Postconditioning (PoC) with brief intermittent ischemia after myocardial reperfusion has been shown to lessen some elements of postischemic injury including arrhythmias and, in some studies, the size of myocardial infarction. We hypothesized that PoC could improve reflow to the risk zone after reperfusion. Anesthetized, open-chest rabbits were subjected to 30 min of coronary artery occlusion followed by 3 h of reperfusion. In protocol 1, rabbits were randomly assigned to the control group (n = 10, no further intervention after reperfusion) or to the PoC group, which consisted of four cycles of 30-s reocclusions with 30 s of reperfusion in between starting at 30 s after the initial reperfusion (4 × 30/30, n = 10). In protocol 2, rabbits were assigned to the control group (n = 7) or the PoC group, which received PoC consisting of four cycles of 60-s intervals of ischemia and reperfusion starting at 30 s after the initial reperfusion (4 × 60/60, n = 7). No reflow was determined by injecting thioflavine S (a fluorescent marker of capillary perfusion), risk zone by blue dye, and infarct size by triphenyltetrazolium chloride. In protocol 1, there were no statistical differences in hemodynamics, ischemic risk zone, or infarct size (35 ± 6% of the risk zone in the PoC group vs. 29 ± 4% in the control group, P = 0.38) between the groups. Similarly, in protocol 2, PoC failed to reduce infarct size compared with the control group (45 ± 4% of the risk zone in the PoC group vs. 42 ± 6% in the control group, P = 0.75). There was a strong correlation in both protocols between the size of the necrotic zone and the portion of the necrotic zone that contained an area of no reflow. However, PoC did not affect this relationship. PoC did not reduce infarct size in this model, nor did it reduce the extent of the anatomic zone of no reflow, suggesting that this intervention may not impact postreperfusion microvascular damage due to ischemia.
- microvascular reperfusion injury
two recent clinical studies have suggested that postconditioning (PoC) may have a beneficial effect on the microcirculation after ischemia. A study by Loukogeorgakis and co-workers (17) showed that PoC protected against endothelial injury after forearm ischemia in humans, and Staat et al. (23) reported an improved myocardial blush grade, an estimate of reperfusion at the myocardial level, in humans who received PoC after coronary angioplasty. These studies suggested that PoC may protect against microvascular reperfusion injury.
First described by Zhao and coworkers (29), and defined as a series of repetitive cycles of brief ischemia and reperfusion initiated shortly after reperfusion of prolonged ischemia, PoC has been shown to reduce myocardial infarct size in some in vivo (4, 15, 18, 25, 28, 29) and isolated heart (5, 6, 26, 28) animal models. In addition to killing myocytes, myocardial ischemia and reperfusion damage the microvasculature, decreasing or preventing reflow at the tissue level after coronary artery occlusion and/or reperfusion. The “no-reflow” phenomenon, observed after transient coronary artery occlusion, is characterized by an anatomic area of hypoperfusion contained within the zone of necrosis. The phenomenon of no or low reflow may represent one element of reperfusion injury that has the potential to be reduced by PoC. The main purpose of the present study was to test the effects of PoC on microvascular reperfusion by assessing the extent of the no-reflow zone. We also assessed the effects of PoC on myocardial infarct size. Two PoC protocols were tested in a rabbit model of 30 min of ischemia followed by 3 h of reperfusion.
This study was approved by the Institutional Animal Care and Use Committee of Good Samaritan Hospital, and the investigation conformed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-230, Revised 1996). The Association for Assessment and Accreditation of Laboratory Animal Care International accredits Good Samaritan Hospital.
Male New Zealand White rabbits (2.2–3.4 kg) were anesthetized with an intramuscular injection of a mixture of ketamine (80 mg/kg) and xylazine (15 mg/kg). Pentobarbital anesthesia was given intravenously during the study (15 mg·kg−1·h−1 or as required to maintain a deep level of anesthesia as assessed by lack of corneal or pedal responses). Rabbits were shaved, intubated, and mechanically ventilated with oxygen-enriched air. Rabbits were placed on a heating pad to maintain body temperature. Fluid-filled catheters were placed in the left jugular vein to administer anesthesia and into the right carotid artery to measure systemic pressure. The thorax was opened through the left fourth intercostal space, and the pericardium was incised, exposing the heart. Near the base of the heart, the first large anterolateral branch of the circumflex artery, or the circumflex artery itself, was encircled with a 4-0 silk suture attached to a nontraumatic needle. The ends of the suture were threaded through a piece of tubing, forming a snare, which was tightened to occlude the artery. Coronary occlusion in this region normally results in ischemia of a large region of the anterolateral and apical ventricular wall. A catheter was placed in the left atrium and used to inject thioflavine S and blue dye at the end of the study.
