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Ann Thorac Surg 1999;67:1708-1713
© 1999 The Society of Thoracic Surgeons

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Original Articles

Transmyocardial laser revascularization: experimental studies in healthy porcine myocardium

^a Divisions of Division of Cardiovascular Surgery, Albert-Ludwigs-University School of Medicine, Freiburg, Germany and Division of
^b Pathology, Albert-Ludwigs-University School of Medicine, Freiburg, Germany

Accepted for publication December 23, 1998.

Address reprint requests to Dr Lutter, Division of Cardiovascular Surgery, Department of Surgery, University of Freiburg Medical Center, 55 Hugstetter Str, D-79106 Freiburg, Germany
e-mail: lutter{at}ch11.ukl.uni-freiburg.de

	Abstract

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Background. Clinical studies have demonstrated a significant reduction of cardiac index shortly after transmyocardial laser revascularization in patients with low ejection fraction. We analyzed the influence of transmyocardial laser revascularization on healthy myocardium in pigs.

Methods. Carbon dioxide channels were created in 20 pigs which were observed for 6 hours. Ten pigs received one laser channel and ten pigs two laser channels per cm² in the left anterior descending artery region. Seven pigs served as controls. Perfusion (microspheres), function, histochemical, and histologic assessments were subsequently performed.

Results. A significant deterioration of left ventricular stroke work index was observed shortly after transmyocardial laser revascularization in both laser groups (p < 0.05). After 6 hours the left ventricular stroke work index did not increase and showed significantly reduced values at rest (p < 0.05) and during stress in the laser groups (p < 0.01). Normal regional perfusion, small ischemic and necrotic areas, open laser channels in the left anterior descending artery region and significantly increased myocardial water content were observed in the laser groups (p < 0.01).

Conclusions. Carbon dioxide laser channels significantly decrease global heart function shortly after transmyocardial laser revascularization in healthy porcine myocardium. This myocardial tissue showed no recovery 6 hours postoperatively.

	Introduction

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Transmyocardial laser revascularization (TMLR) is a new surgical technique of indirect revascularization for patients with symptomatic end-stage coronary artery disease who are not good candidates for conventional revascularization [1]. The clinical experience of single- and multiinstitutional studies with TMLR [2, 3] indicates that angina is significantly relieved, perfusion and treadmill tolerance improved, and hospital admissions decreased. Despite its increasing surgical use careful experimental and clinical validation of TMLR is required to prove its effectiveness in treating regional myocardial ischemia.

Patients with unstable angina and reduced left ventricular (LV) function represent a high-risk group for TMLR, with significantly higher mortality and morbidity compared with stable patients with normal ejection fraction so that TMLR is considered contraindicated for this high-risk group [4–6] at some centers. We have used the surgical strategy of inserting an intraaortic balloon pump before the TMLR procedure, because our hemodynamic data in this patient cohort (ejection fraction < 0.35) have revealed a significant decrease in cardiac index in the first hour after TMLR [7]. In contrast, patients with an ejection fraction greater than 0.35 did not have any reduction in cardiac index. Few studies have explored the hemodynamic changes after TMLR during the perioperative course [7, 8]. Therefore, this study was initiated to evaluate TMLR in healthy porcine myocardium in terms of its prolonged short-term effects on hemodynamic factors. Furthermore, perfusion, histochemical, and histologic parameters were analyzed after TMLR treatment.

	Material and methods

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Animals and anesthesia
All procedures were done in conformity with the "Principles of Laboratory Animal Care" formulated by the Institute of Laboratory Animal Resources and the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985). German Landrace pigs weighing 29 to 42 kg were premedicated and anesthetized, as reported elsewhere [8].

Experimental groups
Hemodynamic and perfusion measurements were performed after thoracotomy. All animals were randomly assigned to one of three experimental groups. In the control group, after thoracotomy 6 pigs were kept anesthetized for 6 hours after a 30-minute observation period (an equal time period compared to laser groups) to determine the effect of thoracotomy and anesthesia. In laser group 1, 10 pigs received the same treatment as the control group, except for additional treatment by TMLR after thoracotomy. They were treated by creating one laser channel (1 mm in diameter) per cm² in the area of the left anterior descending artery (LAD) beyond the first diagonal branch. Animals were monitored for 6 hours after the TMLR procedure. In laser group 2, 10 pigs were treated with the same regimen as laser group 1; however, these animals received two laser channels (of 1 mm diameter) per cm². The monitoring was done as reported elsewhere [9].

