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Circulation. 1996;93:143-152

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(Circulation. 1996;93:143-152.)
© 1996 American Heart Association, Inc.

Articles

Transmural Channels Can Protect Ischemic Tissue

Assessment of Long-term Myocardial Response to Laser- and Needle-MadeChannels

Peter Whittaker, PhD; Karel Rakusan, MD, PhD; Robert A. Kloner, MD, PhD

From the Heart Institute, Good Samaritan Hospital, and Department of Medicine, Section of Cardiology, University of Southern California, Los Angeles; and Department of Physiology, University of Ottawa, Canada (K.R.).


*    Abstract
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 *Abstract
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Background We previously found that transmural laser channels failed to acutely increase myocardial blood flow. Nevertheless, this method is being used to treat patients with coronary artery disease who are unable to undergo angioplasty or bypass graft surgery and in cases in which previous surgery has failed. To reconcile the lack of an acute increase in blood flow with beneficial effects claimed in patients, our hypothesis was that the channel-making process might, over time, stimulate a protective effect, possibly by the growth of new vessels linking channels to the existing circulation. We tested this hypothesis in rat hearts, which have little native collateral circulation.

Methods and Results We made six transmural channels in the left ventricle of each heart using a 400-µm-diameter optic fiber coupled to a holmium:yttrium-aluminum-garnet laser or a 400-µm-diameter syringe needle. Two months after the channels were made, rats were randomized to either an infarct-size study or analysis of myocardial capillary density. We challenged any induced protective mechanism by acutely occluding the left coronary artery for 90 minutes, followed by 4.5 hours of reperfusion. The artery was then reoccluded, and pigment was injected into the circulation to delineate tissue perfused by the occluded vessel and to detect perfusion via the channels. We used triphenyltetrazolium staining to determine the amount of muscle necrosis and the location of muscle protection. Infarct size in needle-treated hearts was smaller than in controls (15±6% versus 40±3% of the left ventricle, P<.01). Infarct size in laser-treated hearts (27±5%) did not differ significantly from controls; however, all eight laser-treated hearts showed evidence of muscle protection in areas adjacent to channels. We found that the laser-made channels were associated with more fibrosis than the needle-made channels (mean width of fibrosis 430±50 versus 180±30 µm, P<.0001), and, in tissue remote from channels, fibrosis was increased more in laser-treated hearts (3.6±0.3%) versus both control (2.5±0.2%) and needle-treated (2.5±0.3%) hearts (P<.05). In addition, muscle disarray was seen adjacent to channel-associated fibrosis. We observed injected pigment within fibrosis associated with the channels and in surrounding myocardium. We also found vessels that appeared to be connected to channels; however, there was no overall increase in capillary density.

Conclusions We were able to protect the heart against coronary artery occlusion by making transmural channels 2 months before occlusion. Channels created by a needle provided greater protection than channels created by a laser, probably because they caused less initial injury. Our results are consistent with the concept that the channels were able to provide blood flow to the tissue directly from the ventricular cavity; however, we cannot rule out the possibility that other mechanisms of protection may be involved.


Key Words: ischemia • lasers • revascularization • capillaries • collateral circulation • remodeling


*    Introduction
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 *Introduction
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The possibility of restoring adequate coronary blood flow and relieving ischemia in patients with coronary artery disease via channels made directly through the myocardium from the epicardial surface to the ventricular lumen is being examined currently in clinical trials.1 2 3 Such a technique would be particularly useful in patients who are unable to undergo angioplasty or bypass graft surgery, in cases in which previous surgery has failed, or in patients with intractable angina.4 However, it is unclear from published animal studies how, or even if, such channels can provide blood flow to ischemic tissue. The published animal experiments have tested the ability of channels to supply blood to ischemic tissue by occluding the vessel that would normally supply that tissue.5 6 7 8 There are two problems associated with these experiments: (1) the channels were made either just before or just after the coronary artery occlusion, which means that to be effective, the channels would have to supply blood immediately to the ischemic tissue; and (2) most of the studies examined the effects of channels made in dog hearts,5 6 7 8 a model known to possess variable collateral perfusion, yet failed to measure collateral blood flow. Although there is considerable evidence that the channels cannot immediately supply blood flow,9 10 11 there is anecdotal evidence that transmural channels improve cardiac perfusion in patients.3 12 13 To reconcile this apparent paradox, our hypothesis was that making channels might, over time, stimulate a protective effect, possibly by the growth of new vessels that could connect the ventricular lumen to the existing circulation via the channels. Such a stimulatory process would not occur immediately; therefore, we chose to make the channels 2 months before a coronary artery occlusion was induced to challenge any protective system that might have developed. If the channels could supply blood to ischemic tissue, then we would expect to see smaller infarcts in treated than in nontreated hearts. We made the channels in rat hearts, which have little or no native collateral circulation. Although most recent studies have made channels by use of laser energy, earlier experiments used biopsy or hypodermic syringe needles.14 15 16 17 Thus, in our experiment, we sought to examine the efficacy of both needle- and laser-made channels.