After the surgical preparation, baseline hemodynamic parameters and rectal temperature were measured. Rabbits were then subjected to 30 min of coronary artery occlusion. Rabbits were randomly assigned to treatment groups at 20 min after coronary artery occlusion. After 30 min of coronary artery occlusion, the clamp was released, and the artery was reperfused. Rabbits randomized to the control group received no further intervention. Rabbits randomized to the PoC group received one of two PoC protocols. PoC was achieved by gently tightening and releasing the snare (see PoC Protocols). All hearts were then reperfused for 3 h. Heart rate, systemic arterial pressure, and rectal temperature were monitored and recorded before coronary artery occlusion, at 25 min of coronary artery occlusion, and at 30, 60, 90, 120, and 175 min of reperfusion. Data were digitized and recorded using software from Advanced Digital Instruments (Grand Junction, CO).
After 3 h of reperfusion, 1 ml/kg of a 4% solution of thioflavine S (Sigma Biochemicals, sigma-aldrich.com) was injected into the heart via the left atrial catheter to define the region of no reflow. Thioflavin S, a fluorescent yellow-green dye, stains intact endothelium and serves as a marker of perfusion. With the use of this dye, areas perfused by blood fluoresce and areas not perfused appear dark. The coronary artery was reoccluded, and the ischemic risk region was delineated with 4 ml of a 50% solution of Unisperse blue (Ciba-Geigy, Hawthorne, NY) injected into the left atrium. The deeply anesthetized rabbit was killed by an injection of KCl (12 meq) into the left atrium, and the heart was excised.
The effects of PoC were tested in two separate experiments. In protocol 1, rabbits were randomly assigned to the control group (no further intervention after reperfusion) or to the PoC group, which consisted of four cycles of 30-s reocclusions with 30 s of reperfusion in between starting at 30 s after the initial reperfusion (4 × 30/30). In protocol 2, rabbits were randomly assigned to the control group or to PoC group, which consisted of four cycles of 1-min intervals of ischemia and 1-min intervals of reperfusion starting at 30 s after the initial reperfusion (4 × 60/60).
Analysis of Risk Zone, No-Reflow Zone, and Necrosis
Hearts were sliced transversely into six to eight sections and photographed. Slices were digitally photographed using an ultraviolet light (254 nm) and a yellow filter to identify the region of no reflow (Fig. 1, B and E) and then under halogen lighting to identify the area at risk (Fig. 1, A and D). Slices were then incubated in a 1% solution of triphenyltetrazolium chloride (Sigma Biochemicals) for 15 min, immersed in formalin, and rephotographed (Fig. 1, C and F). The areas of no reflow, ischemic and normally perfused regions, and areas of necrotic and non-necrotic regions in each slice were determined using Image J (19). Areas in each slice were multiplied by the weight of the slice, and the results were summed to obtain the weights of the no-reflow, risk, and infarcted areas.
Data were calculated and tabulated using Excel work sheets. All data summary and statistical analyses were performed using SAS (version 9.0, Cary, NC). Weights, infarct size, area at risk, area of no reflow, and differences in water content were compared using Student's t-test. Changes in hemodynamic variables over time were analyzed by repeated-measures ANOVA. Analysis of covariance was used to test for a group effect on the regression models of necrotic myocardium with the risk zone and no-reflow zone. Data are expressed as means ± SE.
Twenty-two animals were entered into protocol 1. One rabbit (control) died at 75 min of reperfusion due to hypotension, and one heart (control) was excluded due to poor demarcation of the risk region due to excessively low arterial pressure at the end of reperfusion. Data are reported on 10 rabbits in the control group and 10 rabbits in the PoC group. In protocol 2, 18 rabbits were entered into the study. One rabbit (control) died at 135 min of reperfusion due to hypotension, and three hearts (two control and one PoC) were excluded due to failure to occlude an artery. Data are reported in seven control hearts and seven PoC hearts.
Risk Zone and Infarct Size
The extent of the ischemic risk zone was similar in both the control (31 ± 3% of the left ventricle) and PoC group (32 ± 3% of the left ventricle; Fig. 2). Infarct size expressed as a percentage of the risk zone was 29 ± 4% in the control group and 35 ± 6% in the PoC group (P = 0.38); thus, the 4 × 30/30 PoC protocol conferred no cardioprotection.
There were no significant differences in the extent of the ischemic risk zone in the control group (31 ± 3% of the left ventricle) and in the PoC group (31 ± 6% of the left ventricle; Fig. 2). The 4 × 60/60 PoC protocol also failed to reduce infarct size (42 ± 6% in the control group and 45 ± 4% in the PoC group, P = 0.75).
Anatomic Area of no Reflow
The anatomic zone of no reflow was delineated by thioflavine S staining. Thioflavine S binds to intact endothelium, causing perfused tissue to fluoresce when exposed to ultraviolet light (Fig. 1, B and E).
In protocol 1, the zone of no reflow, expressed as a percentage of the necrotic zone, was 56 ± 9% in the control group and 48 ± 8% in the PoC group (P = 0.48). When expressed as a percentage of the ischemic risk zone, the zone of no reflow was 17 ± 4% in the control group and 19 ± 4% in the PoC group (Fig. 2).