Experimental preparation
After anterolateral thoracotomy 150 IU/kg heparin and 1 g magnesium were given intravenously to all pigs. Lidocaine was applied at 40 to 60 µg/kg per minute in a continuous drip. The heart was suspended in a pericardial cradle. A 7-F cannula was placed through the left hemiazygos vein into the sinus coronarius for venous blood gas analysis and enzyme probes. Complications such as atrial fibrillations were treated with local application of 50 to 100 mg of lidocaine hydrochloride and ventricular fibrillations with electrical countershocks (10 to 20 Joule) and intervening open-chest cardiac compressions.

Laser procedure
TMLR was accomplished with an 800 W (spot size, 1 mm; mean pulse energy, 41 Joule) pulsed carbon dioxide (CO₂) laser (Laser Engineering, Milford, MA) as described previously [9]. Channels were created in a distribution of 1 (laser group 1) or 2 (laser group 2) channels/cm² in the LAD territory beyond the first diagonal branch. On average 21 channels were created in laser group 1 and 30 channels in laser group 2 (p < 0.001). Drilling took an average of 39 minutes per animal (laser group 1) and 47 minutes per animal (laser group 2) to complete.

Hemodynamic measurements
Baseline hemodynamic measurements were recorded before and shortly after thoracotomy and TMLR. Further hemodynamic assessments were performed hourly. Cardiac output was measured by the thermodilution technique. To induce myocardial stress, cardiac preload was increased by stepwise volume loading and controlling left atrial pressure. Frank-Starling curves were registered in all groups shortly after thoracotomy and at the end of the 6-hour observation period. Left ventricular stroke work was calculated and normalized for heart weight as stroke work index in mJ/g as follows:

where SWI is the LV stroke work index (mJ/g); MAP is the mean arterial pressure (mm Hg); LAP, the left atrial pressure (mm Hg); CO, the cardiac output (L/min); HR, the heart rate (beats per minute); and HW, the heart weight (g). The maximal achieved LV stroke work index (LVSWI_max) was used for comparison between the experimental groups.

Perfusion
Perfusion measurements were performed in 6 animals from each experimental group. Regional myocardial blood flow of the LAD and right circumflex artery (RCX) territory was measured based on the microsphere and arterial reference sample technique as described previously [9]. The microsphere suspensions were injected into the left atrium shortly after thoracotomy in all groups and 0.5 hour after the TMLR procedure in the laser groups.

Electrocardiographic findings
After thoracotomy, changes in the electrocardiogram (monitored every hour) included ST-segment changes, T wave inversion, and abnormal Q waves.

Histochemical assessment and histology
Monastryl blue vital dye (2% solution, 0.25 mg/kg body weight) was injected via the left atrium into all animals after proximal LAD occlusion to delineate histologically the area perfused by the laser channels. LAD occlusion was only done 15 seconds before heart arrest (hyperkalemic solution at 4°C) to determine whether perfusion occurs through open laser channels. The ventricles were cut into 5-mm thick transverse sections and incubated in 1% solution of TTC (triphenyl tetrazolium chloride), as reported elsewhere [9]. Thus prepared, the sections of the LV free wall underwent computerized, planimetric analysis to determine the total area of the LV, ischemia, and necrosis [10]. Myocardial samples were fixed [9], sectioned mostly perpendicular and longitudinal to the apex-base axis at a thickness of 5 µm, stained with hematoxylin-eosin, Lie, and Luxol fast blue. To assess the effect of creating channels in healthy myocardial structure two investigators masked to treatment group status measured the following five parameters: (1) ischemia associated with the channels; (2) channel patency; (3) channel diameter and bordered rim of carbonization, necrosis, and myofibrillary degeneration; (4) fibrin content, monastryl blue vital dye, and erythrocyte content in the channels; and (5) the boarded tissue for the presence of monastryl blue vital dye, erythrocytes, and neutrophils. This analysis was done in 30 histologic sections with laser channels in the LAD territory. Furthermore, in 243 histologic samples, the entire anterior wall was analyzed for signs of ischemia and compared with control samples of remote myocardium.

