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Eur J Cardiothorac Surg 2000;18:228-232
© 2000 Elsevier Science NL

Cannulation of the cardiac lymphatic system in swine

Jaime F. Vazquez-Jimeneza, Marie-Christine Seghayeb, Ma Qingb, Oliver J. Liakopoulosa, Marcia L. Rosenbauma, Bruno J. Messmera

a Thoracic Cardiovascular Surgery, Universitätsklinikum RWTH Aachen, Pauwelsstrasse 30, 52057 Aachen, Germany
b Pediatric Cardiology, RWTH Aachen, Germany

Received 13 December 1999; received in revised form 11 February 2000; accepted 15 February 2000.

Corresponding author. Tel.: +49-241-808-9975; fax: +49-241-888-8454
e-mail: jvazquez-jimenez{at}post.klinikum.rwth-aachen.de


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Cardiac lymph is the most direct medium for analyzing metabological changes in the myocardial cell. Currently, dogs are the animals used for investigation of myocardial lymphatic function. However, questions arise when comparing and interpreting the human system to the experimental model, since the dog coronary anatomy is different from human anatomy and pulmonary lymph contamination is found in up to 81% of the cases. Swine, having similar coronary anatomy to humans, are a proven model for cardiovascular research. The purpose of this study was to investigate the cardiac lymphatic anatomy of the swine and to develop a reliable cannulation technique to collect the lymph. Methods and results: The lymphatic anatomy of 60 pigs was studied and classified and a new technique for lymphatic cannulation was developed. The cannulation success rate was 55%. In addition, no pulmonary lymph contamination was found at the cannulation site. Conclusion: We conclude that porcine myocardial lymphatics can be successfully cannulated for the investigation of myocardial lymphatic function.

Key Words: Cardiolymph • Heart lymphatics • Porcine lymphatic anatomy


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Cardiac lymph is a sensitive indicator for evaluating many pathophysiological changes of the myocardium [1]. The potential of the cardiac lymph as an index of the cardiac ‘milieu interieur’ has been widely proofed in multiple studies and the myocardial lymphatic function has been investigated for its involvement in myocardial edema formation, mediator release following myocardial reperfusion injury and neurotransmitter release from the heart [2].

For over 50 years, dogs have been used for cardiac lymphatic studies [3]. Their lymphatic system is well understood, cannulation and sampling techniques are standardized and widely used [4]. However, in contrast to the human heart the coronary system of the dog shows a very rich network of collaterals which compensate ischemia [5] and, therefore, lymphatic studies concerning ischemia in dogs may vary from that of humans.

Lymph samples in dogs are usually collected from the ‘major prenodal cardiac lymphatic’ (MPCL) which drains into a lymph node located between the superior vena cava and the innominate artery and which is commonly referred to as ‘cardiac node of Drinker’ [3]. Assessment of myocardial lymphatic function is based on the assumption that the samples obtained from the MPCL contain pure cardiac lymph. However, anatomical studies in dogs have shown that there is contamination with pulmonary lymph in up to 81% of the cases due to connections with the tracheobronchial node [2].

These two factors (i.e. the abundance of collaterals and contamination with pulmonary lymph) may have a substantial impact on the interpretation of the data obtained from the canine model.

The porcine end-artery coronary anatomy is similar to that of the human heart [6] and pigs are an established animal model for studies of cardiac physiology and myocardial ischemia. To the best of our knowledge there is no publication describing the cannulation of the main efferent lymph trunk in swine.

The purpose of this study was to investigate the cardiac lymphatic anatomy of the swine and to develop a reliable cannulation technique for cardiac lymph collection.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Animal preparation
The animal experiments were conducted according to the guidelines of the European Convention of Animal Care and the protocol was approved by the state agency supervising animal experimentation.

