Eur J Cardiothorac Surg 1999;16:144-149
© 1999 Elsevier Science NL
Transmyocardial laser revascularisation has no beneficial effect on high energy phosphates and lactate content during acute myocardial ischaemia in pigs
Oliver Reuthebucha,
Thomas Podzuweitb,
Sylvia Thomasb,
Klaus Binzb,
Matthias Rotha,
Wolf-Peter Klövekorna,
Erwin Philipp Bauera
a Max-Planck-Institute, Kerckhoff-Clinic, Department for Cardio-Thoracic Surgery, Benekestraße 2-8, 61231 Bad Nauheim, Germany
b Max-Planck-Institute, Department for Experimental Cardiology, Benekestraße 2, 61231 Bad Nauheim, Germany
Corresponding author.
e-mail: oliver.reuthebuch{at}kerckhoff.med.uni-giessen.de
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Abstract
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Objective: Transmyocardial laser revascularisation (TMLR) is used to treat endstage coronary heart disease. There is evidence that angina is significantly reduced after TMLR. However, the precise mechanism by which symptoms disappear remains unknown. The objective of the present study was to examine the potential effects of TMLR on high-energy phosphates and myocardial perfusion in an acute ischaemic model. Method: Five male landrace pigs (42±1.8 kg) had TMLR of the anterolateral wall of the left ventricle using a 1000 W CO2 laser (PLC, USA). Thereafter the anterior descending coronary artery was occluded with a tourniquet. After 90 min of ischaemia, drill-biopsies were taken from ischaemic and non-ischaemic areas as well as from laser channels. The specimens were snap-frozen in liquid nitrogen. Subsequently, methylene blue was injected into the left atrium to study tissue distribution. The hearts were excised and the patency of channels was examined visually. Results: Coronary artery occlusion resulted in immediate blue discoloration in both TMLR and control areas. There was no subendocardial methylene blue staining around laser channels. Inspection of hearts showed occlusion of laser channels due to thrombus formation at both endo- and epicardial levels. ATP-metabolites significantly increased in ischaemic areas compared to non-ischaemic areas. Furthermore there was significant upregulation of purine-content in ischaemic regions even in areas with laser channels. Conclusions: In our acute model there was early occlusion of the channels after TMLR. We suggest that clinical improvement after this procedure is not due to increased myocardial oxygen delivery, since high energy phosphate levels and lactate content remained unchanged.
Key Words: TMLR • Animal models • High energy phosphates
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1. Introduction
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TMLR is supposed to be a reasonable therapy for patients with endstage coronary heart disease. Especially with regard to reduction of angina associated pain, it seems to have a clinical benefit. However, there are still contradictory results concerning improvement of left ventricular ejection fraction (EF) [1]. Yet it is still unknown why TMLR has positive clinical effects. At present there are three major theories with regard to the underlying mechanism. (1) The laser-channels remain patent and connect to intramyocardial sinusoids and/or vessels. As a consequence ischaemic myocardium is supplied by oxygen-enriched blood during systole. (2) The laser-channels induce an inflammatory response with immigration of fibroblasts, monocytes and macrophages. Fibroblasts form fibrotic tissue whereas macrophages and especially monocytes produce angiogenic growth factors. These factors may form new vessels (angiogenesis, arteriogenesis) [2,3]. New arteries connect to pre-existing vessels and sinusoids resulting in improved perfusion of the ischaemic area. (3) The high-energy CO2-laser produces local tissue necrosis around the laser channels including destruction of myocardial nerve fibres. This denervation may result in reduction of angina [4]. The open-channel and denervation theory may explain immediate pain relief after operation, whereas the inflammation theory does not, since it takes some weeks until new vessel formation is completed.
The aim of this study was to evaluate myocardial metabolites during acute ischaemia. It is well known that adenosine triphosphate is a high-energetic chemical compound. Each dephosphorylation, resulting in adenosine diphosphate or adenosine monophosphate, releases a certain amount of energy for muscle contraction. Splitting off a ribose inosine turns into hypoxanthine, which itself oxidates and transforms to xanthine. Vice versa, to phosphorylate AMP to ATP, the oxygen-dependent aerobic respiratory chain is mandatory. These compounds have purine as an elementary base in common [5].
As a result of synthesis of ATP under anaerobic conditions lactate is generated. Thus the amount of lactate is an indicator for lack of oxygen.
Therefore, we have investigated the purine and lactate shift in non-ischaemic, ischaemic and lasered myocardium. Furthermore we tried to demonstrate myocardial perfusion via channels by injecting methylene blue into the left atrium.
