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Eur J Cardiothorac Surg 1998;13:694-701
© 1998 Elsevier Science NL
a Division of Cardiovascular Surgery, Albert-Ludwigs-University, School of Medicine, Freiburg, Germany
b Division of Cardiovascular Surgery, Jikei University Medical Center, Tokyo, Japan
c Division of Thoracic Surgery, Sapporo University Medical Center, Sapporo, Japan
d Division of Nuclear Medicine, Albert-Ludwigs-University, School of Medicine, Freiburg, Germany
Received 10 November 1997; received in revised form 9 March 1998; accepted 17 March 1998.
Corresponding author. Division of Cardiovascular Surgery, Department of Surgery, University of Freiburg, Hugstetter Strasse 55, D-79106 Freiburg, Germany. Tel.: +49 761 2702818; fax: +49 761 2702550; e-mail: lutter@ch11.ukl.uni-freiburg.de
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Abstract |
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 Top Abstract Introduction
Materials and methodsMiscellaneous measurementsDiscussion
Appendix A. Conference...References |
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Key Words: Laser • Coronary disease • Ischemia • Myocardium • Transmyocardial laser revascularization
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Introduction |
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TopAbstractIntroduction
Materials and methodsMiscellaneous measurementsDiscussion
Appendix A. Conference...References |
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However, the exact mechanism based on that TMLR is facilitating these subjective and objective improvements remains unknown yet. Therefore, this study was initialized to evaluate the short-term effectiveness of TMLR in the setting of an acute coronary occlusion in pig hearts.
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Materials and methods |
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TopAbstractIntroduction
Materials and methodsMiscellaneous measurementsDiscussion
Appendix A. Conference...References |
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Pigs of the `German Landrace' weighing 27–35 kg were premedicated with an intramuscular injection of 0.2 mg/kg flunitrazepam and 7 mg/kg ketamine hydrochloride. An ear vein was cannulated and anesthesia was induced with 0.1 mg/kg flunitrazepam and 10 mg/kg ketamine hydrochloride by titrated intravenous injection.
After muscular relaxation has been induced with 0.1 mg/kg i.v. vecuronium bromide an endotracheal tube was inserted and mechanical ventilation begun. Tidal volume and respiratory rate were adjusted maintaining oxygen tension >100 mmHg, carbon dioxide tension 35–42 mmHg and pH 7.3–7.4. Anesthesia was maintained by continuous intravenous infusion of 0.06 mg/kg per h vecuronium, 0.004 mg/kg per h fentanyl dihydrogen citrate, 5.2 mg/kg per h propofol, and 0.04 mg/kg per h flunitrazepam.
At the end of the experiment the animals were sacrificed by an intravenous injection of potassium chloride.
Experimental groups
After thoracotomy, hemodynamic and perfusion-measurements were performed and all animals were randomized into one of four experimental groups.
Control group, no ischemia
Six pigs underwent isolation of the left anterior descending artery (LAD) without occlusion and were kept anesthetized for 6 h (after thoracotomy) to determine the effect of thoracotomy and anesthesia.
Ischemic group
In seven pigs myocardial infarction were created by isolated occlusion of the LAD beyond the first diagonal branch after an 30 min observation period after thoracotomy (as an equal time period compared with laser groups). The occlusion were left in place for 6 h. No laser treatment was performed in these cases.
Laser group 1
Nine pigs received the same treatment as the ischemic group except prophylactical treatment by TMLR half an hour prior to coronary occlusion. They were treated by one laser channel (of 1 mm diameter) per cm2 in the ischemic LAD area. To define this area of risk LAD has been occluded for 10 s prior to laser treatment. After LAD occlusion animals were monitored for 6 h.
Laser group 2
Eight pigs were treated with the same regimen as laser group 1. However, in contrast to laser group 1 these animals received two laser channels (of 1 mm diameter) per cm2.
