INTRODUCTION

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THE SEQUENTIAL HISTOLOGICAL and physiological changes that take place in the rat myocardium after occlusion of the left anterior descending (LAD) coronary artery in experimental animals, including rats, have been well documented (10, 11, 25, 26). It has been shown that, after chronic myocardial infarction (MI) in the rat, an impairment of the left ventricular function (reduced power output and elevated filling pressure) occurs that is related to the loss of viable myocardium (11, 26).

The possible involvement of myocardial stress proteins in myocardial protection after ischemic injury has been recently reviewed (4). The anti-ischemic (22) and antiapoptotic (34) properties of the most important of these proteins, the heat shock proteins (HSP) [specifically, the inducible 70-kDa HSP (HSP70i)], have been demonstrated by using the transgenic mouse model that overexpresses this protein (22).

Novel approaches for enhancing angiogenesis in the ischemic myocardium by introducing growth factors (mainly of the family of vascular endothelial growth factors) were adapted and found to have beneficial effect on patients with severe angina (20). Furthermore, the significance of angiogenesis with regard to ischemic heart disease has been addressed in a recent review (32). Low-energy lasers have been found to modulate various biological processes in tissue cultures and animal models (12, 16, 17). For example, laser irradiation has been found to increase mitochondrial respiration and ATP synthesis (24, 38), accelerate wound healing (12), and promote the process of skeletal muscle regeneration after injury (5, 7, 37). Inflammatory response was markedly decreased by laser irradiation (6), and neoformation of blood vessels in the injured zone of skeletal muscles was elevated (5). Recently, our laboratory has shown that low-energy laser irradiation (LELI) induces cell cycle regulatory protein synthesis in satellite cells from skeletal muscles because of activation of early cell cycle regulatory genes (2).

The effect of LELI on the heart muscle and cardiomyocytes has been studied to a limited extent. Zhu et al. (39) recently showed that argon dye laser (660-nm wavelength) improves the functional recovery of cold-stored, isolated rat cardiomyocytes. Those authors also found that the end-storage ATP and end-reperfusion catalase activities in isolated cardiomyocytes that were irradiated with the argon dye laser were significantly higher than those in the untreated cells.

We therefore hypothesized that 1) an increase in the content of any cytoprotective molecule, such as HSPs in the myocardium, as a consequence of LELI immediately post-LAD coronary artery ligation, might have a beneficial effect on cardiomyocytes under ischemic conditions; and 2) exposing the heart to a second LELI (on day 3 after LAD ligation) might cause enhancement of new blood vessel formation and thereby contribute to a better recovery of cardiomyocytes under ischemic conditions.

	METHODS

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Experimental Procedures

Chronic MI. Studies were performed on 65 eight- to ten-week-old (250-350 g) male Sprague-Dawley rats. The rats were anesthetized with Avertin (1 ml of 1.25% tribromoethanol in saline per 100 g body wt), and the operative technique for LAD coronary artery occlusion was carried out essentially as described previously (30). Anesthesia with Avertin was found to be superior to other common drugs for anesthesia and led to better survival of rats. In brief, thoracotomy was performed after longitudinal incision of the left side of the thorax and insertion of a silk thread (4-0) through the intercostal muscles around the area (~15 mm in diameter) of the thoracotomy in the chest. A thoracotomy was made between the fifth and sixth ribs, and the heart was exteriorized by exerting pressure on the chest (rats were not ventilated during surgery). The LAD was occluded 3 mm distally from where it branches off the aorta by using 5-0 polypropylene thread (Ethicon, Cincinnati, OH). The exact location of ligation of the LAD coronary artery (distance from where it branched off from the aorta and its anatomy) was determined after preliminary studies. The chest was then squeezed to inhibit pneumothorax and closed by using the prepared guided suture as described above. The above procedure from thoracotomy up to its closure lasted 30-60 s. Postoperatively, food and water were supplied ad libitum. Mortality rate after the above procedure was 42%.