*    Methods
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 *Methods
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The protocol was approved by the Hospital's Animal Care and Use Committee and conforms to the principles of the American Physiological Society. The Heart Institute, Good Samaritan Hospital is accredited by the American Association for Accreditation of Laboratory Animal Care.

Channel Surgery
Ninety-three female, retired-breeder, Sprague-Dawley rats (body mass range, 305 to 505 g) were entered into the study. The rats were anesthetized with ketamine (100 mg/kg) and xylazine (40 mg/kg) given intramuscularly, intubated, and ventilated with room air. A thoracotomy was performed through the fifth intercostal space to expose the apical region of the heart, and the parietal pericardium was removed. At this point, we randomized the rats to one of three groups: laser treatment, needle treatment, or control.

In the laser-treated rats, we made six channels from the epicardial surface to the ventricular lumen in the anterior left ventricular wall, which is the region of the heart that becomes ischemic when the left coronary artery is occluded. We avoided regions in which surface vessels could be seen and tried to space the channels evenly, at least 3 mm apart. We used a flash-lamp pulsed holmium:yttrium-aluminum-garnet (YAG) laser (Laser 1-2-3, Schwarz Electro-Optics), which produced multimode radiation at a wavelength of 2.1 µm and a pulse width of 250 µs. The laser beam was focused into a flat-tipped, 400-µm-diameter optic fiber. The output power at the tip was 200 mJ per pulse, and the repetition rate was 2 Hz. We wanted to produce the channels by tissue ablation and not by pushing the optic fiber through the heart, so only slight pressure was applied to keep the fiber tip in contact with the tissue. Less than six pulses were required to cross the wall. Passage into the ventricular cavity was marked by a loss of resistance to the applied pressure and by pulsatile bleeding when the fiber was withdrawn. The bleeding was stopped by application of pressure via a cotton-tipped swab for a few minutes, and the chest was closed.

In the needle-treated group, the six channels were made in the same location as described above but by use of a 25-gauge hypodermic needle with an external diameter of 400 µm. Passage of the needle into the ventricular lumen was indicated by a pulsatile flow of blood into the body of the needle. There was more bleeding associated with needle-made than with laser-made channels; however, the bleeding appeared to stop as before when pressure was applied with a cotton-tipped swab.

In the control group, there was no intervention, but the chest was left open for a similar duration as for the other groups (approximately 20 minutes).

After the rats regained consciousness, they were returned to their cages. Two months later, the rats were reanesthetized and randomized to undergo either (1) coronary artery occlusion and subsequent histological analysis of myocardial morphology or (2) analysis of capillary density.

Coronary Artery Occlusion
After the rats were anesthetized with ketamine and xylazine (doses given above), we performed a tracheostomy and ventilated the lungs with room air. Additional anesthetic was given as required throughout the protocol. A catheter was placed in the left femoral artery to measure blood pressure. A thoracotomy was performed via the fourth intercostal space, and the basal region of the heart was exposed. We placed a stitch using a C-1 taper needle and 5-0 polypropylene suture from the atrioventricular groove to the pulmonary cone. Additional sutures were tied to each arm of the stitch suture to enable the occlusion knot to be untied.18 We then tied a single knot in the stitch suture to occlude the coronary artery. Occlusion of the artery was confirmed by an increase in amplitude of the ECG signal and development of a deep S wave within the first minute after occlusion. At 90 minutes after occlusion, the artery was reperfused by pulling the releaser sutures to untie the occlusion knot. The artery was reperfused for 4.5 hours. Heart rate and mean arterial blood pressure were recorded before occlusion, 5 minutes after occlusion, and at the end of the experiment. At the end of the experiment, the artery was briefly reoccluded, and 0.5 mL of blue pigment (Unisperse Blue, Ciba-Geigy Corporation) was injected into the circulation via the left femoral vein to delineate the tissue perfused by the occluded vessel. The nonperfused tissue is referred to as the area at risk of infarction. The heart was arrested in diastole by injection of 3 mL of a saturated potassium chloride solution while the animal was under deep anesthesia. The heart was then cut into four or five slices parallel to the atrioventricular groove and photographed. To assess the amount of muscle necrosis, heart slices were incubated for 15 minutes in a 1% solution of triphenyltetrazolium chloride (TTC) at 37°C and rephotographed. TTC stains viable muscle red, whereas necrotic muscle does not stain and so appears pale.19 20 We used computer-assisted planimetry to determine the size of the area at risk (expressed as a percent of the area of the left ventricle) and the size of the area of necrosis (expressed as a percent of both the left ventricle and the area at risk) as described previously.21