Monitoring
The right and left external jugular vein were surgically exposed and cannulated with a sheet. An intravenous line (14-gauge) was placed into the left external jugular vein to provide access for fluid and medications. A 7-French Swan-Ganz thermodilution catheter (Baxter Healthcare Corporation, Irvine, CA, USA) was inserted through the right external jugular vein into the pulmonary artery in order to record pulmonary artery pressure, cardiac output, pulmonary capillary wedge pressure and central venous pressure. The left carotid artery was cannulated with a 14-gauge polyurethane catheter to monitor systolic and mean arterial pressure (MAP) and to facilitate arterial blood gas analysis. Both catheters were connected to pressure transducers (SX 509597; Medex Medical GmbH, Ratingen, Germany) which drove an amplifier monitor (Sirecust 404–1; Siemens, Erlangen, Germany). The right femoral artery was cannulated with a 14-gauge polyurethane catheter to take blood samples for measurement of the perfusion. A 7F polyurethane catheter was inserted into the left atrium through the left atrial appendage to monitor left atrial pressure and to inject the microspheres. Electrocardiographic leads were affixed to the left thorax and extremities, and connected to a monitor. Sinusrhythm and any kind of rhythm disturbance were registered.
Experimental preparation
After anterolateral thoracotomy 150 IE/kg heparine and 1 g magnesium were given i.v. to all pigs. Lidocaine was applicated at 40–60 µg/kg per min in a continuous drip. The heart was suspended in a pericardial cradle. A 7F cannula was placed through the left hemiazygos vein into the sinus coronarius for venous blood gas analysis and enzyme probes. The LAD was dissected, isolated immediately distal to the bifurcation of the first diagonal branch and a snare occluder was placed loosely around the LAD for subsequent occlusion to produce an area at risk of about 20–30%. Complications such as atrial fibrillations were treated with local application of 50–100 mg lidocaine hydrochloride, ventricular fibrillations with electrical countershocks (10–20 J) and intervening open-chest cardiac compressions.
Laser procedure
Transmyocardial laser revascularization was accomplished with a 800 W (spot size 1 mm, wavelength 10.6 µm) pulsed carbon dioxide laser coupled to an articulating arm (Laser Engineering, Milford, MA, USA). The maximal output is 80 J. and the pulse width can be adjusted from 5 to 99 ms. Laser activation was synchronized with the R-wave of the electrocardiogram to avoid creating arrhythmias by stimulating the heart during the electrically vulnerable ventricular repolarization period. The carbon dioxide laser was aimed with a helium-neon laser (5 mW, wavelength 632.8 nm). The articulating arm connected to a focusing probe was placed against the epicardium of the heart within the area of risk and fired, creating a transmyocardial channel. Channels were created in a distribution of 1 (laser group 1) or 2 (laser group 2) channels/cm2. After each channel was formed, manual pressure with gaze was gently applied to the epicardial surface to secure hemostasis. Drilling took an average of 17 min/animal (Laser group 1) and 40 min/animal (Laser group 2) to complete (Table 2.).
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Miscellaneous measurements |
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TopAbstractIntroduction
Materials and methodsMiscellaneous measurementsDiscussion
Appendix A. Conference...References |
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where SWI is the stroke work index (mJ/g), MAP the mean arterial pressure (mmHg), LAP the left atrial pressure (mmHg), CO the cardiac output (L/min), HR the heart rate (beats/min), HW the heart weight (g).
For comparison between the experimental groups we the maximal achieved left ventricular stroke work index (LVSWImax) was used.
Perfusion
Regional myocardial blood flow of the LAD and RCX territory was measured based upon the microsphere and arterial reference sample technique. Fifteen±3 µm microspheres labeled with 95Nb, 103Ru, 141Ce were injected over a 30 s period into the left atrium while withdrawing a reference sample from the descending aorta over 2 minutes at a rate of 10 ml/min starting 5 s before the injection of the spheres as described previously
[6]
[7]. The spheres were suspended before injection in blood with 0.01 % Tween 80 and were vigorously agitated prior to their use. The number of spheres injected was calculated so that a 0.5 g sample of myocardium contained 800 spheres. The microsphere suspensions were injected into the left atrium shortly after thoracotomy in all groups, after TMLR procedure (only in the laser groups), and at the end of the study (after the 6 h observation period in all groups). The heart was removed after the experiment and samples of endocardial, mid-myocardial, and epicardial muscle were obtained and placed into tared vials. Radioactive content of each nuclide was determined by 
-spectrometry and blood flow was calculated as described previously
[6]
[7].