Ischemia-reperfusion injury. LAD coronary artery occlusion in the ischemia-reperfusion injury to the rat heart was performed on 18 rats similarly to that described in the chronic MI experimental model. The LAD coronary artery was ligated with 4-0 silk thread (Ethicon) by using a flat and tight (as for the chronic LAD coronary artery occlusion) knot suture. One tip of the thread was left protruding from the chest muscles during closure after LAD coronary artery occlusion to enable release of the occlusion. At 45 min post-MI, the rats were lightly anesthetized with halothane and the suture around the LAD coronary artery was released and removed by gently pulling the protruding tip of the thread. In ~20% of the rats the "pull force" was very low, indicating release of the flat suture after occlusion. These rats were discarded from the study. In preliminary experiments, the above process was performed, and blood flow in the occluded artery after suture removal was evident by injection of yellow dye into the aorta. Halothane was found to be the most effective short-term anesthetic in terms of reduced mortality compared with ether or chloroform. Rats were killed 2 wk post-MI. In a review article (8) discussing the point of perfusion and reperfusion time periods in experimental animal, models of 90 min of LAD coronary artery occlusion and 3 and 6 h of reperfusion were discussed. Thus a 45-min period in a smaller animal (rat) is sufficient to cause similar damage to cardiomyocytes.

Laser Irradiation

Five anesthetized rats whose chests had been opened at the midline were used to determine the dispersion and power of the laser beam after penetration through the intercostal muscles. An infrared viewer (Lasotronic, Zug, Switzerland) and infrared-sensitive detecting card (Newport, Irvine, CA) were used to trace the infrared irradiation area. A NOVA power-energy laser monitor equipped with a special detecting probe (Ophir Optronics, Jerusalem, Israel) was used to measure the power output of the laser and the power of the laser irradiation after penetration through the chest muscles. The laser monitor probe and the infrared sensor card were placed in the rats' chests at the same distance and angle of the heart. This was performed to detect, as closely as possible, the area of irradiation and its power on the heart during laser irradiation of a closed chest, as described above. The area of irradiation (1.1 cm²) was elliptic in shape (1.8-cm long axis × 1.1-cm short axis) and was determined by planimetry. The measured power of irradiation in the above area was 5.0 ± 0.7 mW; thus the power density of the irradiation on the myocardium was 4.5 ± 0.6 mW/cm². It could be assumed, therefore, that the dispersed laser beam would cover the infarcted area, including most of the lateral wall of the left ventricle (LV) of the rats. Laser irradiation was performed as described above 10-15 min after LAD coronary artery occlusion for a duration of 1 min after the rats were breathing regularly. Total energy given to the tissue thus was 0.27 J/cm². On day 3 post-LAD coronary artery occlusion, the rats were lightly anesthetized with halothane (ICI, Cheshire, UK), the skin sutures over the chest were removed, and the intercostal muscles below were exposed. Laser irradiation was performed as above, but time of exposure was lengthened to 3 min. On the basis of the above data, the total energy given on the third day was 0.81 J/cm². Control infarcted sham-operated rats underwent the same process as the treated rats (anesthetized and chest exposed), and the laser was applied to the chest but was not connected to a power source. Irradiation immediately post-LAD coronary artery ligation was aimed at enhancing ATP content (24, 38) or stimulating other factors that induce cytoprotective effects in the cardiomyocytes and, at 3 days postligation, at promoting angiogenesis, which commences in the rat at this time interval (13). The ischemic-reperfused rats were laser irradiated at the same power density and timing as the chronic MI rats. The above regime of laser irradiation (energy and length of irradiation, timing and number of irradiations, and so forth) was determined after several preliminary experiments to yield optimal beneficial effects of irradiation on infarct size (Table 1). However, not all possible combinations of the above parameters of variation of laser irradiation (i.e., exposure time, energy, wavelength, and so forth) on the reduction of infarct size were investigated. Experiments to explore the effect of single laser irradiation on the infarct size were performed using 12 rats. In these experiments, laser irradiation was performed as described above 30 min post-LAD coronary artery occlusion at power densities of 5 and 12 mW/cm². Infarct size was determined as described below.

Histology and Immunohistochemistry

The area at risk was determined on nine rats essentially as described by Black and Rodger (8) Rats were anesthetized with Avertin, and the LAD coronary artery was occluded as described in Chronic MI. Four rats (randomly chosen) served as control NLI rats, whereas the rest (5 rats) served as LI rats. Laser irradiation was performed after LAD coronary artery occlusion as described in Laser Irradiation. A 1.5% (wt/vol) solution of Evans blue in saline was injected into the aorta. The hearts were then excised and fixed in 4% neutral buffered formalin. Transverse slices (width of 2 mm) were prepared from each heart. The percentage of the area at risk out of the total area of the left ventricle (LV) was calculated for the section in the middle of the area at risk by using computerized histomorphometric methods as described in Laser Irradiation for the infarct size (see Fig. 5). It was found that the area at risk was 60.3 ± 6.4 (SD) and 62.4 ± 9.3% in the NLI and LI rats, respectively.