Histology
To assess the effect of channel making on myocardial structure, we measured four parameters: (1) fibrosis associated with the channels, (2) myocyte orientation, (3) myocardial collagen content in tissue remote from the channels, and (4) capillary density in the myocardium. In addition, we examined the tissue for the presence of blue pigment in and around the channels and for vascular connections to the channels. This analysis was performed on the hearts used in the infarct-size study, except for analysis of capillary density, which was performed on an additional set of 17 hearts that were randomized to this part of the study 2 months after the channels were made. For analysis of the first three structural parameters, heart slices were fixed in a solution of 10% neutral buffered formalin, processed for paraffin embedding, and sectioned perpendicular to the apex-base axis at a thickness of 5 µm. The sections were stained with hematoxylin and eosin and with picrosirius red.22

Channel-Associated Fibrosis
We measured the maximum width of fibrosis associated with the channels from bright-field examination of picrosirius red–stained sections using a x10 objective lens and a calibrated eyepiece graticule. The red-stained collagen is easily differentiated from the yellow-stained muscle.

Myocyte Organization
We previously found that viable muscle adjacent to healed infarcts no longer possessed the normal highly aligned parallel organization.23 We therefore sought to determine if a similar disruption occurred adjacent to fibrosis associated with the channels. We measured the two-dimensional orientation of 50 longitudinally sectioned, picrosirius red–stained myocytes adjacent to channel-associated fibrosis in both treated groups and also from comparable locations in the control group. Measurements were made according to previously published methods by use of a polarized light microscope equipped with a rotating stage.23 24 A x40 objective lens was used. In two hearts from both the laser- and needle-treated groups, we were unable to locate regions of longitudinally sectioned muscle, and these four hearts were excluded from this analysis. We used circular statistics to calculate the angular deviation of each measured distribution (the circular statistics equivalent of standard deviation) and then calculated the average angular deviation for the muscle orientation distributions obtained in each group.

Collagen Content
It is known that the presence of a myocardial infarct results in an increase in the collagen content of noninfarcted tissue.25 We speculated that the focal muscle necrosis caused by making the channels might result in similar changes. Therefore, we measured myocardial collagen content in the left ventricular free wall in areas remote from the channels and also in the interventricular septum. Collagen content was measured from picrosirius red–stained sections examined by use of a video image analysis subtraction method described previously.26 In each heart, three separate areas were analyzed (each 0.33x0.24 mm) in both the left ventricular free wall and the interventricular septum by use of a x20 objective lens. We then calculated average collagen content expressed as a percent of total area and average collagen content in each of the groups in each of the areas examined.

Capillary Density Analysis
After fixation by immersion in a 10% solution of neutral buffered formalin, the hearts used in this analysis were dehydrated in alcohol, embedded in historesin, sectioned at a thickness of 1 µm, and stained by Avallone's modification of Jones' silver methenamine method for staining basement membranes.27 Previous study has shown that the capillary densities calculated from immersion-fixed hearts do not differ from those obtained from hearts fixed by perfusion with glutaraldehyde.28 Photomicrographs were taken of myocytes cut approximately in cross section in three areas of subendocardial tissue: (1) adjacent to scar tissue associated with the channels, (2) in the left ventricular free wall remote from scar tissue, and (3) in the interventricular septum. The average density of capillaries and myocytes and the capillary-myocyte density ratio was calculated for each region as previously described.27

Statistics
We used ANOVA to test for differences between the three groups. Subsequent comparisons were made by use of Tukey's test. Mortality was evaluated using a {chi}2 test. Values were expressed as mean±SEM and were considered to be significantly different if the probability was less than .05. Analysis of myocyte orientation was performed by use of circular statistics.29


*    Results
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*Results
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Mortality
Table 1Down shows the mortality associated with both the initial surgery and the coronary artery occlusion. Although both lasers and needles caused ectopic beats when the channels were made, these arrhythmias were not responsible for any deaths. The needle-treated group had the highest initial mortality (57%; P<.05 versus control).


