lung lobes. The right lung is divided into a cranial, an intermediate and a caudal lung lobe. The left lung consists of a cranial and a caudal lung lobe. The position of the different pulmonary veins draining the corresponding lung lobes is visualized in Fig. 3. The position of both pulmonary vein ostia in relation to their surrounding structures is visualized in Figs 4–7. Both ostia (I and II) are located ventral to the pulmonary arteries. From a dorsal view of the heart, the right pulmonary artery separates antrum I from antrum II (Fig. 4b). The larger pulmonary ostium, referred to as
Fig. 1. Left image: schematic drawing of the branching pattern of the pulmonary veins (cranial view). Middle image: equivalent intra-atrial view of the left atrium. Right image: overview of the branching pattern of the pulmonary veins and the corresponding lung lobes (ventrocaudal view). For abbreviations, see Fig. 7.
Fig. 2. Different branching patterns of the pulmonary veins (cranial view). Type I: 32 of 57 cases (V7 single) and 2 of 57 cases (V7 double); types II, III, IV and VIII: respectively 1, 14, 3 and 1 cases of 57 cases (V7 single); types VI and VII: both 1 of 57 cases (V7 double); type V: 2 of 57 cases (V7 single or double). For abbreviations, see Fig. 7.
4
© 2013 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 1–12
T. Vandecasteele et al.
The Pulmonary Veins of the Pig
Fig. 3. Cranial view of a silicon cast of the porcine pulmonary veins (left image) and schematic drawing of the pulmonary veins (right image). For abbreviations, see Fig. 7.
(a)
(c)
(b)
(d)
(e)
Fig. 4. Different views on silicone casts of the porcine heart and the major vessels (a, b, c, d, e) and schematic representation (b). (a) Right lateral cranio-dorsal view. (b) Dorsal view. (c) Right lateral view. (d) Right caudo-lateral view. (e) Caudal view. (1) Right pulmonary artery; (1′) left pulmonary artery; (2) pulmonary veins draining into ostium I; (3) pulmonary veins draining into ostium II; (4) cranial vena cava; (5) aorta; (6) right auricle; (7) left auricle; (8) caudal vena cava; (9) pulmonary trunk; (10) left azygos vein.
ostium I, is adjacent to and separated from the left auricle by the left azygos vein (Fig. 4b and e), while the caudal vena cava is located caudally and on its right side (Figs 4c and d). Ostium II is located more cranially towards the septal side of the left atrium (Fig. 4a), just above the oval fossa, at the level of the intervenous tubercle (Fig. 4b). © 2013 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 1–12
The venous ostium I is located ventral to the left pulmonary artery and more distally, the antrum slightly deflects to the right side of the left pulmonary artery (Figs 5 and 6). The bifurcation of this antrum into V4 and V5 is situated ventral to the left principal bronchus. V6 is situated ventral to the left pulmonary artery, cranial to the bronchus of the left caudal lung lobe and caudal to the bronchi of the cranial
5
The Pulmonary Veins of the Pig
and caudal parts of the left cranial lung lobe. V7, on the other hand, is located cranial to the latter bronchi (Fig. 7). The venous ostium II is situated ventral to the origin of the pulmonary artery of the cranial right lung lobe (A1) (Figs 5–7). Its corresponding venous antrum is located between this artery (A1) and the more caudal
Fig. 5. (left image) Ventral view of a silicon cast of the lungs showing the pulmonary veins (blue), pulmonary trunk (red) and trachea (white).
Fig. 6. (right image) Ventral view of a silicone cast of the lungs showing the pulmonary veins (blue), pulmonary trunk (red) and bronchi (white).
T. Vandecasteele et al.
pulmonary artery irrigating the right middle (A2), accessory (A3) and caudal (A4) lung lobes (Figs 5–7). After giving off the bronchus trachealis to the right cranial lung lobe, the trachea bifurcates into the primary bronchi at the level of the heart base, dorsal to the left atrium and to the right side of the median plane. The trachea is located dorsal to and on the right side of the pulmonary trunk. Both pulmonary veins ostia are located at the left side of the trachea, ostium II being situated closer to the trachea as compared to ostium I. Similar to the relative position of the trachea, the bronchi are also located dorsal to the pulmonary arteries, except in three places specified below (Fig. 7). 1. A branch of the right primary bronchus bends ventrally and crosses the right pulmonary artery ventrally, just cranial to the separation between the pulmonary arteries of the right accessory (A3) and middle (A2) lung lobes. Subsequently, the right pulmonary artery continues into A4. 2. The left primary bronchus divides into two large branches. The cranial branch enters the cranial part of the left cranial lobe, while the caudal branch ramifies in the caudal part of the left cranial lung lobe and the left caudal lung lobe. The caudal branch of the left bronchus is located ventral and between the pulmonary arteries, which irrigate the caudal part of the left cranial (A6) lung lobe and the left caudal (A5) lung lobe. This caudal bronchial branch is ventrally accompanied by V6. 3. The cranial branch of the left bronchus crosses the left pulmonary artery ventrally, just cranial to the origin
Fig. 7. Ventral view of the lung showing the pulmonary veins (blue), pulmonary trunk (red) and trachea (white) with abbreviations of the pulmonary veins, arteries and bronchi.