In protocol 2, the zone of no reflow, expressed as a percentage of the necrotic zone, was 64 ± 5% in the control group and 55 ± 7% in the PoC group (P = 0.34). When expressed as a percentage of the risk zone, the zone of no reflow was 27 ± 5% in the control group and 26 ± 5% in the PoC group (P = 0.89). Thus, neither PoC protocol reduced the extent of the no-reflow region.
There was a strong correlation in both protocols between the size of the necrotic zone and the portion of the necrotic zone that contained an area of no reflow (Fig. 3). The coefficient of correlation was 0.89 (P < 0.001) in protocol 1 and 0.97 (P < 0.001) in protocol 2. PoC had no effect on this relationship in either protocol.
Heart Rate and Blood Pressure
Heart rate and mean arterial pressure were similar at baseline in both groups in protocols 1 and 2 (Table 1). There were no significant differences in either variable between group or over time (repeated-measure ANOVA) in either protocol.
Our goal in this study was to test whether PoC had a protective effect on the coronary vasculature recovering from ischemia and reperfusion, as had been suggested by two recent clinical studies (17, 23). Results from our study indicated that in our model, two different protocols of PoC after acute ischemia failed to reduce the extent of the anatomic no-reflow zone and, in addition, these PoC regimens had a neutral effect on infarct size.
We have shown previously that ischemic preconditioning is effective in reducing infarct size in our model (9), and, although preconditioning has been shown to be effective in virtually all species tested, the effects of PoC seem to be less consistent. Studies in intact and isolated rabbits have, for the most part, shown benefits of PoC in models of coronary artery occlusion and reperfusion(1–4, 12, 13, 18, 25, 27). However, effectiveness seems to be highly dependent on the exact PoC protocol used. For example, Chairi and co-workers (2) tested two protocols of PoC in pentobarbital-anesthetized rabbits. They found that three cycles of 10-s PoC ischemia failed to reduce infarct size but that three cycles of 20-s PoC resulted in a significant cardioprotective effect (2).
Some laboratories have shown that four 30-s cycles of PoC ischemia (the same protocol that we tested in the present study) reduces infarct size in the rabbit (18, 25, 27); however, this has not been the case in other laboratories. In agreement with our data, Iliodromitis (13) found that four 30-s cycles of PoC ischemia did not significantly reduce infarct size in the intact rabbit heart; however, in their study, six 10-s cycles were effective in reducing necrosis. Argaud and co-workers (1) found that four 60-s cycles were required for infarct size reduction. In contrast, in our laboratory, four 60-s cycles did not reduce infarct size, nor did three 60-s cycles in another study (3) that investigated PoC in isolated, buffer-perfused rabbit hearts.
This variability is not limited to the rabbit. In rats, some studies have shown that various PoC protocols reduce infarct size (15, 24), whereas others failed to show a reduction (7, 14). In fact, Kaljusto and co-workers (14) found that the results were different in two laboratories doing the same protocol. The same is true in pigs. Data from Schwartz and Lagranha's study (22) showed no benefit from PoC, whereas data from Iliodormitis’ study (12) showed a reduction in infarct size. The variability in response is intriguing and suggests that the ultimate protocol with the associated signaling pathways has not yet been found.
A previous study (16) from our laboratory showed that PoC was very effective in reducing postischemic ventricular arrhythmias after 5 min of ischemia in rats. However, the same PoC protocol of four cycles of 20-s reocclusion had no effect on infarct size, nor did four cycles of 10-s PoC (7). Therefore, the data we obtained in rabbits agree with our observations in the rat model in that PoC did not reduce infarct size. We did not assess the effects of PoC on arrhythmias in our rabbit model as ketamine-xylazine-anesthetized rabbits rarely exhibit ischemia- or reperfusion-induced arrhythmias.
Data from the present study showed that the region with a no-reflow defect comprised a large percentage of the area of necrosis (average values ranging between 48% to 64% of the necrotic region). We have observed from other experiments performed in our laboratory that with a coronary artery occlusion of 30 min followed by reperfusion of 3 h, the zone of no reflow can comprise as much as 75% (11) to 85% (21) of the necrotic zone. In another study of rabbit hearts, Genda and co-workers (8) assessed the zone of no reflow after a 30-min coronary artery occlusion and found an average of 34% of the necrotic zone to exhibit a no-reflow defect. However, in that study, infarct size and no reflow were measured at 90 min of reperfusion rather than at the 180 min of reperfusion we used in our study. Refflemann et al. (20) have shown that the anatomic zone of no reflow exhibits rapid expansion from minutes to hours after reperfusion.
In conclusion, in the present study, two PoC protocols failed to reduce necrotic damage. The extent of no reflow was highly correlated with the extent of necrosis, but PoC had no effect on this relationship, suggesting that this intervention does not impact microvascular damage from ischemia. Whereas ischemic preconditioning is effective in reducing infarct size in our rabbit model of coronary artery occlusion and reperfusion, the two protocols of PoC that we tested lacked efficacy.
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