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2. Materials and methods
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The experimental protocol described in this study was approved by the Bioethical Committee of the district of Darmstadt, Germany. Furthermore all animals were handled in accordance with the guiding principles in care and use of animals approved by the American Physiological Society and the investigations conformed with the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health.
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2.1. Experimental protocol
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Five male German landrace pigs with a median weight of 42±1.8 kg were included in this study. All animals were premedicated with an intramuscular injection of 2 mg/kg of BW azaperone (Stresnil, Janssen Pharmaceutica, Neuss, Germany). Thirty minutes later an intravenous line was placed into the ear vein to administer 600 mg of pentobarbital sodium (Nembutal, Serva Pharmaceutica, Heidelberg, Germany). This line was also used to continue the i.v. anaesthesia with 9.82 mg/min nembutal, as well as for volume replacement. By means of a tracheotomy a 32 Ch tube was inserted into the trachea. Ventilation was performed with a pressure-controlled respirator (Stephan Respirator ABV, F. Stephan GmbH, Quickborn, Germany) with a ratio air:oxygen of 3:1. Then the carotic artery and jugular vein were dissected. Haemodynamic parameters such as arterial pressure were measured in the carotic artery via a 7F arterial sheath catheter. This was connected to a Shathman transducer (P23XL, Shathham, Puerto Rico). A second line was placed for high volume-replacement and i.v. medication into the jugular vein. ECG was continuously monitored and documented. Besides blood samples were measured for glucose, ph, pCO2, pO2, natrium, potassium and calcium. There were no antiarrhythmic drugs given. Hearts were exposed via median sternotomy. A retractor was inserted, the pericardium then opened and the edges fixed with sutures. After completion of operation anaesthesia was deepened and the animals were sacrificed.
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2.2. Surgical technique
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Laser-channels (1/cm2) were created by a 1000 W CO2-laser (PLC, Medical Systems, USA) with an energy of 20 J. Laser shots were ECG-triggered and fired within a period of 0.15 s after R-spike. Transmural penetration was supposed when there was bleeding out of laser channels. In most cases bleeding was terminated by moderately pressing a swab onto the laser hole. The number of laser channels per animal was 26.2±1.85. Of these 19.8±0.97 were located in the ischaemic area. Five out of 131 channels did not perforate. After TMLR LAD was ligated with a tourniquet, approximately midpoint and distal to large diagonal branches in order to prevent fatal myocardial ischaemia. The adjacent vein was not occluded. Within seconds livid discoloration sharply demarcated ischaemic from non-ischaemic regions. Ventricular fibrillation was treated by electrical defibrillation (20 J).
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2.3. Myocardial biopsies
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Drill biopsies were taken 90 min after LAD-occlusion in four areas of interest (Fig. 1). A special driller was used with exchangeable tips, which varied in diameter and length. With the proper driller tip it was possible to harvest most of the myocardial wall without penetrating into the ventricle. The samples were snap-frozen in liquid nitrogen and stored at -80°C. All biopsies were tested for adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (ADO), inosine (INO), hypoxanthine (Hyp) and lactate (LAC).Therefore specimens were extracted into 6% perchloric acid, using a Branson B-15 ultrasonic disintegrator and 1.5 ml polypropylene reaction tubes. Following 2 min centrifugation at 15000g (in an Eppenorf model 5414 C centrifuge), the acid supernatants were neutralised with 5 N KOH against universal indicator (BDH Chemicals Ltd., UK). Myocardial content of nucleotides, nucleosides and nucleobases was analysed by reversed phase HPLC (column LIChrospher RP-18e (endcapped) 250x4 mm, 5 µm packing) (Merck, FRG) with UV (254 nm) or electrochemical detection [5]. Lactate content of tissue extracts was quantitated spectrophotometrically by an adaptation of the method of Hohorst et al. [6].
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Fig. 1. Areas of interest. 1 non-ischaemic; 2 non-ischaemic lasered; 3 ischaemic; 4 ischaemic lasered.
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2.4. Injection of methylene blue
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Methylene blue was injected into the left atrium. With the heart still beating the dye was supposed to be enriched in perfused myocardium. The heart was excised after discoloration of myocardium. Drill-biopsies were taken again and macroscopically examined.
The left ventricle was opened and endocardial and epicardial orifices of the laser-channels inspected.
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2.5. Statistical analysis
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All data are shown as mean value ±SEM. The statistical significance of differences between groups was determined by the Student t-test. A P value <0.05 was considered statistically significant.