Myocardial water content
Transmural myocardial samples were obtained from transverse sections of the LV free wall after cardiac arrest [11]. Samples were dried at 85°C for 24 hours to achieve constant weight, and water content (%) was determined by the following formula:

Statistical analysis
Statistical analysis was done by two-way analysis of variance, unpaired Student’s or Welch’s t test. Bonferroni corrections for repeated measurements over time were used as appropriate. The ² test was used to compare different electrocardiographic changes between the experimental groups. Values were considered to differ significantly if p was less than 0.05. Results are expressed as mean value ± standard deviation.

	Results

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One pig in the control group died of intractable ventricular fibrillation. In the control group, compared with baseline values, global hemodynamics (LVSWI_max) were minimally changed after 6 hours. Mean arterial blood pressure remained at control levels, electrocardiographic measurements did not change. Histochemical and histologic assessment did not reveal any ischemia. In the electrocardiographic findings, there was mild T-inversion in five cases from laser group 1 (significantly increased compared with control, p < 0.05) and in one case from laser group 2 (not significant). Five to 6 hours after TMLR they reverted to normal (not significant). Four of 10 pigs (laser group 1) had ventricular fibrillation during the TMLR procedure (not significant). In contrast, ventricular fibrillation occurred less often in laser group 2 (10%, 1/10) and in the control group (17%, 1/6).

Systemic hemodynamics
The LVSWI changes during the perioperative course of TMLR and LVSWI_max at baseline and at the end of study are summarized in Figures 1 and 2 for all study groups.

View larger version (24K): [in this window] [in a new window]   Fig 1. Left ventricular function in healthy porcine myocardium during the perioperative course of transmyocardial laser revascularization (TMLR) (left ventricular stroke work index in mJ/g). Shortly after TMLR, a significant deterioration of left ventricular stroke work index compared with baseline values was observed in laser groups 1 and 2. Even after a 6-hour observation period left ventricular stroke work index did not increase and demonstrated significantly reduced values in laser groups 1 and 2 compared with baseline (*p < 0.05, p < 0.001) (B = baseline; AT = after thoracotomy; A TMLR = after TMLR; h = hours).

View larger version (22K): [in this window] [in a new window]   Fig 2. The maximal left ventricular stroke work index was assessed after a 6-hour observation period in all study groups and compared with baseline values. In laser groups 1 and 2, a comparable reduction in left ventricular stroke work indexmax. was observed at the end of the experiment, with a significant reduction compared with control group (p < 0.01 and baseline, *p < 0.05). Mean values (column height) and standard deviation (error bars) are shown.

View larger version (22K): [in this window] [in a new window]   Fig 3. Comparison of the total amount of the left ventricular myocardium, ischemia, and necrosis (of a transversal section of the left ventricular free wall) for controls and laser groups 1 and 2. The area of the left ventricle in the control and laser groups did not differ significantly. The areas of ischemia and necrosis of laser groups 1 and 2 were not significantly higher than in control group. Mean values (column height) and standard deviation (error bars) are shown.

View larger version (21K): [in this window] [in a new window]   Fig 4. Myocardial water content in the analyzed left anterior descending artery (LAD) and left circumflex artery (LCX) territory of healthy porcine myocardium after transmyocardial laser revascularization and an observation period of 6 hours for controls and laser groups 1 and 2. Myocardial water content of laser groups 1 and 2 differed significantly in the LAD and LCX territory compared with controls (*p < 0.01). Mean values (column height) and standard deviation (error bars) are shown.

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
This experimental model demonstrated that CO₂ laser revascularization in healthy porcine myocardium significantly deteriorated global left ventricular function (LVSWI) in laser groups 1 and 2 (p < 0.05; Fig 1, 2). After 6 hours LVSWI did not increase and at rest (p < 0.05) and under stress (p < 0.01) values were significantly lower in the laser groups compared with baseline. Laser groups 1 and 2 did not differ significantly. Normal regional perfusion values were achieved 0.5 hour after TMLR treatment (Table 1). Six hours after the procedure, only small ischemic and very small necrotic areas, open laser channels in the LAD region (Fig 3), and significantly increased myocardial water content were observed (Fig 4) compared with control in the laser groups (p < 0.01). Despite mild T-inversion which was observed in five cases from laser group 1 shortly after TMLR (significantly increased compared with control for 2 hours, p < 0.05), neither significant ST changes nor increased arrhythmias were observed in the laser groups during the subsequent observation period.