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  		Table 1. Postoperative and Postocclusion Mortality

Mean time between the original surgery and coronary artery occlusion was 64 days in each group (range, 62 to 68 days). The number of rats that died after occlusion was four in the control group, none in the needle-treated group, and three in the laser-treated group. The hearts of two of the laser-treated rats that died contained healed scars. Presumably these scars were the result of a channel perforating or occluding a coronary artery during the initial surgery. One of these scars was large, occupying approximately 40% of the circumference of the left ventricle. This heart was excluded from all further analysis. The second heart contained a small apical scar and was transferred to the capillary density analysis group. The four control rats that died during occlusion were also transferred to the capillary analysis group; however, good-quality sections were obtained from only one of these hearts. The other three were excluded from further analysis. The number of rats analyzed in each group and the number of exclusions are shown in Table 2Down.


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  		Table 2. Exclusion of Hearts From Analysis

Hemodynamics
There were no differences in either heart rate or mean arterial blood pressure between the three groups at any of the time points examined (Table 3Down).


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  		Table 3. Hemodynamic Parameters

Area at Risk
The margins of the area at risk of infarction were defined as the lateral borders of the areas of the heart slices stained deep blue by in vivo pigment injection. It is possible that the presence of the channels could have reduced the size of the area at risk; however, there was no statistically significant difference between the groups (control, 60±3%; needle-treated group, 51±5%; laser-treated group, 53±7%; expressed as percent of the left ventricle).

Infarct Size
The area of necrosis, expressed as percent of the left ventricle, was significantly smaller in the needle-treated group than in the controls (15±6% versus 40±3%, P<.01). Necrosis in laser-treated hearts (27±5%) was not significantly different from the other groups (Fig 1Down). However, there was evidence of localized myocardial salvage adjacent to channels in all eight laser-treated hearts.



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  		Figure 1. Bar graph showing area at risk (AR) as a percent of the left ventricle (LV), area of necrosis (AN) as a percent of LV, and AN as a percent of AR for each group. AN was significantly reduced in needle-treated hearts (*P<.01 vs control).

Because a reduction in necrosis expressed as a percent of the left ventricle could be achieved simply by a more distal occlusion of the artery rather than by a treatment effect, it is usual to express the amount of necrosis as a percent of the area at risk (AN/AR). However, because the channels could have reduced the size of the area at risk, such calculation might not provide a true reflection of infarct size. For example, this calculation might mask a protective effect (if area at risk is reduced, then AN/AR will be larger than it should be); however, it would not give a false-positive result. Infarct size, expressed as a percent of the area at risk, was smaller in needle-treated hearts than in the control group (28±9% versus 68±6%, P<.01). Infarct size in the laser-treated hearts (46±7%) was not significantly different from the other groups. Therefore, the reduction in necrosis found in the needle-treated hearts appears to be a treatment effect and not a function of a more distal occlusion.

Evidence for Flow via Channels
Although there was no quantitative difference in area at risk between the groups, macroscopic inspection of the tissue slices revealed qualitative differences in pigment perfusion within the risk region. In control hearts, we observed the expected sharp boundary between the perfused and nonperfused regions. In contrast, the area at risk in some of the needle-treated hearts had a blue tinge. In one of the slices from a control heart, we observed a blue-stained region in the subepicardial muscle within the lateral boundaries of the area at risk that did not become necrotic. The heart had adhered to the chest wall at this location, and we speculate that new blood vessels grew into the subepicardial tissue from the chest wall. We also observed pale blue regions within the area at risk in some of the laser-treated hearts. These macroscopic observations were confirmed by microscopic analysis. Fig 2ADown shows a high-magnification view of a channel from a needle-treated heart. A bifurcating vessel containing red blood cells and blue pigment can be seen within the scar tissue. Pigment can also be seen in the surrounding tissue. We observed vessels connected to the channels; for example, Fig 2BDown shows a vessel aligned perpendicular to a needle-made channel that contains both red blood cells and pigment. The channels were all smaller than the 400-µm-diameter optic fiber or needle used to create them (Figs 2BDown and 3ADown). In fact, some of the laser-made channels appeared to be completely occluded by fibrosis (Fig 3BDown).



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  		Figure 2. A, Photomicrograph of a section from a needle-treated heart stained with picrosirius red. Both pigment and red blood cells can be seen in a vessel with a bifurcation within a band of fibrosis (collagen appears red and muscle appears yellow). B, Photomicrograph of a section from a needle-treated heart stained with hematoxylin and eosin. A channel runs from left to right. Several vessels aligned perpendicular to the channel contain blue pigment and red blood cells (arrows). Some of the vessels appear to connect directly to the channel. Bar indicates 50 µm in both A and B.