Histochemical assessment and histology
Monastryl blue vital dye (2% solution, 0.25 mg/kg body weight) was injected via left atrium in all animals with LAD occlusion to delinate the area at risk. The area of the left ventricle that did not receive monastryl blue vital dye was measured by planimeter and considered to be the area at risk. After 15 s the heart was arrested with a hyperkalemic solution at 4°C, excised, inspected, and the heart weight assessed. The ventricles were cut into 5 mm thick transverse sections. The slices were then incubated in 1% solution of TTC (triphenyl tetrazolium chloride) made up in 8 mmol/l dibasic and 2 mmol/l monobasic phosphate buffer (pH 8.4) for 30 min at 37°C
[8]
[9]. Thus prepared, the sections underwent planimetric analysis to determine the area at risk (AR of the left ventricle, expressed as the ratio AR/LV) and the area of necrosis in the area at risk (AN, expressed as the ratio AN/AR). The area of necrosis is defined as the TTC-unstained area (infarct size). Normal (viable) and abnormal (infarcted) myocardium could be sharply delineated by the TTC technique
[10]. Myocardial samples were fixed in 10% formalin, embedded in paraffin, cut and stained with hematoxylin/eosin.
Statistical analysis
Statistical analysis was performed based upon Student's- or Welch's- t-test or the Wilcoxon test to compare either paired or unpaired data as appropriate. A P-value less than 0.05 was considered statistically significant. Results are expressed as the mean±SD. Only data are used of animals who survived the whole 6 h observation period. Myocardial flow heterogeneity occured in the laser group 1 and ischemia group at baseline and at the end of experiment. The flow ranged from one third to more than three to six times the mean. Consequently the values of the RMBF which were greater as three times of the standard deviation had been excluded from analysis (<3% of the RMBF – data)
[11]
[12].
Results
The results are shown in Table 1Table 2Table 3 and
Fig. 1
Fig. 2 . Before randomization into the experimental groups, four pigs died of intractable ventricular fibrillation.
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Ischemia, laser I and 2 groups
Seven of seven pigs (ischemia group) had ventricular fibrillation (100%) during the first hour of LAD occlusion (without TMLR). In contrast, ventricular fibrillation occurred significantly less often in the laser group 1 (56%; 5/9, P<0.05) and laser group 2 (38%; 3/8, P<0.05) (Table 1).
In each of the study groups one pig died of intractable ventricular fibrillation during the 6 h observation period and could not be studied. All pigs (in whom ventricular fibrillation occured) required electrical countershocks and intervening open-chest cardiac compressions (2–5 min). Intraoperative data are shown in Table 2.
Regional myocardial blood flow (RMBF, Table 3)
Data indicated that (prior to ischemia) LAD and LCx territory showed normal regional myocardial blood flow for all study groups. Even after the laser procedure, RMBF in both laser groups demonstrated normal values. Occlusion of the LAD caused a profound and persistent reduction in transmural flow to regional myocardium supplied by the LAD in the ischemia-and laser groups after 6 h compared to baseline (ischemia group: from 187±139 to 4±5 ml/100 g/min, P<0.001; laser group 1: from 199±141 to 3±10 ml/100 g/min, P<0.001, and laser group 2: from 102±46 to 2±10 ml/100 g/min, P<0.001) and to the control group (from 120±59 to 175±149 ml 100 g/min; P=n.s). Thus, there was no significant difference between laser-treated and ischemia hearts regarding the amount of blood flow into the infarcted LAD region after 6 h (P=n.s). In contrast, normal RMBF in remote myocardium supplied by the LCx could be assessed in all study groups.