Sixty healthy female ‘Deutsche Landrasse’ pigs (body weight 41.9±3.9 kg) were studied. Premedication was performed with azaperon (4 mg/kg) i.m. and ketamine (4 mg/kg) i.m. General anesthesia was induced intravenously with pentobarbital (5 mg/kg) and maintained with boluses of pentobarbital (5 mg/kg) and ketamine (1 mg/kg) as required. The trachea was intubated with a cuffed intratracheal tube and respiration was controlled with a Servo A ventilator (Siemens 900B, Solna, Sweden). Fluid-filled catheters were placed into the right carotid artery and jugular vein for arterial pressure monitoring, arterial blood sampling and fluid administration, respectively.

A median sternotomy was performed and the anterior wall of the pericardial sac was incised. Evans blue (0.1 ml, 0.5% aqueous solution) was injected subepicardially into the right atrial wall and both ventricles (Fig. 1) , taking care not to contaminate the pericardial fluid and the lymphatic system of the pericardium.



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  		Fig. 1. Epicardial lymphatic vessel of the anterior portion of the left ventricle running close to the left anterior descending artery after injection of Evans blue. l, left ventricular epicardial lymphatic vessel; a, left anterior descending artery.

 
2.2. Cannulation technique
In all pigs an efferent cardiac lymph vessel was identified. A 4-0 (Ethicon) ligature was placed on the cranial portion of the lymphatic proximal the cardiac lymph node. Within 10–15 min, the vessel expanded to a diameter of ca. 0.5–1.0 mm. The expanded vessel was dissected to free the longest possible section of the vessel between the aortic root and the cranial ligature, usually 2.0–2.5 cm. After injecting Heparin (3 mg/kg) intravenously for systemic anticoagulation, a silk tape was placed around the ascending aorta for future traction. The pericardium overlying the right pulmonary artery was cut just to the left of the superior vena cava (SVC) and silk tape was wrapped around the SVC for snaring. Throughout the procedure, caution must be taken not to injure the lymphatic vessel.

A 18G Cavafix-Certo-Basilica® catheter (Braun, Melsungen, Germany) with a inner diameter of 0.8 mm was shortened to 14 cm. The last 8 cm were wrapped with adhesive film (Opsite®, T.J. Smith and Nephew, UK) leaving a flap 2 cm wide on both sides of the catheter for subsequent fixation to the pericardium and epicardium (Fig. 2) . After wrapping, the catheter was flushed with heparin.



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  		Fig. 2. 2 A 18G Cavafix-Certo-Basilica® catheter (Braun, Melsungen, Germany) prepared for subsequent fixation to the epi- and pericardium.

 
A 6-0 Prolene ligature was placed at the distal edge of the lymphatic collector, 3 mm proximal to the first ligature and the lymphatic collector was divided between the two ligatures. The other end of the 6-0 Prolene was passed through the prepared Cavafix catheter until the end of the ligature arrived at the ‘connector-end’ of the Cavafix catheter. Keeping tension on the Prolene ligature, the Cavafix catheter was gently pulled down until the dissected lymphatic collector was completely INSIDE the catheter (Figs. 3 and 4) . Maintaining tension, the Cavafix catheter containing the lymphatic vessel was sutured on both sides through the flap to the pericardium, adventicia of the SVC and the subcutaneous tissue with four 6-0 Prolene sutures. The catheter should form a soft and anatomically appropriated curve. The connection-end of the catheter was placed outside of the thoracic incision. Special care must be taken to maintain tension on the lymphatic vessel in order to prevent kinking at the entrance of the catheter after fixation.



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  		Fig. 3. The Cavafix catheter is pulled down until the dissected lymphatic collector is completely inside of the catheter. l, main lymphatic collector; c, Cavafix catheter; s; superior vena cava; a, aorta.

 


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  		Fig. 4. Schematic drawing of the inclusion technique as described in the text. SVC, superior vena cava; lymph, main lymphatic efferent trunk; cat, 18G Cavafix-Certo-Basilica® catheter.