6
© 2013 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 1–12
T. Vandecasteele et al.
of the pulmonary artery (A7) irrigating the cranial part of the cranial left lung lobe. The position of the oesophagus in relation to the surrounding structures is demonstrated by the silicone cast presented in Fig. 8a and b. In the caudal part of the neck, the oesophagus is situated slightly to the left side of the trachea. More caudally, it is situated completely dorsal to the trachea in the mediastinum until the level of the tracheal bifurcation. At this point, the oesophagus is located to the right side of the aorta. Both pulmonary veins ostia are separated from the oesophagus by the pulmonary trunk and the trachea. To the right of the venous ostium II, the most adjacent structure in ventral direction is the sinus venarum cavarum. While dissecting in dorsal direction, there can be passed between A1 and the pulmonary artery leading to A2, A3 and A4. Following this path, the trachea perfectly shields the oesophagus, which lies on the dorsal side of the trachea. To the right of the venous ostium I the caudal vena cava, while to the left side, the left azygos vein is reached. In dorsal direction, on the other hand, the passage is partially shielded by the left pulmonary artery on the right side, while the aorta and oesophagus are located more dorsally and the left primary bronchus is encountered more caudally. This indicates that intravascular ablation in both venous ostia in the pig is not likely to induce atriooesophageal fistulae, although ostium II may constitute more risks compared with ostium I (Figs 5, 6, 8a and b). The position of the phrenic nerves in relation to the pericardium at the level of the left and right auricle is shown in Fig. 9. Both nerves lie dorsal to the auricles and the level of the ostia. The left phrenic nerve crosses ostium I, which might include a potential risk of causing phrenic nerve injuries during intra- and extra-ostial ablation procedures at this level (Fig. 9a–d). The right phrenic nerve lies lateral to the cranial and caudal vena cava (Fig. 9e and f). The right phrenic nerve is not lying adjacent to the pulmonary ostia, but the nerve crosses ostium I, ostium II, V1 and V2 ventrally and the right atrium dorsally, which might compromise an extra-ostial ablation procedure (Fig. 9g and h).
Fig. 8. Overview of the casted porcine lungs. (a) Ventral view of the casted porcine lungs. (b) Dorsal view of the casted porcine lungs.
© 2013 Blackwell Verlag GmbH Anat. Histol. Embryol. 44 (2015) 1–12
The Pulmonary Veins of the Pig
Myocardial sleeve Myocardial sleeves were localized in both antra. The myocardial sleeve in the wall of the venous antrum draining through ostium II is visualized in Fig. 10. The measurement indicates a relative length of the myocardial sleeve of approximately 13 mm at this sampling spot. The immunohistochemical staining of myocardial sleeve tissue clearly distinguishes the cardiac muscle tissue from the surrounding smooth muscle tissue and connective tissue (Fig. 11a). The staining of the ex-vivo-heated myocardial tissue (referring to ablation procedures) with the myosin marker demonstrated the disappearance of the myosin fibres caused by the high temperatures at the heated side of the sample (Fig. 11b). In contrast, the non-heated tissue of the sample was still clearly stained, indicating that the myosin fibres were still present (Fig. 11c). Discussion A complication incidence after ablation procedures for atrial fibrillation of 4.5% has been reported by Anderson (2012) in 16309 patients studied between 2003 and 2006. Catheter manipulations can cause several complications, of which the majority are not directly caused by the delivery of radiofrequency energy (Angkeow and Calkins, 2001). Apart from the complications, such procedures can fail and cause conduction recurrence. Conduction recurrence may occur when the targeted pulmonary veins are not completely isolated, when the induced lesions are not completely transmural or when the pathological conduction is caused by stimuli which originate from ectopic foci located outside the pulmonary veins (Haissaguerre et al., 1996, 1998; Chen et al., 1999; Hsieh et al., 1999; Pappone et al., 2000; Cappato et al., 2003; Gerstenfeld et al., 2003; Nanthakumar et al., 2004). An easily executable, fast ablation procedure with a high success rate, which can be repeated with low impact on the patient, if necessary, would be a big leap forward in terms of quality of the surgical technique and applicability in various
(a)
(b)
7
The Pulmonary Veins of the Pig
T. Vandecasteele et al.
(a)
(b)
(d)
(c)
(f) (e)
(g)
Fig. 10. Histologic section of the myocardial sleeve of a left pulmonary vein (HE staining).
patients. The continuous search for new ablation techniques requires appropriate animal models, in which the left atrium-pulmonary vein junction is documented in detail. In this investigation, we described all important characteristics of the pulmonary veins and their ostia, including the topography and the branching pattern of the pulmonary veins, the histological structure of the myocardial sleeves and their destruction as visualized by immunochemistry after heating.
8
(h)
Fig. 9. Overview of the topography of the phrenic nerves in relation to the pulmonary veins. (a and b) Left view of the topography of the left phrenic nerve (indicated in yellow) before (left image) and after (right image) fenestration of the pericardium. (c and d) Left view of the topography of the left phrenic nerve (indicated in yellow) in relation to casted pulmonary veins with ostium I. (e, f, g and h) Right view of the topography of the right phrenic nerve (indicated in yellow) before (e) and after (f–h) fenestration of the pericardium in relation to the casted pulmonary veins (g and h).
Because of the adverse effects that may occur after an ablation procedure in man, this article has focused mainly on the risk for atrio-oesophageal fistulae and phrenic nerve injuries, because these are two of the most important complications. This emphasizes the need of having information about such possible risks in pigs. While the human oesophagus covers the posterior side of the left atrium (Ho et al., 2012), the porcine oesophagus passes dorsal to the base of the heart, hereby separated from the pulmonary veins ostia by the pulmonary trunk and the trachea. While Barone (1997) already discussed in detail the topography of the porcine oesophagus and trachea, the present paper elaborates on their positions relative to the ostia of the pulmonary veins. In 40% of human cases, the distance between the oesophagus and the endocardium is