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  		Figure 3. Photomicrographs of representative examples of fibrosis associated with needle-made channels (A) and laser-made channels (B). Tissue was stained with picrosirius red and viewed in bright field. Collagen appears red and muscle appears yellow. The epicardial surface is at the top and the ventricular lumen at the bottom of the photomicrographs. We captured almost the entire length of the fibrosis associated with the needle-made channel within the section but only about 50% of the length in the laser-treated heart. Fibrosis associated with a second laser channel can be seen in the subendocardium. Although pigment-containing vessels can be seen within the laser-associated fibrosis, there is no obvious channel. In contrast, a portion of a channel can be seen in the subepicardial region of the needle-treated tissue. Bar indicates 500 µm.

Channel-Associated Fibrosis
We were able to locate 45 of the 96 channels made in histological sections. Of these, 25 were from laser-treated hearts and 20 were from needle-treated hearts. At least one measurement of fibrosis was made from each heart. The average width of fibrosis was significantly greater in the laser-treated hearts (430±50 versus 180±30 µm in needle-treated hearts, P<.0001 by use of Mann-Whitney test, based on the total number of channels examined). Fig 3Up shows representative examples of channel-associated fibrosis in needle- and laser-treated hearts, and Fig 4Down shows the distribution of channel-associated fibrosis with both treatments. The maximum width of fibrosis associated with the majority of channels was in the 200- to 400-µm range; however, only channels created by needles had <200 µm of fibrosis, and only laser-created channels had >600 µm.



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  		Figure 4. Bar graph shows frequency distribution of the maximum width of fibrosis associated with 45 laser- and needle-made channels. The average width of fibrosis was significantly greater for laser-made channels (430 µm) than for needle-made channels (180 µm).

Myocyte Orientation
In control hearts, myocytes exhibited the expected parallel alignment (Fig 5Down), resulting in orientation distributions with small angular deviations. In contrast, myocytes adjacent to laser-made channels were no longer aligned in parallel; thus, the orientation distributions were broadened, and the angular deviations of the distributions were higher (Fig 6Down). The mean angular deviation in the laser-treated group was 9.4±0.7°, which was greater than in the control group (5.0±0.3°, P<.01). The mean in the needle-treated group was 6.9±1.1°, which did not differ from either the control or laser-treated group. Thus, normal organization of myocytes was disrupted by the presence of the channels, and the degree of disruption was greatest in laser-treated hearts.



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  		Figure 5. Examples of muscle alignment (A) in the midmyocardium of a control heart and (B) adjacent to a laser-made channel (channel not shown in photomicrograph). Sections were stained with picrosirius red and viewed with polarized light. Collagen fibers appear bright orange/yellow whereas muscle appears green. In contrast to the parallel organization in the control heart, the muscle adjacent to the laser channel is disorganized. These photomicrographs also show increased collagen content in the laser-treated heart. Bar indicates 50 µm.



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  		Figure 6. Bar graphs of representative muscle orientation distributions from control, needle-treated (needle), and laser-treated (laser) groups. The orientation angle is given on the x axis and the percent frequency of cells with a particular orientation on the y axis. Orientation angles are in 5° intervals, and each distribution is presented so that the mean of the distribution is in the middle of the x axis. The number in the upper right area of each panel is the angular deviation of the distribution (a measure of the spread). Thus, muscle adjacent to laser-associated fibrosis was less organized than that in control hearts.

Collagen Content
Collagen content in the left ventricular free wall (in tissue away from the channels) was significantly greater in laser-treated hearts (3.6±0.3%) than in either control hearts (2.5±0.2%, P<.05) or needle-treated hearts (2.5±0.3%, P<.05). However, there was no difference in collagen content measured in the interventricular septum between the groups (control, 2.3±0.3%; needle-treated hearts, 2.7±0.2%; laser-treated hearts, 2.8±0.2%). Thus, laser treatment resulted in a significant increase in interstitial fibrosis in the left ventricular free wall away from the channels; however, the increase did not extend into the interventricular septum.

Capillary Density
There was no difference between average capillary densities in each group in each of the regions examined (Table 4Down). The calculated values of capillary density are similar to those reported for 9-month-old rats,30 which was the approximate age of animals in the present study. Similarly, there was no difference in myocyte density between groups (Table 4Down), and hence capillary-myocyte ratios were unchanged by channel making.


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  		Table 4. Capillary and Myocyte Density


*    Discussion
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 up arrowMethods
up arrowResults
*Discussion
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We found that the presence of transmural myocardial channels protected the heart against coronary artery occlusion applied 2 months later. Channels made with a needle provided greater protection than those made with use of a holmium:YAG laser. Infarct size was reduced by an average of >50% in needle-treated hearts, and four of the infarcts occupied <10% of the area at risk. We also observed muscle protection adjacent to channels in all eight laser-treated hearts. This localized protection was insufficient to give a statistically significant reduction in infarct size; however, its location and consistency are compatible with channel-related protection.