 
When the catheter was properly fixed, the 6-0 ligature was pulled out by pinching the catheter containing the lymph vessel with a vascular forceps 3 mm below the ligature (Fig. 4(2)) thereby breaking off the tied end of the lymphatic collector. After removing the 6-0 Prolene ligature attached to the broken end of the vessel, lymph filled the catheter (Fig. 4(3)). Next, the Cavafix catheter was exteriorized through the skin incision between neck and sternum and kept in place with one polyester suture. This prevents distortion of the catheter after closure of the chest. The exposed portion of the Cavafix catheter was extended with a Heparin flushed connecting tube (osol 1.2x2.2 mm, 30 cm, Braun, Melsungen Germany) so that the free end of the catheter hung below the thorax level, thereby increasing the gravitational drainage force of the lymph. Once a stable flow of cardiac lymph was established, cardiopulmonary bypass was started with one aortic and two venous cannula placed in the superior and inferior vena cava. After 60 min of cardioplegic cardiac arrest and 30 min of reperfusion cardiopulmonary bypass was discontinued and the aortic and venous cannula were removed taking care to reduce cardiac manipulation in order to prevent lymph catheter dislocation. Fixation of the lymph catheter on the cranial portion of the wound allows chest closure without lymph flow interference.

Possible pulmonary contamination of the collected cardiac lymph was investigated by two methods. Firstly, Evans blue dye was injected in both lungs after successful cannulation of the cardiac lymph vessel and did not show any staining of the collected lymph. Secondly, alterations of tidal volume through the respirator during cardiac arrest did not result in a change of lymph flow rate assuming that pulmonary lymph flow depends on respiratory movement [2].


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
We found that in 31 of 60 pigs (51.6%), the common efferent trunk runs along the dorsal side of the superior vena cava and drains into a cardiac lymph node between the superior vena cava and trachea (these we classified as ‘right drainage type’) (Fig. 5A) . Cannulation of the trunk was successful in 22 of 31 pigs (70.9%) utilizing the new technique described in this article.



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  		Fig. 5. ‘Right drainage type’ (A) and ‘left drainage type’ (B) of the porcine heart after subepicardial injections of Evans blue in the right atrium, right and left ventricle. Ao, Aorta; SVC, superior vena cava; IVC, inferior vena cava; PA, pulmonary artery; LA, left atrium; RA, right atrium; AVT, anterior interventricular trunk; OMT, obtuse marginal trunk; LCT, left coronary trunk; RCT, right coronary trunk; MCT, main coronary trunk; CLN, cardiac lymph node.

 
In six of 60 pigs (10%), upon opening the left pleura, the lymphatic trunk was found cranial to the left pulmonary artery. The trunk ascends and drains into the left subclavian vein (these we classified as ‘left drainage type’) (Fig. 5B). This vessel was larger and was cannulated in one pig (17%) successfully by punction with a 20 GA intravenous cannula (Medicate®, Shrewd Medical, Toolmaker, Ireland).

In 23 of 60 pigs (38.4%), there was no common trunk. The drainage occurred via two or three smaller vessels, one of which passes directly towards the left pretracheal lymph node between the brachiocephalic artery and trachea (these we classified as ‘small drainage type’). Cannulation of this type was possible in 10 pigs (43.5%).

