Weimar, Timo MD; Lee, Anson M. MD; Ray, Shuddhadeb MD; Schuessler, Richard B. PhD; Damiano, Ralph J. Jr MD
From the Division of Cardiothoracic Surgery, Department of Surgery, Washington University School of Medicine, Barnes-Jewish Hospital, StLouis, MO USA.
Accepted for publication December 8, 2012.
Supported, in part, by the National Heart, Lung and Blood Institute at the National Institutes of Health (grants RO1 HL032257, RO1 HL085113, and T32 HL07776).
Disclosures: Richard B. Schuessler, MD, receives research support from AtriCure, Inc, Cincinnati, OH USA. Ralph J. Damiano, MD, is a consultant for AtriCure, Inc, Cincinnati, OH USA, and Medtronic, Inc, Minneapolis, MN USA and has received research grants from AtriCure, Inc. Timo Weimar, MD; Anson M. Lee, MD; and Shuddhadeb Ray, MD, declare no conflict of interest.
Address correspondence and reprint requests to Ralph J. Damiano, Jr, MD, Division of Cardiothoracic Surgery, Washington University School of Medicine, Barnes-Jewish Hospital, Suite 3108 Queeny Tower, 1 Barnes-Jewish Hospital Plaza, St Louis, MO 63110 USA. E-mail: firstname.lastname@example.org.
Atrial fibrillation (AF) is the most common sustained arrhythmia worldwide. The first successful nonpharmacological treatment of this arrhythmia was introduced at our institution in 1987 by Dr James Cox. The final version of his cut-and-sew technique, termed the Cox-maze procedure III, has been proven to be highly efficacious.1–3 However, this procedure was technically demanding and associated with a considerable morbidity and was not widely adopted by cardiac surgeons. The development of alternative energy sources has simplified and shortened the procedure by replacing most of the incisions of the Cox-maze procedure III with linear lines of ablation.4–7 These alternative energy sources included radiofrequency, focused ultrasound, microwave, laser, and cryothermy.
See accompanying editorial on page 387
Cryoablation destroys cardiac tissue by the formation of intracellular and extracellular ice crystals, which disrupt cell membranes and organelles. With the development of specially designed cryoprobes, discrete lines of ablation could be achieved with homogenous scar formation.8,9 This made this energy source potentially useful for the treatment of cardiac arrhythmia. As opposed to unipolar radiofrequency energy, which resulted in complications such as pulmonary vein stenosis and esophageal perforation,10 cryoenergy has an excellent safety profile.11 It is the only available energy source that does not disrupt tissue collagen, thus preserving normal tissue architecture and allowing a safe ablation close to valvular tissue or the fibrous skeleton of the heart with a low arrhythmogenic potential.12–14 To be effective for the treatment of AF, an energy source must reliably create transmural lesions, leading to conduction block. Studies investigating the efficacy of endocardial cryoablation in producing reliable transmural lesions on the beating heart and their impact on atrial activation have been limited, and many of the commonly used devices have not been investigated.
The purpose of this chronic animal study was to examine the ablation performance of two cryoprobes on the beating heart: a newly introduced malleable cryoprobe and a rigid linear probe, both of which are clinically available for the surgical ablation of AF.
Six Hanford miniature swine weighing 50 to 70 kg were used in this study. The protocol was in compliance with the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC USA, 1996) and approved by the Washington University School of Medicine Animal Studies Committee.
Two different cryoprobes were used in this study. Both devices were cooled using nitrous oxide delivered through a controlling console (AtriCure, Inc, Cincinnati, OH USA). The newly developed Cryo1 probe is a disposable malleable aluminum device with a 10-cm probe length. The nitrous oxide gas is evenly dispersed to avoid temperature variations along the shaft, and the use of aluminum provides a high thermal conductance. The rigid 3011 Maze Linear probe is reusable, with a probe length of 3.5 cm consisting of gold-plated copper that is already widely used for the surgical ablation of arrhythmias.
Each animal underwent biatrial ablation on the beating heart. After overnight fasting, the animals were premedicated intramuscularly with ketamine (15 mg/kg) and anesthetized with endotracheally applied isoflurane (2%–4%) after intubation. Preoperative intramuscular antibiotic treatment with ceftiofur (2 mg/kg) was given to all animals. Continuous monitoring throughout the procedure consisted of electrocardiogram, invasive arterial blood pressure from the left internal thoracic artery, and routine blood gas analyses. The animals were heparinized intravenously (initial bolus, 250 U/kg, additional boluses as needed) to reach and maintain an activated clotting time of, longer than 350 seconds.