For desmin immunostaining, the peroxidase staining kit (no. 9543, Zymed, San Francisco, CA) was applied on paraffin serial sections of formaldehyde-fixed tissue. In brief, after sections were deparaffinized and hydrated in alcohol, they were incubated with 3% peroxidase followed by blocking solution, and then they were incubated with mouse anti-desmin (diluted 1:100) for 30 min at room temperature after being washed with PBS-Tween 20 solution. Horseradish peroxidase-streptavidin conjugate was then applied for 30 min. Chromogen solution was then applied for 10 min, and sections were counterstained with hematoxylin for immunofluorescence. Immunostaining for identification of 5-bromo-2'-deoxyuridine (BrdU)-positive nuclei was performed by using the commercial BrdU staining kit according to the manufacturer's instructions (no. 93-3943, Zymed).

Histomorphometric Measurements and Statistics

Analysis of Newly Formed Blood Vessels

Western blotting analysis of HSP70i. For HSP determination, chronic MI was induced in a group of 10 rats as described in Chronic MI. Six rats were laser irradiated immediately after LAD coronary artery occlusion, and the rest (4 rats) served as control NLI rats. Five hours post-LAD coronary artery occlusion (this time interval was found to be optimal on the basis of preliminary studies at 1 and 24 h post-LAD coronary artery occlusion), the rats were killed with an overdose of chloroform and the hearts were excised and washed briefly in cold saline to remove excess blood. Transverse sections were then prepared in the middle of the ischemic area. This area could be delineated from the nonischemic region by the change in the color of the ischemic portion of the anterior and lateral walls of the LV. The ischemic portion was removed and homogenized (10% wet wt/vol) in 0.01 M Tris · HCl (pH 7.4) buffer containing 2 mM CaCl₂, 0.01% Triton X-100, and proteinase inhibitor cocktail 2 for mammalian cells (Sigma Chemical). The homogenate was then centrifuged at 7,000 g for 15 min, and the supernatant was stored at 80°C until used. Protein was determined by using the Bradford method (9) with bovine serum albumin as a standard. For each sample, 140 µg of protein were mixed with 250 mM Tris · HCl (pH 6.8), 4% SDS, 40% glycerol, and 0.2% bromophenol blue (1:4 vol/vol) and heated to 95°C for 4 min and 32 µl of each sample were loaded on 7.5% SDS-polyacrylamide gel electrophoresis gel (19). After electrophoresis, the proteins were transferred to ProtranBA 85 nitrocellulose membranes (Schleicher & Schuell, Keene, NH) by electroelution. After transfer and blocking with 5% nonfat dry milk in Tris-buffered saline-Tween (TBST) containing 50 mM Tris · HCl, 150 mM NaCl and 0.1% Tween 20, pH 7.5, the nitrocellulose membranes were incubated for 1.5 h with rabbit anti-HSP70, which recognizes HSP70i in rat (Calbiochem-Novabiochem, La Jolla, CA), in TBST with 2% dried low-fat milk, at a dilution of 1:10,000. The secondary antibody was horseradish peroxidase conjugated to goat anti-rabbit IgG (Zymed) at a dilution of 1:7,500. The membranes were developed using enhanced chemiluminescence (Biological Industries. Beit Haemek, Israel) and exposed to X-ray film for an appropriate time. A densitometer software (Image master1D prime ver. 3.01, Amersham Pharmacia, Buckinghamshire, UK) was used to determine the optical density of the blots.