Experimental studies
In contrast to LV function, regional myocardial blood flow was not impaired shortly after TMLR, signifying that major intramyocardial coronary branches were not destroyed. Furthermore, ischemic and necrotic areas were too small (and not even observed in some animals, which also demonstrated significant LVSWI reduction) to induce deterioration of the LV function. Nevertheless, the very sensitive parameter of myocardial water content, which was increased 6 hours after TMLR compared to control (p < 0.01), demonstrated global myocardial edema in the LAD and LCX territory which might have caused this functional deterioration as an injury to the entire myocardium (Fig 4). There is little experimental and clinical information in the literature as to whether TMLR reduces short-term LV function [5–7, 16]. In a short-term study conducted in canine hearts, Yano and coworkers [16] and Kadipasaoglu and colleagues [17] found no significant reduction in LV function, despite severe regional dysfunction, after laser channel creation and acute LAD ligation in lased and control animals in the 6-hour observation period. This finding suggests global compensation of LV function, which can be explained by the intrinsic collateral blood flow in canine hearts.

Study limitations
The main limitation of this study is that it was performed in a short-term model, and we therefore do not know when LV function would have been recovered. Further measurements of regional contractility [16] would have provided additional information, especially concerning the quantity of laser channels.

Channel density and evidence for flow via channels
The number of channels that should be drilled is a topic that remains controversial. We drilled 1 and 2 CO₂ laser channels per cm² in laser groups 1 and 2, respectively (p < 0.001). One channel per cm² is the most widely used channel density in clinical and experimental studies [2–7, 9, 14, 15, 17–20]; however, a general threshold of channel density should be defined for clinically treating myocardial ischemia.

In laser groups 1 and 2, we observed typical features of CO₂ laser channels with marginal necrotic and thermal damage as described by Hardy and colleagues [13] and Fisher and coworkers [14]. Furthermore, small amounts of pigment could be seen in the channels and their surrounding myocardium, indicating that blood could flow from the LV cavity to the endocardium via the channels [12].

The ongoing decline of LV contractility 6 hours after TMLR in this study could be caused by the combination of a generalized myocardial edema, laser carbonization, necrosis, and myofibrillary degeneration in the border zone of the microchannels as an inflammatory response [7, 15].

Clinical studies
Although TMLR significantly reduces short-term LV function in healthy porcine myocardium, TMLR is done clinically mainly in areas where regional wall motion is reduced to hypokinesia or akinesia, because of the underlying chronic ischemia in viable myocardium. After TMLR, a decrease of wall motion movement in these hypokinetic to akinetic regions or a reduction of the EF by perioperative transesophageal echocardiography [7] or follow-up ventriculography has not been observed [19, 20]. In patients with unstable angina and low EF we inserted an intraaortic balloon pump preoperatively to provide cardiac support during the phase of reversible myocardial damage induced by TMLR, because hemodynamic data in these patients only have shown a significant reduction of LV function shortly after TMLR and a significant increase after 6 hours [7]. To treat the recoverable form of LV dysfunction after TMLR the intraaortic balloon pump assistance might be required in some patients. However, these experimental results from a short-term model in healthy porcine myocardium cannot be transferred directly to chronic ischemic injury in the human myocardium.

In conclusion, we demonstrated that CO₂ laser channels significantly decrease global heart function shortly after TMLR in healthy myocardium. This myocardial tissue shows no recovery during a 6-hour observation period. Therefore, the possibility of a reduction of global contractile function cannot be ruled out if viable myocardial areas maintaining LV function are lased, especially in cases with low ejection fraction.

	Acknowledgments

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This study was supported by the Clinical Cardiovascular Research Center II at the Medical Center, Albert-Ludwigs-University of Freiburg, Germany, the German Research Foundation, Bonn, the Research Fellows from the Department of Thoracic Surgery, Sapporo University Medical Center, Sapporo, Japan, and the Department of Cardiovascular Surgery, Jikei University Medical Center, Tokyo.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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