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3. Results
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All five animals survived the operation. Mean lasing-time per pig was 17±1.5 min. Ligated areas immediately turned cyanotic, including areas around laser channels. After ligation, ventricular arrhythmias occurred consistently in 2 phases in all animals (Fig. 2). Most arrhythmias were seen after 8 and 27 min. Internal defibrillation was necessary in all animals (1x in three; 2x in one; 7x in one). Forty minutes after LAD ligation there was stable heart rate and haemodynamics.
Purine content is depicted in Tables 1–4. There was no significant difference (P<0.05) between ATP and metabolites in non-ischaemic compared with non-ischaemic lasered areas, except of adenosine (Table 1). The same results were seen when comparing ischaemic-lasered with ischaemic regions (Table 2). However, comparing non-ischaemic with ischaemic regions all values showed significant differences, except of 
purines (Table 3). Ischemia resulted in significant decrease of energy enriched phosphates. The same results were seen when comparing non-ischaemic lasered with ischaemic lasered zones (Table 4).
Lactate content of biopsies showed that lactate in non-ischaemic area was 7.62±1.6 µmol/g dw, in non-ischaemic/lasered 9.8±3.0 µmol/g dw, in ischaemic area 215±5 µmol/g dw and in the ischaemic/lasered area 222±4.4 µmol/g. There was no statistical significance between ischaemic and ischaemic/lasered specimens as well as between non-ischaemic and non-ischaemic/lasered areas. However there was high statistical significance between non-ischaemic and ischaemic and non-ischaemic/lasered and ischaemic/lasered regions with regard to lactate content.
Injection of methylene blue into the left atrium resulted in a moderate discoloration of the non-ischaemic area after several heart beats due to native coronary flow. However no methylene blue staining was observed in the ischaemic area including surrounding tissue of laser channels.
There was no passage of methylene blue through channels. Inspection of left ventricular endocardium showed thrombosed laser channels in both ischaemic and non-ischaemic regions.
Macroscopically all channels seemed to be occluded.
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4. Discussion
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It is well known, that ATP concentration is higher in non-ischaemic compared to ischaemic myocardial tissue. In contrast, values of ATP-metabolites are higher in ischaemic tissue. For this reason we thought that analysis of purines after TMLR would bring some new information concerning the function of laser channels. If blood from within the ventricle perfusated the channels and supply the surrounding myocardium with oxygen, the ATP concentration would be higher in the vicinity of the channels. However, the value of this high energy phosphate did not increase in ischaemic lasered areas. These results show that oxygen supply of tissue around laser channels is not better in ischaemic myocardium, at least not in this acute animal model. Metabolites of ATP (AMP, adenosine, inosine, hypoxanthine) were significantly higher around laser channels after 90 min in ischaemic myocardium. To the best of our knowledge we are the first group showing different effects of TMLR on purines. Values of ADP and hypoxanthine were identical in ischaemic lasered and ischaemic non-lasered areas. We assume that statistical reasons and chemical instability of detergents are mainly responsible for this result. Lactate concentration was significantly higher in ischaemic myocardium. This finding is not surprising, since anaerobic glycolysis is increased in hypoperfused areas. However, there was no difference between lasered and non-lasered ischaemic myocardium, indicating, that lasering did not increase myocardial perfusion. Macroscopic view showed most channels occluded by thrombus on endocardial surface. These findings are supported by the fact that discoloration due to injection of methylene blue was not observed in lasered areas as well as in ischaemic regions. Only normally perfused myocardial tissue showed such lividness. From these findings we can conclude that laser channels in acute ischaemic areas do not improve oxygen supply of myocardial tissue. It is possible that TMLR in chronic ischaemic myocardium acts differently, since there are intramural collaterals in hearts with highly stenosed or occluded coronary vessels. Thus, in theory laser channels could connect to such collaterals. This would explain the findings of others, which observed open laser channels after up to 1 year in patients with endstage coronary artery disease undergoing TMLR [7–12]. Yet, other authors found fibrotic tissue filling out the entire channels [13–16]. But why do patients have less angina after TMLR? One explanation would be that CO2 laser not only destroys myocytes but also intramural nerve fibres. Kwong and colleagues showed in dogs, that TMLR destroys cardiac nerve fibres, which may result in reduced angina pain [4]. However, it seems improbable that the relatively small number of laser channels would be sufficient to destroy enough nerve fibres to result in angina relief.
At present most authors discuss a third theory. Laser channels induce an inflammatory response, which causes an immigration of lymphocytes, macrophages and fibroblasts, resulting in angiogenesis or arteriogenesis. This hypothesis may explain long-term benefit, but does not give an answer to the acute pain relief. Yamamoto et al. show in a canine model of chronic ischaemia, that TMLR significantly enhances angiogenesis. This is evidenced by the increased number of vessels lined with smooth muscle cells, increased vascular proliferation and increased blood flow capacity during stress [17].