	RESULTS

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Effect of laser irradiation on infarct size and LVD. The effect of laser irradiation on infarct size at various time intervals post-LAD coronary artery ligation is presented in Fig. 1. Infarct size in the LI rats was significantly smaller than in NLI rats. At 14, 21, and 45 days post-LAD coronary artery ligation, infarct sizes in the NLI rats were 52 ± 12 (SD), 47 ± 11, and 34 ± 7%, respectively, whereas infarct sizes in the LI rats were 20 ± 8, 15 ± 6, and 10 ± 4%, respectively. Thus the reduction of infarct size after LELI was 64-70% at 14-45 days, respectively. LVD was also significantly lower at 14, 21, and 45 days post-LAD coronary artery occlusion in LI rats compared with NLI rats (Fig. 2). At 14, 21, and 45 days post-LAD coronary artery occlusion the LVD of the NLI rats was 36 ± 7, 30 ± 9, and 25 ± 7%, respectively, whereas in the LI rats the values were 18 ± 4, 10 ± 3, and 14 ± 3% for the respective time intervals. At 14, 21, and 45 days post-LAD coronary artery occlusion, LVD in the MI-induced LI rat hearts was not significantly different from the noninfarcted intact rat hearts in which the LVD was 11 ± 4% (Fig. 2). The infarct size and LVD of the ischemic reperfused rat hearts are presented in Figs. 3 and 4. It can be seen that, at 2 wk after ischemia-reperfusion injury to the heart, infarct size in the NLI rats was 19.8 ± 7% (SD), whereas in the LI rats it was 11.7 ± 3.4% (not statistically different). However, LVD in the LI rats was only 12 ± 2.5%, which was significantly lower than the 18 ± 3% found in the NLI rats (Fig. 4). When laser irradiation at 3 days was omitted and the irradiation was performed only immediately post-LAD coronary artery occlusion (at a power density of 12 mW/cm²), the reduction in infarct size (2 wk post-MI) was only 46% (significant, P < 0.05) compared with 68% when two irradiations were applied. Single laser irradiation (power density of 5 mW/cm²) 30 min post-LAD coronary artery ligation resulted in 21% reduction (not statistically significant) of infarct size.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Histograms of the infarct size of myocardial infarction (MI)-induced non-laser-irradiated (NLI; hatched bars) and laser-irradiated (LI; solid bars) rats at different time intervals post-MI. Values are means ± SD of 10-12 rats at each time interval. LV, left ventricle. Significantly different from NLI rats: *P < 0.05; **P < 0.01.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Histograms of LV dilatation of intact (open bar), MI-induced NLI (hatched bars), and LI (solid bars) rats at different time intervals post-MI. Values are means ± SD of 10-12 rats at each time interval. Significantly different from NLI rats: *P < 0.05; **P < 0.01.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Histograms of the infarct size of ischemic-reperfused NLI (hatched bar) and LI (solid bar) rats 2 wk post-MI. Values are means ± SD of 10-12 rats.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Histograms of LV dilatation of intact rat (open bar), ischemic-reperfused NLI (hatched bar), and LI (solid bar) rats 2 wk post-MI. Values are means ± SD of 10-12 rats. *Significantly different from NLI rats, P < 0.05.

Effect of Laser Irradiation on Histology, Desmin Expression, and HSP Synthesis in the Infarcted Area

View larger version (74K): [in this window] [in a new window]   Fig. 5.   Light micrographs of cross sections of intact heart (a) and area at risk staining (b and c) of NLI (b and d) and LI (c and e) hearts 2 wk after MI (d and e) or reperfusion injury (f and g). Note similar area at risk in NLI (b) and LI (c) hearts. Note also the large infarct (stained in green color) in the anterior and lateral wall of the LV in d compared with small infarct in e. Note similar LV dilatation in the intact (a) and LI hearts (e and g). Typical diffused scarring (stained in green color) in the LV is presented in the hearts after ischemia-reperfusion injury (f and g). Masson's trichrome stain was used. RV, right ventricle. Bar = 5 mm.

View larger version (113K): [in this window] [in a new window]   Fig. 6.   Light micrographs and immunohistochemic desmin localization of infarcted zones of NLI (a, c, and e) and LI hearts (b, d, and f) 3 wk post-MI. Note that the infarcted area in the NLI is filled with scar connective tissue (a and c) as opposed to aligned fused cells (arrowhead) in the infarcted area of the LI rats (b and d). Cells in the infarcted zone of the LI hearts are positively stained for desmin (arrowhead) in f as opposed to no staining in the infarcted zone (IF) of the NLI ones in e. ED, endocardium; EP, epicardium. Masson's trichrome stain was used. Bars in a and b = 2 mm; bars in c-f = 100 µm.

The effect of LELI on the amount of HSP70i 5 h postirradiation is presented in Fig. 7. It can be seen that, in the noninfarcted myocardium, there is a small amount of HSP70i, which becomes elevated by 5 h postinduction of MI. However, in LI, rats the content of HSP70i is much higher. Densitometric analysis of the Western blots confirmed that the amount of HSP70i in LI rats was 2.2-fold (significant, P < 0.05) higher than in the NLI rats.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Western blot of samples prepared from ischemic portion of the heart harvested from LI (B and D) and NLI (A and C) rats that were killed 5 h after infarction. The samples were probed with a polyclonal antibody recognizing inducible heat shock protein 70.