The overall success rate was 55% (33 of 60 pigs). The flow rate varied from 0.9 to 2.2 ml/h at baseline and rose to 7.5 to 12.0 ml/h directly after unclamping the aorta and progressively decreased during the hours after cardiopulmonary bypass.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Julien et al. described and classified the porcine cardiac lymphatic anatomy [7]. They found that both ventricles contain dense regional networks of subepicardial lymph capillaries, which group near to the coronary arteries and converge into two larger left and right efferent lymph vessels. These efferent vessels run separately in the atrioventricular sulcus close to the coronary arteries and then converge towards the root of the aorta to form a common efferent lymph trunk behind the aorta. In 10 of 15 (67%) pigs the common efferent trunk follows the dorsal side of the superior vena cava and drains into a cardiac lymph node between the superior vena cava and the trachea. This anatomical drainage type (‘right drainage type’) was found in our study in 31 of 60 pigs (51.6%). In the remaining five pigs (33%) Julien et al. described a common trunk which divided into two or three smaller vessels, one of which passed directly towards the left pretracheal lymph node between the brachiocephalic artery and trachea. In our study 23 of 60 pigs (38.4%) present this pattern of lymphatic drainage (‘small drainage type’). Furthermore, we found that in six of 60 pigs (10%), upon opening the left pleura, the lymphatic trunk was found cranial to the left pulmonary artery. The trunk ascends and drains into the left subclavian vein (‘left drainage type’).

Since cardiac lymphatic cannulation in pigs has never been described, we assume that this has been the major hindrance for using pigs as the experimental model for cardiac lymphatic studies. In order to develop a standard method for cannulation in pigs, trial and error was necessary. First we attempted to cannulate directly the lymphatic collectors in seven pigs (four right and three ‘small drainage’) similar to the cannulation technique in the dog, but the results were discouraging because punction was either impossible or dislocation of the cannula occurred early. The next approach was ‘vessel inclusion’, resulting in 22 successful cannulations of 31 ‘right drainage’ and ten of 23 cases of ‘small drainage type’ pigs. The cannulation of the ‘small drainage type’ was only successful after having much experience with the inclusion technique because the vessels leaving the heart surface went directly posterior in the thoracic plane to the pretracheal lymph nodes and therefore, the length of the available vessel for cannulation was very short.

In the ‘left drainage type’, cannulation by puncture with a appropriate cannula (20 GA) is easier because of the anatomical position and the larger diameter of the collector.

In conclusion, we think that this new technique for the cannulation of porcine cardiac lymphatics offers a reasonable success rate. We hope that our results will encourage researchers to use the porcine model for cardiac lymphatic studies since, as shown by dogs, the cardiac lymph is the most direct medium for analyzing the metabological changes in interstitium, therefore examination of the lymph will lead us to better quantification of myocardial protection techniques and better understanding of the immunological response of the myocardium to the ischemia and extracorporeal circulation.


   Acknowledgments
 
This study was supported by a START-grant of the Technical University of Aachen (RWTH), Germany. We wish to thank Dr H.J. Geissler, Cologne for the correction of this manuscript and Dr R.E. Drake, Houston for his stimulating comments.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

  1. Michael L.H., Hunt J.R., Weilbaecher D., Perryman M.B., Roberts R., Lewis R.M., Entman M.L. Creatinine kinase and phosphorylase in cardiac lymph: coronary occlusion and reperfusion. Am J Physiol 1985;248:H350-H359.
  2. Geissler H.J., Davis K.L., Laine G.A., Brennan M.L., Mehlhorn U., Allen S.J. Contamination of lymph from the major prenodal cardiac lymphatic in dogs. Am J Physiol 1999;267:H1795-H1800.
  3. Drinker C.K., Warren M.F., Maurer F.W., McCarrel J. The flow, pressure, and composition of cardiac lymph. Am J Physiol 1940;130:43-55.[Free Full Text]
  4. Michael L.H., Lewis R.M., Brandon T.A., Entman M.L. Cardiac lymph flow in conscious dogs. Am J Physiol 1979;237:H311-H317.
  5. Schaper W., Jagenau A., Xhonneux R. The development of collateral circulation in the pig and dog heart. Cardiologia 1967;51:321-335.
  6. Horneffer P.J., Vincent L.H., Gardner T.J. Swine as a cardiac surgical model. In: Tumbleson M.E., ed. Swine in biomedical research. New York, London: Plenum Press, 1986:321-325.
  7. Julien P., Downar E., Angel A. Lipoprotein composition and transport in the pig and dog cardiac lymphatic system. Circ Res 1981;49:248-254.[Free Full Text]



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