Further investigations have to be done, since different groups have reported that certain growth factors like vascular endothelial growth factor (VEGF) or fibroblast growth factor (FGF) have the potential to create new vessels [18–20]. Both therapies, TMLR and gene-therapy, in combination may be the future treatment.
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5. Study limitations
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The present study is an acute model quite different to man, where lasering is performed in chronically diseased hearts. Therefore it is likely that the significance of collaterals, created due to ischaemia, is not sufficiently considered. However it was the aim of the study to show the benefit of short-circuits between laser-channels and vessels, without regarding their origin. So we think it is unimportant whether the vessels are native (sinusoids) or consecutively grown (collaterals) to transport blood.
In this pig model we have seen most of the laser-channels closed. This could be due to the heat- or shock-trauma caused by the laser. On the other hand it is well known that pigs tend to hyper-coagulability, so that the channels coagulated immediately. This phenomenon will be investigated in future studies when heparin will be administered.
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Footnotes
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Presented at the 12th Annual Meeting of the European Association for Cardio-thoracic Surgery, Brussels, Belgium, September 20–23, 1998.
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Appendix A
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Conference discussion
Professor T. Treasure (London, UK): Thank you for bringing an animal experimental work to the meeting. It strikes me as interesting. Apart from the conclusion you gave, there is another one: the pig is not a good model for this preparation. There is yet another one: the technique does not work at all. You opened your talk and your abstract by saying it is a good way of relieving angina. Could you tell us where you think the evidence is at present that this is clinically effective in the treatment of end stage angina, because I have had reason to look it up and I remain unimpressed. What is your evidence?
Dr Reuthebuch: Well, this is a statement I have because of clinical experience. This is not a statement due to these experiments.
Professor Treasure: No, no, I understand. Is it your clinical experience or is it a trial?
Dr Reuthebuch: In our clinical experience, we have got the impression that TLMR improves the angina classification of patients, nothing else, but this helps people to live a better life.
Professor Treasure: I wanted to know what the evidence was, and you said you have a clinical impression. Have you got better evidence to persuade us that this very expensive equipment and very elaborate technique actually works in patients, I mean evidence like trials that I have missed?
Dr Reuthebuch: We published a couple of months ago a study. We tried to detect laser channels by means of TEE, and we injected into the left ventricle contrast medium, and this contrast medium was distributed through laser channels and perfused the myocardium. And in this case we were quite convinced that there are channels that helped to perfuse the myocardium and to reduce the angina.
Dr D. Maass (Kreuzlingen, Switzerland): In my opinion, your experimental approach to study the effects of transmyocardial laser revascularization (TMLR) has no clinical relevance. You investigated acute ischaemia following an occlusion of a great coronary artery in an acute experimental setting in pigs. Why? Nobody wants to treat acute myocardial infarctions with TMLR. Clinically we are dealing with chronic ischaemia in a severely diseased myocardium with fibrosis, scarring and mostly with a pre-existing collateral network. This is an important difference compared to acute ischaemia. We treated 300 patients with severe angina with TMLR and found an improvement of the perfusion in 40–50% of our patients studied with szintigrams or PET-scans. In 10 autopsy cases intensive angiogenesis and anatomical connections between laser channels and native vessels could be demonstrated. But we found no perfusion via the left ventricular cavity. Therefore, the hypothesis of the "crocodile heart" seems not to be true. Angiogenesis and development of collaterals both need time. Acute experiments cannot demonstrate these effects. Consequently we should develop an experimental model with chronic myocardial ischaemia and a longer follow-up to study the pathophysiology of TMLR.
Dr R. Dion (Brussels, Belgium): Originally every firm marketing these laser devices was swearing that all the channels were keeping open, which was not the case if you used the concurrent device. So I believe that your experiment is interesting in that it confirms that laser certainly doesn’t work through the open channels but through the angioneogenesis initiated by the reaction to the channels.
Dr Reuthebuch: This is what we wanted to show. The first theory of why laser works is that there is a short-circuit between pre-existing sinusoids and vessels, between the laser channels and the sinusoids, and I think with this model, whether it is acute or chronic, we have proved that there is no short-circuit between these channels and the pre-existing vessels. Perhaps in the chronic model after angiogenesis has begun, there is another factor that helps to improve myocardial perfusion, but not due to the connecting between channels and pre-existing sinusoids.
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Received November 23, 1998;
received in revised form April 6, 1999;
accepted April 13, 1999.
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Abstract
1. Introduction