Systemic hemodynamics
The maximal LVSWI at baseline and end of study are summarized in
Fig. 1 for all groups. After 6 h maximal LVSWI in the ischemia and laser group 1 and 2 were reduced significantly
[13] as compared to their preoperative value (36 vs. 38 and 41%; P<0.05) and to the 6 h value of the control group (P<0.01). At the end of the study maximal LVSWI in the ischemia-, laser 1- and laser 2 group did not show any difference.
Macroscopic and microscopic pathology
The area at risk of the left ventricle for the ischemic (18±5%) and laser groups (laser group 1, 18±3%; laser group 2, 12±3%) did not differ significantly (
Fig. 2). The amount of necrosis in the area at risk of the laser group 1 and 2 were smaller compared to that in the ischemic group (23%; 14 vs. 31%, P<0.01). Longitudinal and transverse sections of myocardium demonstrated typical features of open laser channels
[14].
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Discussion |
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TopAbstractIntroduction
Materials and methodsMiscellaneous measurementsDiscussion
Appendix A. Conference...References |
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This model of acute regional ischemia demonstrates that CO2-laser revascularization diminish significantly the incidence of ventricular fibrillation and necrosis in the area at risk, and does not change regional myocardial perfusion and global left ventricular function.
TMLR limits expansion of the myocardial infarction zone after acute coronary occlusion: laser group 2 showed a significant smaller area of necrosis in the area at risk (laser group 1 did not reach significancy) than in the ischemia group (triphenyl-tetrazolium chloride technique: laser group 1, 23%; laser group 2, 14%; vs. ischemia group, 31%; P<0.01). This is in agreement with the results of Jeevanandam et al. [18], Whittaker et al. [21], and Jing-xuan et al. [22] who observed in their acute ischemic settings a significant smaller area of necrosis (in the area at risk) in the transmural channel group than in the control group [18] [21] [22]. Horvath et al. [19] demonstrated minimal necrosis in the area at risk (6%) compared to control (39%) in sheep, using the same technique as we have used. Their smaller necrosis area can not be explained by better coronary collateralization in sheep, because their area at risk was nearly the same compared to this study (14 vs. 16%) and the control animals showed high amount of necrosis (39%).
The maximal left ventricular stroke work index in the ischemia-, laser 1- and laser 2 group did not show any difference at the end of the experiment. All groups showed in average a mean reduction of 39% of their baseline value [13]. It might be speculated that the similarity in measurements of global left ventricular function after a 6 h observation period between the three ischemic groups was due to a too short time period (half an hour) between creating CO2-channels and setting of the infarction. The new possible collateralization by the microchannels (direct blood flow) did not improve the myocardial function in the laser groups compared to the ischemia group. These hemodynamic results mirror those seen in the regional myocardial blood flow measurements. No significant difference between laser-treated (laser group 1, 3±10 ml/100 g/min; laser group 2, 2±10 ml/100 g/min) and ischemia hearts (ischemia group, 4±5 ml/100 g/min) regarding the extremely low blood flow into the infarcted LAD region after 6 h (P=n.s.) could be seen. Even in the laser group 2 (with a double number of laser channels) a higher blood flow compared with laser group 1 has not been observed. A longer time period of existing microchannels could have improved the blood flow through angiogenesis and consecutively the global contractility of the lased myocardium. In two separate acute studies conducted on dog hearts, Landreneau et al. [17] and Whittaker et al. [15] concluded, in agreement with our results, that CO2 lasing of the acute ischemic myocardium does not change regional myocardial perfusion (analysed by radiolabeled microspheres). Our functional results are consistent with the results of the acute ischemic study of Kadipasaoglu et al. [23] who also did not find any differences of the hemodynamic response in two different experimental groups after 6 h occlusion of the LAD.
There is little experimental evidence in the literature whether blood flow through the microchannels is the mechanism of action. In most studies the regional myocardial blood flow has not been determined. In contrast, Kohmoto et al. [24] measured regional myocardial blood flow (by colored microspheres) after using a holmium:yttrium-aluminum garnet laser in the ischemic canine hearts. They found only a very low blood flow (<1 ml/100 g/min) and the amount of collateral flow into the channel region was not increased compared to non-treated myocardium [24], as we have observed in this study.