After median sternotomy, the lesion set diagrammed in Figure 1 was applied to all animals. After measuring pacing thresholds with a pen, a bipolar radiofrequency clamp (Isolator Synergy; AtriCure, Inc, Cincinnati, OH USA) was used to circumferentially isolate the right atrial appendage and the inferior vena cava. Electrical isolation was documented by pacing, with stimulus strength of twice the previous measured threshold to confirm exit block. Epicardial unipolar electrograms from the right atrium were recorded in sinus rhythm and during pacing at 120 beats per minute (bpm). These two lesions, representing areas of known conduction block, were connected by creating an endocardial line of ablation using the malleable cryoprobe Cryo1. Epicardial mapping was then performed to determine whether conduction block was present along the length of the lesion. The probe was inserted into the right atrium via a purse-string suture placed at the right atrial appendage. The ablation was performed for 2 minutes at −60°C, which was confirmed by thermocouples placed along the surface of the device. Epicardial activation mapping of the right atrium was repeated during sinus rhythm and pacing at 120 bpm. Through a separate purse-string suture, a parallel endocardial line of ablation on the free right atrial wall was performed for 2 minutes at −40°C using the 3011 Maze Linear probe. An additional lesion was performed with each cryoprobe on the left atrial free wall through two separate purse-string sutures for 2 minutes at −40°C. All lesions were created by a single application of the assigned device. The ending of each ablation line was marked with Prolene sutures.
After recovery from the procedure, all animals were allowed to survive for 14 days. Two doses of ceftiofur (2 mg/kg) were given intramuscularly within 48 hours postoperatively. Starting on the day of operation, each animal received a daily oral dose of 325 mg of aspirin. No antiarrhythmic drugs were administered during postoperative care.
On postoperative day 14, all animals were anesthetized and intubated and underwent redo sternotomy. Simultaneous recording of epicardial voltage at the right atrium was repeated during sinus rhythm and while pacing with bipolar pacing at 120 bpm.
The animals were then euthanized using an infusion of 3 M of potassium chloride. The aorta was cross-clamped, the coronary arteries were perfused with a solution of 2,3,5-triphenyl-tetrazolium chloride (1%), and the heart was then removed en bloc for inspection and histologic evaluation.
Epicardial Conduction Mapping
Custom-made silicon patches with 128 embedded unipolar electrodes were used to simultaneously record epicardial electrograms. The electrodes were separated by 5 mm. The patch was secured to the epicardium of the right atrium using Prolene suture. Recordings were made for at least 10 seconds during normal sinus rhythm and during atrial pacing at 120 bpm from the medial side of the lesion. Data were recorded at a frequency response of 0.5 to 500 Hz and digitized at 1000 Hz.
The epicardial electrograms were acquired and analyzed by two programs developed in our laboratory (Data Acquisition System Host and Get Look Analyze Save, respectively; Washington University, St Louis, MO USA). Time of epicardial activation was selected by identifying the peak negative derivative of voltage over time. Automatically selected activation times were manually reviewed and edited for accuracy. Activation maps and activation movies were then generated and displayed on a three-dimensional anatomical porcine model constructed by a software developed in our laboratory (Getpic3 and Map3; Washington University, St Louis, MO USA) based on porcine computed tomographic scans.
Microscopic and Histologic Analysis
After resection, the heart was thoroughly inspected for intra-atrial thrombus formation and then incubated in 2,3,5-triphenyl-tetrazolium chloride (1%) at room temperature for 45 minutes. Each marked lesion was sectioned at 5-mm intervals perpendicular to the longitudinal axis of each ablation line. The cross-sections were examined under the microscope, and each slide was scanned with a high-resolution scanner and evaluated for transmurality, lesion depth, and lesion width using Adobe Photoshop (Version 7; Adobe Systems, Inc, San Jose, CA USA).
All cross-sections were fixed in formalin, molded in paraffin, and stained in hematoxylin and eosin, and trichrome. A histologic examination of the specimen was performed by a veterinary pathologist.
All continuous data were expressed as mean ± SD, unless otherwise specified. Categorical data were expressed as absolute numbers and proportions. Comparisons were made with the unpaired t test. All data analyses were performed with the SYSTAT system for statistics (SYSTAT version 13; SYSTAT, Inc, Chicago, IL USA).
All animals survived the surgical ablation procedure and the 14-day postoperative period. There was no intraoperative or postoperative cardiac arrhythmia. All animals remained in sinus rhythm until they were killed. There were no signs of neurological dysfunction in the postoperative setting in any animal.