Effect of Laser Irradiation on Angiogenesis

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Histograms of the number of blood vessels (top), 5-bromo-2'-deoxyuridine (BrdU)-labeled blood vessels (middle), and BrdU-labeled endothelial cells (bottom) in the area of the LV in a histological section of the LV of NLI (hatched bars) and LI (solid bars) rats 6 days after left anterior descending coronary artery occlusion. Values are means ± SE of 12 and 10 histological sections in NLI and LI rats, respectively. Significantly different from NLI rats: *P < 0.05; **P < 0.01

View larger version (162K): [in this window] [in a new window]   Fig. 9.   Representative light micrographs of neoformation of blood vessels (BV) in NLI (a) and LI (b) infarcted rats. Note blood vessels and BrdU-immunostained endothelial cells (arrows) indicating newly formed blood vessels. Blood vessels are more numerous in the LI than in the NLI rats. CP, capillary. BrdU immunostaining and hematoxylin counter staining. Magnification: ×220.

	DISCUSSION

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The infarct size in control NLI rats decreased with time after infarction from 57 to ~34%. This phenomenon corroborates with previous studies that found gradual shrinkage of the relative volume of scar tissue with time after LAD coronary artery occlusion (13). The results of the present study indicate that the beneficial effects of LELI on infarct size and LVD in the rat after chronic MI or ischemia-reperfusion injury progressed with time post-LAD coronary artery occlusion in the rats and that they were most prominent at the longest time interval tested. Thus, at 45 days post-LAD coronary artery occlusion, reduction of infarct size in the LI rats was 70% compared with NLI rats. The fact that the LVD in the LI rats completely returned to the normal value of an intact noninfarcted rat (from 2 wk on post-MI) further supports the profound enhancement in recovery of the myocardium of the LI rats post-MI compared with NLI rats. It was previously demonstrated that reduction in stroke volume in rats occurs only when the infarct size is larger than ~46% of the myocardium (27). Thus it may be postulated that, if laser irradiation in the present study caused reduction of infarct size from a range of 34-52% (2-6 wk post MI) to 10-20%, the irradiated rats may show a better functional performance of the heart over the NLI rats. LELI given within a short time interval after occlusion of the coronary arteries may attenuate the very rapid decrease in ATP and the consequent irreversible adverse effects that take place in the cardiomyocyte mitochondria in the ischemic zone. Indeed, LELI has been found to increase mitochondrial respiration and ATP synthesis (24, 38). Thus the injured laser-irradiated cells may have a much slower rate of degeneration because of an increase in ATP production in the LI rats. It was previously demonstrated that, after coronary occlusion in rats, the basic coronary flow was completely normalized within 7 days (23). Thus it may be postulated that there is a rapid gradual growth of new blood vessels in untreated ischemic heart. In the LI rats, in which formation of new blood vessels is enhanced as shown in the present study, angiogenesis is even faster enabling some portion of the cardiomyocytes to synthesize ATP. The decrease in the number of injured cardiomyocytes may also markedly reduce the inflammatory response after MI in the myocardium, as was previously shown by our laboratory for toad skeletal muscles after injury (6). This phenomenon may, in turn, reduce the adverse effects that have been attributed to leukocyte infiltration as part of the complex sequential processes that occur post-MI (31). Furthermore, we show in the present study that LELI given post-LAD coronary artery ligation caused a rapid elevation in the content of HSP70i in the ischemic area over the nonirradiated myocardium. The HSP family has been shown to play a major role in cytoprotection of cells and enhanced protection against ischemic injury in the heart (4, 22). Thus laser irradiation, via elevation of HSP70i content in the cardiomyocytes, could lead to salvage of a higher percentage of cells in the ischemic zone of LI rats compared with NLI rats. Indeed, it was previously shown that infarct size was negatively correlated with myocardial HSP70i content (15). Furthermore, it should be emphasized that the elevation of HSP70i synthesis in the ischemic myocardium by LELI is a novel phenomenon that is demonstrated in the present study for the first time. So far it has been shown that induction of HSP in vivo could only be achieved by heat stress.

The present study also indicates that LELI has beneficial effects when ischemia in the myocardium is transient and followed by reperfusion injury. Indeed, in the LI rats, there was a marked reduction of the diffused infarct in the myocardium and a return to normal values (as in noninfarcted heart) of LVD as soon as 2 wk post-MI. These results indicate that LELI may have a mitigating influence on the deleterious effects of the free radicals that are formed in the ischemic zone after reperfusion or reduce their content. Indeed, it was recently shown that argon dye laser caused elevation of catalase activity in isolated cardiomyocytes (39). Thus it can be postulated that the direct or indirect elevation of catalase activity, and/or of other enzymes that act as antioxidants, by LELI may contribute to a decrease in the content of superoxides in the reperfused injured or ischemic heart. Indeed, brief periods of ischemia (as in ischemic preconditioning) have been recently suggested to trigger production of low levels of reactive oxygen species, which induce antioxidant defenses that can reduce subsequent ischemic damage (21).