Mechanism of Protection
The protection against cell death that we observed is related, in general terms, to oxygen supply and demand. Either oxygen supply was increased or oxygen demand was decreased. There were no differences in heart rate and blood pressure between groups, so these hemodynamic factors can be eliminated as potential explanations for the decrease in infarct size.

One possible explanation for increased oxygen supply is that blood was able to flow from the ventricular cavity to the ischemic tissue via the channels. Although we do not have a direct measurement of blood flow, there was indirect evidence consistent with flow via the channels. There was evidence in both laser-treated and needle-treated hearts that small amounts of pigment were able to enter at least some of the area at risk, rendering the region pale blue. Histological examination supported the idea that some pigment gained access to the ischemic tissue via the channels. Pigment could be seen in fibrosis associated with the channels and in surrounding myocardium. Because the rat heart has little or no native collateral circulation, the pigment most likely entered the area at risk via the channels.

How did the blood and pigment gain access to the area at risk via the channels? Our original hypothesis was that the channel-making process might stimulate the growth of new vessels. However, there was no overall increase in capillary density (Table 4Up). Measurements were made over a relatively large area, and so we cannot exclude the possibility that capillary growth was confined to localized regions, nor can we exclude the possibility that there was an increase in larger-diameter vessels, such as arterioles. With a capillary density in the rat hearts >2000/mm2 (the same density found in adult human hearts28 ), it is inevitable that a considerable number of capillaries would have been intersected when the channels were made. However, such intersections do not appear to allow immediate blood flow to the tissue, as demonstrated by the failure of laser-created channels to provide protection against acute ischemia. Therefore, to explain a later increase in blood flow, either new vessels grew in the scar tissue around the channels, or vessels sprouted from the damaged ends of the transected capillaries and connected to the channel lumens. We observed vessels containing pigment and red blood cells that had direct connections with the channels (Fig 2BUp). The diameter of these vessels ranged from 2 to 10 µm, which is comparable to the range of capillary diameters in rat hearts measured from corrosion casts.31 We also noted pigment in the subepicardial tissue of a control heart at the site of adhesion to the chest wall. Both channel making and adhesion elicit a wound-healing response, a known stimulus for vessel growth.32

We found structural changes in hearts containing channels that might be compatible with decreased oxygen demand. Muscle cells in the immediate vicinity of channel-associated fibrosis lacked the normal parallel organization found in control hearts. Such disorganization is likely to impair contractile function and could result in areas of hypokinesis adjacent to the channels. This situation may be similar to the disorganization of contractile elements found after unloading cardiac papillary muscle by cutting chordae tendineae.33 Cardiac unloading produces a rapid and severe loss of contractile function and may result in metabolic downregulation and a resultant reduction in oxygen demand.34 Similar metabolic downregulation of disorganized muscle adjacent to the channels might enable it to survive coronary artery occlusion. In addition, oxygen could be supplied via diffusion from a patent channel even if there was no blood flow directly to the tissue. The protection afforded by such downregulation with or without oxygen diffusion would be limited by the extent of downregulation and by the limit of passive diffusion. Such a mechanism of protection would not be consistent with the significant reduction in infarct size found in the needle-treated hearts but would be consistent with the bands of viable muscle observed next to some of the laser-created channels.

There is a third possible protective mechanism that is worth considering: the tissue had an increased tolerance to ischemia. Several studies have demonstrated that stress—for example, in the form of increased temperature—can stimulate the production of endogenous protective agents called heat shock proteins.35 Whether heat shock proteins or other protective substances are produced in response to the channel-making process is unknown, as is the potential for such agents to provide protection 2 months after application of the stress.

In hearts that possessed little or no collateral blood flow, the finding of pigment within the area at risk is suggestive of a direct link, via the channels, between the ventricular cavity and the tissue. Nevertheless, additional studies will be required to determine the exact contribution of these proposed mechanisms to the observed reduction in infarct size.

Comparison of Needle- and Laser-Made Channels
The obvious physical difference between the two channel-making methods is that needle channels are made mechanically, whereas laser channels are associated with a thermal process. The influence of thermal injury on channel patency is unclear. Owen et al36 examined the temporal response of rat myocardium to the presence of channels made by use of a carbon dioxide laser and found that thermal injury produced an `intense inflammatory response,' which they concluded was inconsistent with provision of an alternative circulation. Hardy et al37 examined channels made in dog hearts by use of either a carbon dioxide laser or an 18-gauge hypodermic syringe needle. The authors stated that needle-made channels were completely occluded within 2 days by cellular infiltrate, which was eventually replaced by scar tissue.37 Laser-created channels remained patent slightly longer before they too were occluded, first by fibroblasts and macrophages and subsequently by collagen. The authors37 speculated that thermal injury to myocardium adjacent to the channels delayed healing and thus maintained channel patency, albeit briefly. In the present study (2 months after the channels were made), we found that channels were 40 µm wide at most and that some of the laser-made channels did not appear to be patent. However, the lack of an obvious channel in histological sections does not preclude blood flow in vivo via the channels. Thus, the challenge of coronary artery occlusion and injection of pigment are useful additional tests of the functional ability of the channels.