No thrombus formation was found at any location in the right or left atria when macroscopically inspected. The ablation lines of both devices appeared equally discrete, linear and pale, and could be visualized endocardially and epicardially.
Epicardial Conduction Mapping
Electrical isolation was documented for the circumferential lesions around the right atrial appendage and around the inferior vena cava and was confirmed by exit block with stimulus strength of twice the previous measured pacing threshold in all animals. Thus, two areas of known conduction block were established by the bipolar radiofrequency clamp.
The activation maps of the right atrium generated from surface electrogram recordings identified a significant conduction delay from 20 ± 2 milliseconds before creation of the ablation line to 51 ± 8 milliseconds afterward (P < 0.001) between two defined points on either side of the ablation line created by the Cryo1 probe. All six animals were found to have complete electrical block across the Cryo1 lesion. Fourteen days postoperatively, this conduction block remained unchanged in all animals, and the right atrial conduction delay was similar (52 ± 10 milliseconds) to the acute postablation activation map (P = 0.88, Fig. 2).
Microscopic and Histologic Examination
After the biatrial lesions created by the 3011 Maze Linear probe were sectioned at 5-mm intervals, 41 cross-sections were inspected microscopically. A well-marginated transmural lesion was confirmed in all but one cross-section (97.6%; Fig. 3). The only section that failed transmurality was located at the very end of the left atrial ablation line (Fig. 5).
The biatrial lesions created by the Cryo1 probe were sectioned at 5-mm intervals as well. Microscopic examination of 84 cross-sections also revealed transmurality in all but one specimen (98.8%; Fig. 3). Again, the only cross-section that could not confirm transmurality was located at the end of the left atrial ablation line.
The atrial wall thickness encountered was not significantly different for the two devices (Cryo1, 4.5 ± 1.8 mm; 3011 Maze Linear, 4.7 ± 1.5 mm; P = 0.74). There was also no significant difference in lesion depth (Cryo1, 4.5 ± 1.7 mm; 3011 Maze Linear, 4.6 ± 1.5 mm; P = 0.74) or lesion width (Cryo1, 10.7 ± 3.5 mm; 3011 Maze Linear, 10.0 ± 3.9 mm; P = 0.31) between the Cryo1 probe and the 3011 Maze Linear probe (Fig. 4).
The histologic examination revealed no significant differences between the Cryo1 and 3011 Maze Linear probes. Both probes caused a distinct, well-marginated transmural ablation. The endothelial layer was intact in almost all cases for both probes. A significant observation for both probes was the development of necrotic cardiomyocytes in the center of the ablation site. These sites were characterized by occasional pyknotic cells and loss of the vessel endothelium and, presumably, loss of function of the vasculature. Mild lymphocyte infiltration was often present at the margins of these areas (Fig. 5).
The advent of ablation technology has resulted in the development of less-invasive surgical approaches for AF by replacing surgical incisions with transmural lines of ablation.5–7,15 Tissue injury and cell death are caused by either heat (ie, laser, microwave, or focused ultrasound) or freezing when cryoenergy is applied. Cryogenic techniques have been reliably used in surgery for decades to treat cardiac arrhythmias. The mechanisms of cell injury during cryoablation are complex. Extracellular fluid freezes at −20°C, creating a hyperosmotic environment that causes cell shrinkage and, ultimately, cell death. Rapid freezing to −40°C causes expansion of intracellular ice formation that disrupts organelles and cell membranes before osmotic imbalance occurs16 and is the mode of ablation in both devices used in this study. Because of its unique method of cell destruction, cryoenergy has an excellent safety profile and is the only available energy source that does not disrupt tissue collagen. Thus, it can be applied safely in the vicinity of the heart valves. Although Doll and colleagues17 reported mild esophageal lesions when applying epicardial cryoenergy in a sheep model, the risk for collateral damage of surrounding tissue is minimal as opposed to reports for unipolar radiofrequency.10
To be a treatment option, any alternative energy source must reliably create transmural lesions. The efficacy of cryoenergy depends mainly on the cooling capability of the device, on the thermal conductivity of the probe material, and on established surface contact. Of three earlier published animal studies investigating the effectiveness of cryoenergy on the beating heart, only one reported all samples to be transmural.18 Whereas this study investigated endocardial isolation of the pulmonary veins, the remaining two studies used cryoenergy epicardially. Although electrical isolation and transmurality could be documented for thin-walled structures such as the pulmonary veins and the vena cava, results for thicker tissue at the atrial wall were rather disappointing, with only 25% to 84% of the lesions being transmural.17,19 One reason for the failure rate of epicardial linear cryoablation on the beating heart might be the heat-sink effect caused by the blood flow in the cardiac cavum. To overcome this effect, we chose an endocardial approach to mimic the endocardial application of cryoenergy that is routinely performed in concomitant cardiac surgery and stand-alone procedures. Because of the complexity and the morbidity of surviving animals after cardiopulmonary bypass, this study was performed on the beating heart. This provided an additional thermal load to the cryoprobes because the flowing blood pool impacts device performance. The goal of this study was to evaluate the ablation performance of a new malleable aluminum probe (Cryo1) using a nitrous oxide cooling system when used endocardially within the beating heart and to compare it with a cryoprobe that has been successfully used for surgical cryoablation for many years (3011 Maze Linear).