Seven pigs in the ischemia group had ventricular fibrillation (100%) during the first hour of LAD occlusion (without TMLR). In contrast, ventricular fibrillation occurred significantly less often in the laser group 1 (56%; 5/9, P<0.05) and laser group 2 (38%; 3/8, P<0.05) Laser group 2 with a double number of laser channels was less prone to ventricular fibrillation and demonstrated a smaller area of necrosis compared with laser group 1. These results may signify that the density of creating two channels per cm2 has to be preferred in terms of preventing ischemia. This is consistent with the results of Whittaker et al. [25] and Okada et al. [26]. Okada et al. [26] had in the acute setting of a myocardial infarction significantly more intractable ventricular arryhthmias in the control group (80%) than in the laser group (0%) with the creation of 3–4 channels per cm2 by a CO2 laser.
Goda et al. [27] used in their porcine long term study a CO2 laser (creating 0.2 mm laser channels) and did not see a difference in the amount of the ischemic and necrotic area in the laser and control group, but observed stronger regression of ischemic ST-elevations in the laser group [27]. Nagata et al. [28] found in their acute CO2-laser study a significant higher stroke work index and regional myocardial blood flow in the laser group compared to control [28].
Occlusion of the LAD coronary artery distal to the first diagonal branch produced an area at risk of 18±4%, which does not involve sufficient myocardium to cause cardiac shock (i.e. 40% of LV mass [29]). Therefore, this experimental model did not reflect the natural history of single-vessel disease with more proximal LAD occlusion. This more limited area at risk was chosen to allow evaluation of the ischemic myocardium. Other influences in affecting our results such as general anesthesia with barbiturates, thoracotomy, lidocaine to reduce arrhythmias, and coronary dissection remain unclear. Myocardial flow heterogeneity occurred in the laser 1 and ischemia group at baseline and end of experiment. The flow ranged from one third to three to six times the mean in these groups. It is presumably related to inadequate mixing which can occur with any number of injected spheres or due to the degree of spatial heterogeneity [11] [12]. Consequently the values of the RMBF which were greater at 3x SD had been excluded from analysis (<3% of the RMBF data).
Theoretically, it has been stated that it is impossible for blood to flow directly from the ventricle into the myocardium during either systole or diastole [30] [31]. In normal myocardium the endomyocardial pressure approaches left ventricular pressure during systole and diastole [32]. This endocardial pressure permits blood flow into the myocardium, but this flow may be enhanced by diminished tissue pressure due to ischemia.
Various animal models have provided contradictory results that have led investigators to opposing conclusions as to patency, perfusion and global function after TMLR. In addition to the physical parameters of the laser (type, energy, duration of laser pulse), the physiologic conditions of our study differed from those traditionally used. In most experimental settings, acute ischemia has been created 0.5–6 h before the creation of laser channels. Allowing that much time to elapse before laser treatment produces the irreversible effects of myocardial ischemia, and ongoing necrosis may impede the beneficial effect of TMLR. We avoided this problem by first performing TMLR throughout the area at risk and then creating ischemic conditions half an hour later. Another reason might be that these models do not reproduce accurately the complex anatomy and pathology of the human atherosclerotic heart disease. The only current clinical indication for TMLR is a chronically ischemic myocardium (myocardial hybernation). Further studies to quantify perfusion through the CO2-channels, to monitor regional and global function, to determine the amount of angiogenesis and metabolic changes in a chronic ischemic study are in progress.
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Acknowledgments |
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Footnotes |
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Appendix A. Conference discussion |
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TopAbstractIntroduction
Materials and methodsMiscellaneous measurementsDiscussion
Appendix A. Conference...References |
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References |
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TopAbstractIntroduction
Materials and methodsMiscellaneous measurementsDiscussion
Appendix A. Conference...References |
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