In a pilot experiment, we found that the laser provided no protection against later ischemia.38 However, we have shown that it is possible to alter the degree of thermal injury associated with the holmium:YAG laser by changing pulse energy or repetition rate.39 We therefore performed an in vitro study to determine the appropriate parameters to minimize thermal injury and yet permit rapid passage through the ventricular wall. We found that the combination of 200 mJ per pulse and a repetition rate of 2 Hz was the best combination (in the pilot experiment, we used 200 mJ and 3 Hz). We assume it was this reduction of thermal injury that improved the results obtained with the laser. The extent of tissue damage associated with creation of the channels should be reflected in the amount of fibrosis produced. Even though we reduced the amount of thermal injury in the current study, the presence of fibrosis indicated that tissue damage still occurred. The maximum width of fibrosis associated with laser-created channels was much greater than for needle-made channels (Figs 3Up and 4Up). We speculate that further reduction of thermal injury, and hence fibrosis, may improve results in laser-treated hearts. Moreover, we propose that needle-created channels provided more protection in our experiments because they caused less damage to surrounding tissue than the relatively extensive thermal injury associated with the holmium:YAG laser. Another potential cause of tissue injury is the production of vapor bubbles by pulsed holmium lasers.40 We found that the degree of myocardial disruption by such `acoustic' injury was slight at repetition rates of 2 and 3 Hz, especially at the pulse energies used in the current study.39 Nevertheless, vapor bubbles may be responsible for tissue injury in excess of that caused by temperature increases.

We found that the process of making channels exerted effects beyond the tissue that was initially injured. Fibrosis associated with the initial injury resulted in disorganization of adjacent myocytes. The observed disarray is similar to that found in viable muscle adjacent to a healed infarct.23 The degree of muscle disorganization may be determined by the amount of channel-associated fibrosis, because more disarray was found in laser-treated than in needle-treated hearts. Furthermore, we found an increase in the amount of interstitial fibrosis in tissue distant from the channels in laser-treated hearts. There are at least two possible explanations for this observation. After myocardial infarction, there is an increase in the amount of fibrosis in noninfarcted tissue,25 and a similar process may occur after the focal necrosis caused by making the channel. Second, because the increase in distant, interstitial fibrosis was found only in laser-treated hearts, it could be a direct stimulatory effect of the laser itself.

The structural changes that we observed in laser-treated hearts could have deleterious effects. For example, muscle disarray could result in diminished function and could also provide a substrate for abnormal electrical conduction and hence increase the potential for the development of arrhythmias. Similarly, the increase in interstitial collagen could also be expected to affect function by making the heart stiffer and could affect electrical conduction by decreasing the amount of cell-to-cell contact. These effects on distant tissue emphasize the need for further investigation of the methods used to create the channels.

Previous Experiments
Animal Studies
It is difficult to interpret many of the previous experiments in which transmural channels were examined because they failed to measure native collateral blood flow. Before blood flow via the channels can be confirmed, it is necessary to take into account collateral blood flow from other coronary artery beds into the area served by the occluded artery, either by measuring collateral blood flow or by using an animal that has little or no collateral flow. Most previous studies have tested channels in dog hearts, which can have variable native collateral circulations. Therefore, it is impossible to know if any beneficial effect was attributable to the channels or if the treated hearts had high intrinsic collateral flow. Such limitations also apply to sheep41 and, to a lesser extent, to rabbit hearts.42 In contrast, pig and rat hearts have little native collateral circulation.

A second confounding factor in many previous studies is the temporal relation between creation of the channels and coronary artery occlusion. In most of the studies, the channels were made either just before or just after coronary occlusion. To obtain a positive effect, the channels would have to provide an immediate increase in tissue blood flow. There is substantial evidence that laser-made channels cannot provide immediate blood flow to ischemic myocardium, either in dog hearts (when collateral flow was measured)9 10 11 or in pig hearts.43 44 The evidence against acute benefit from the laser channels rules out the possibility of blood flow via the myocardial sinusoids, as proposed by those who claim that blood can circulate immediately via the channels.3 The only other study that tested needle-made channels several weeks before coronary occlusion was performed in dogs and did not measure collateral flow.17 Thus, our study is, to our knowledge, the first to examine an ischemic challenge to transmural channels several weeks after the channels were made in a model in which preexisting collateral circulation was not a factor.