In the present study, we were able to reliably create transmural lesions with both devices. The only two cross-sections that could not confirm transmurality were located at the very end of an ablation line, emphasizing the need for overlapping connecting lesions to ensure a complete line of conduction block. At our institution, we mark the endpoints of an ablation line with methylene blue to ensure precise connection and overlapping when creating a lesion set. The ablation with the Cryo1 probe resulted in a significant increase in conduction delay, as would be expected for a transmural lesion. Earlier investigations in our laboratory showed that an acute conduction delay was not an accurate indicator for the evaluation of chronic lesion integrity.20 In this study, both the acute conductance block and delay remained unchanged at 14 days, evidence of chronic line of block.
The thermal conduction coefficient is the predictor of the rate of heat loss through a unit thickness of material and is reciprocally proportional to the thermal resistance. Although the copper used as the medium in the 3011 Maze Linear probe has a twofold higher thermal conduction coefficient than the aluminum used in the Cryo1 probe, we could not find any significant differences in the performance or efficacy between the two probes. However, the length of the probe and its malleability make the design of the Cryo1 probe potentially more useful for some surgical approaches.
There are limitations to this study, and these results must be cautiously applied to the clinical application. There are significant differences between the human and the porcine atrial anatomy. This study was performed on healthy pigs with normal atria, which cannot be thoroughly compared with the diseased and thickened atria of patients with AF. However, the successful use of cryoablation in creating transmural lesions has been reported for clinical applications as well.21 A significant shortcoming for the clinical use is the endocardial application via purse-string sutures. Although this would allow for a minimally invasive approach without the need for cardiopulmonary bypass and could be performed on the right atrium with few difficulties, it would be dangerous and ill advised on the left atrium because there would be a chance for both bleeding and air embolism. The epicardial application of cryoenergy on the beating heart has already failed to demonstrate transmurality on thicker wall structures such as the lateral and the posterior atrial wall because of the heat-sink effect.17 Thus, the creation of a full maze lesion set with epicardial cryoablation is not promising. Rather, the design of this study was to mimic an endocardial approach with cardiopulmonary bypass because it is used for concomitant cardiac surgery and for stand-alone procedures.
The authors thank Diane Toeniskotter and Naomi Still for their technical assistance.
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This timely article from the laboratory of Drs Schuessler and Damiano details the in vivo performance of a new, flexible, disposable cryoprobe with a previously available, rigid, reusable probe. Using nitrous oxide as the refrigerant, the probes were used to form biatrial ablation lines in the beating heart porcine model. The results of this study provide histologic and electrophysiologic data demonstrating that both probes produce reliably uniform, transmural scars that block electrical conduction. The only failures of transmurality were the tissues ablated at the distal end of the probe, emphasizing the importance of overlapping the lesions. Of note, to avoid the complexity of cardiopulmonary bypass, the lesions were formed by applying the probes endocardially via purse strings. Although this technique is well described in clinical applications, the authors do not recommend it, particularly for left atrial lesions, because of concerns of bleeding and air embolism. The authors astutely emphasize that epicardial applications of cryoprobes on beating hearts are unreliable because of the heat-sink effect of the blood stream. Limitations of this study include the fact that the mean lesion depth was only 4.5 mm, substantially less than the wall thickness often encountered in diseased human atria, particularly in the mitral isthmus and the atrioventricular fat pad. This emphasizes our concern that 2-minute endocardial applications of the cryoprobes in this study may be inadequate for producing uniform transmural lesions in some clinical situations. With the knowledge that cryothermia is the only power source that does not destroy tissue collagen, this is an important contribution to our understanding of the effects of cryothermy in producing electrically silent lesions in atrial tissue.
Harold G. Roberts, MD, is the guest editor
Copyright © 2012 by the International Society for Minimally Invasive Cardiothoracic Surgery. Unauthorized reproduction of this article is prohibited.