Another mechanism that might be associated with reduction of formation of scar tissue after MI in the rats is the possible transformation of more presumptive fibroblasts in the infarcted area into myofibroblasts after LELI. Pourreau-Schneider et al. (28) have previously shown, using electron microscopic methods and immunohistochemistry, that a direct and massive transformation of cultured fibroblasts into myofibroblasts was observed in cultures 24 h after He-Ne laser irradiation, whereas control cultures contained only resting and active fibroblasts. Indeed, in the present study, cells that were positively stained for desmin, which is attributed to myogenic cells that synthesize de novo proteins (1), were much more numerous in the infarcted area of the LI rats than in the NLI ones. This phenomenon may suggest increased survival of cardiomyocytes in LI relative to NLI hearts and possible regenerative capability of partially injured cardiomyocytes to synthesize new myogenic proteins and express desmin-positive staining. It may also be postulated that a much higher percentage of presumptive myoblasts or myofibroblasts is transformed into myoblasts due to LELI (28) and positively react to desmin. The possibility that scar tissue formation is inhibited by LELI rather than a mainly cardioprotective effect of the LELI cannot be ruled out. Indeed, it has been previously shown that LELI enhanced the proliferation of fibroblasts in vitro (35) and enhanced wound healing (12). Thus it seems more likely that the complex process of fibrosis post-MI may be attenuated by the LELI rather than inhibited.

Irradiation of the heart on day 3 post-MI was aimed at enhancing the process of angiogenesis that commences on the third day, after occlusion of the left coronary artery in the rat (26). Quantitative methods have shown previously that formation of blood vessels in the regenerating zone after skeletal muscle injury is enhanced twofold by applying laser irradiation (7). The results of the present study also indicate a significant (3.1-fold) increase in the number of newly formed blood vessels and proliferating endothelial cells in the infarcted LV and also in the noninfarcted LV. Such a profound increase may be due to stimulation of endothelial cell proliferation by LELI (Y. Krispel, U. Oron, and N. Mirsky, unpublished data) as was found also for example for satellite cells of skeletal muscle origin (2). Thus cardiomyocytes that were salvaged from the ischemic injury by the first laser irradiation that caused elevation of HSP70i may have benefitted from the better oxygen and nutrient supply through an enhanced neoformation of blood vessels in the noninfarcted area. The significant contribution of the laser irradiation at the third day post-LAD coronary artery occlusion can be explained by the reduction of only 21% (when power density was 5 mW/cm²) in infarct size when this irradiation was omitted. Yet, application of single irradiation at 12 mW/cm² probably has better cardioprotective properties because reduction of infarct size was 46% compared with control.

The results of the present study may also have clinical relevance. On the basis of our laboratory's previous results showing that direct LELI on myoblasts in culture does not affect their differentiation in vitro (2) and that the use of LELI in humans has no known deleterious effects (12, 16), it can be postulated that the use of LELI post-MI would appear to be safe. LELI can be delivered to the myocardium in humans via fiber optics in the catheter of the nonfluoroscopic in vivo navigation and mapping technology currently in use in experimental animals and in humans (3, 14, 18). The LELI energy can also be applied during or after the procedure of balloon angiography, using a catheter with a central canal bearing a fiber optic, through which the laser energy can be transversely delivered (360°) to the infarcted area. As an adjunct procedure to other, increasingly popular approaches of repairing ischemic myocardium (angioplasty, stenting, grafting, transmyocardial revascularization, and so forth), the cardioprotective effects of LELI may similarly be beneficial at minimal or no additional risk.

In conclusion, the results of the present study demonstrate that LELI causes a profound reduction in the size of necrotic and scar tissue and complete recovery of LVD after induction of acute MI in rats. These phenomena can most probably be explained as a result of the cardioprotective effects of the LELI at early stages post-LAD coronary artery occlusion, (most probably as a consequence of elevation of HSP70i content in the irradiated myocardium) and enhancement of neoformation of blood vessels later on. However, the precise molecular mechanisms associated with the above phenomena remain to be further elucidated.