Clinical Studies
The concept of supplying blood to ischemic tissue via alternative channels has historical precedent. Creating channels is in some respects similar to the Vineberg procedure, in which the internal mammary artery was tunneled directly into the myocardium. Although the operation was controversial, there was evidence that the Vineberg procedure worked by stimulating the growth of new vessels connecting the arterial insertion with the existing circulation.45 Recent studies used radioactive microspheres to document flow via the internal mammary artery in dogs 8 weeks after implantation of the vessel into the myocardium46 and found that the flow could be increased by heparin treatment,47 an agent known to play an indirect role in vascular neogenesis.

Although clinical trials designed to evaluate channels made by use of a carbon dioxide laser are in progress, only limited data has been published. The reports claim that patients examined several months after the channels were made had increased myocardial perfusion and less angina.1 2 3 Gradual rather than immediate improvement would be consistent with new vessel growth in response to the channel-making process. Mirhoseini et al13 reported endothelialization of laser-made channels and an increase in the number of vessels in treated myocardium from a patient who died 4 years after the original channels were made. However, it was not clear whether the increase in vessels was because of angiogenesis or the presence of numerous laser-made channels. More recently, Cooley et al48 described the histology of channels made by use of a carbon dioxide laser in a patient who died 3 months later. Vascular connections were observed between the channels and native myocardial vessels. In addition, each channel was surrounded by fibrosis (width, 150 to 500 µm) and the channel diameter (20 to 75 µm) was less than the initial diameter of 1 mm. Both the presence of channel-associated fibrosis and a reduction of channel diameter over time are similar to our results.

Study Limitations
The main limitation of the present study is that we did not document an increase in blood flow via the channels. Although our evidence of reduced infarct size and the observation of pigment within the area at risk is consistent with flow via the channels, it is circumstantial rather than conclusive evidence.

Although we demonstrated that the presence of transmural channels protected the myocardium against later ischemia, the model used—a normal, healthy heart—may not be relevant. In a practical situation, channels would be made through tissue with a compromised blood supply. We do not know if such tissue would respond differently.

We examined only one type of laser. We did so because the reported mechanism of protection (an immediate increase in blood flow via myocardial sinusoids) did not appear to depend on the method of creating the channels. However, we interpret our results to indicate that the method of making the channels is a crucial determinant of success. We speculate that if reduction of thermal injury is important, then it is likely that different lasers or different methods of energy delivery (for example, use of a Q-switch to produce short, powerful energy pulses) will give better results than obtained with the holmium laser we used.

In the present study, creation of channels was associated with high mortality, perhaps because of perforation of coronary arteries resulting in myocardial ischemia. This possibility was supported by the observation of healed scars in two laser-treated hearts. We did not perform necropsies on animals that died, and thus we cannot rule out either pneumothorax or hemothorax as a cause of death. In addition, we cannot exclude the possibility that the high initial mortality after needle treatment might have resulted in a group that was in some way better able to withstand ischemia.

The size of rat hearts limits the number and diameter of channels that reasonably can be made. It is possible that the diameter and density we used was not optimal. It is evident that after 2 months, the diameter of the channels was less than the 400-µm-diameter needle or optic fiber used to create them. Healing of the initial injury may be responsible for this reduction in channel diameter, and it is possible that channels with larger initial diameters may maintain greater patency after the healing process is complete.

Summary
Channels made through the myocardium were able to protect the heart against coronary occlusion 2 months later. In our experiments, needle-made channels provided greater protection than channels made with a holmium:YAG laser. The precise mechanism of protection remains unknown, although the results of our study are consistent with the concept that channels are able to provide blood flow to the tissue from the ventricular cavity. Our study provides some support and rationale for the clinical trials that are currently under way to test the value of laser-made channels in patients. However, our results emphasize that the mechanism of protection is unclear, and therefore we do not know the best method of making the channels.


*    Acknowledgments
 
This study was supported in part by a grant (to K.R.) from the Heart and Stroke Foundation of Ontario. We thank Seda Dzhandzhapanyan and Barbara Hébert for assistance with the histology, Shi-ming Zheng for advice regarding laser operation, and Karin Przyklenk for editorial assistance.


*    Footnotes
 
Reprint requests to Peter Whittaker, PhD, Heart Institute, Good Samaritan Hospital, 1225 Wilshire Blvd, Los Angeles, CA 90017.

Received August 5, 1994; revision received July 10, 1995; accepted August 8, 1995.


*    References
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*References
 

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