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J Thorac Cardiovasc Surg 1999;117:810-817
© 1999 Mosby, Inc.
CARDIOPULMONARY SUPPORT AND PHYSIOLOGY |
From the Department of Surgery, University of Kentucky College of Medicine, Lexington, Ky.
Supported by grant HL34579 from The National Heart, Lung, and Blood Institute.
Received for publication Aug 31, 1998. Revisions requested Oct 30, 1998. Revisions received Dec 4, 1998. Accepted for publication Dec 4, 1998. Address for reprints: M. Salik Jahania, MD, Department of Surgery, University of Kentucky College of Medicine, Albert B. Chandler Medical Center, MN 273 B, 800 Rose St, Lexington, KY 40536-0084.
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Abstract |
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Introduction |
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1 that ischemic preconditioning reduced infarct size, there have been numerous studies documenting the effects of preconditioning in several animal models and species. 2 Ischemic preconditioning has been shown to have numerous beneficial metabolic effects such as reduced rates of high-energy phosphate catabolism and reduced accumulation of myocardial lactate and hydrogen ions during prolonged ischemia. 3-5 There is also clinical evidence indicating human myocardium may be preconditioned during balloon angioplasty 6 and cardiac operation. 7,8
The role of ischemic preconditioning in attenuating reversible postischemic dysfunction, however, remains controversial. Although ischemic preconditioning consistently improves postischemic recovery of function in the isolated perfused rat heart, 9-11 there are conflicting reports about its effects in the globally ischemic rabbit heart. 12-17 In rabbit in vivo regional ischemia models, ischemic preconditioning has been shown to have no beneficial effect on postischemic regional function independent of infarct size reduction. 18 In 2 in vivo large animal studies Ovize, 19 Miyamae, 20 and their colleagues reported that ischemic preconditioning did not exert any beneficial effect on regional segment shortening in canine and porcine myocardium. However in both studies, the substantial decrease in segment shortening, associated with the 5-minute preconditioning cycles, itself may have obscured any protective effect after the subsequent longer occlusion. Furthermore, assessment of regional ventricular function by segment shortening is preload and afterload dependent, which hinders its use as a sensitive measure of cardiac contractility. In contrast, load insensitive measures, such as the end-systolic pressure segment length relationship (ESPLR), preload recruitable stroke work (PRSW) and preload recruitable stroke work area (PRSWA) are more sensitive and reliable indicators of cardiac contractility in intact animals. 21-24 Therefore the purpose of this study was to assess the effects of ischemic preconditioning on both load-sensitive and load-insensitive measurements of contractility in a porcine model of in vivo regional myocardial stunning.
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Methods |
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Animal preparation
Farm pigs weighing 22 to 27 kg were used. Anesthesia was induced with ketamine (20 mg/kg, intramuscularly) and sodium pentobarbital (15-18 mg/kg, intravenously). Anesthesia was maintained with additional sodium pentobarbital (1.5-2 mg/kg, intravenously) every 15 minutes. Ventilation was maintained with a tracheotomy with a mixture of room air and 100% oxygen. Tidal volume, respiratory rate, and fraction of oxygen in inspired air were adjusted to maintain normal arterial blood gas and pH values. Core body temperature was monitored with an esophageal temperature probe and maintained with a heating pad between 37.0°C and 37.5°C. Lactated Ringer's solution was administered through an ear vein or femoral vein, at 5 to 7 mL/kg/min after an initial bolus of 300 to 400 mL. A femoral artery catheter was used to monitor arterial blood pressure and to obtain arterial blood gas samples.
The heart was exposed through a median sternotomy and suspended in a pericardial cradle. Left ventricular pressure (LVP) was measured with a 5F high-fidelity pressure-sensitive tip transducer (Millar Instruments, Houston, Tex) placed in the left ventricular cavity through the apex and secured with a purse-string suture. A segment of the left anterior descending coronary artery (LAD), distal to the origin of the first diagonal branch, was dissected free of surrounding tissue. The area at risk was delineated by a brief (<20-second) occlusion of the LAD with a small vascular occluding clamp. A transit time perivascular flow probe (Transonic Systems Inc, Ithaca, NY) was placed around this segment to measure coronary blood flow. Pairs of piezoelectric segment-shortening crystals (Crystal Biotech, Houston, Tex) were placed in the LAD and left circumflex coronary artery perfused beds to measure regional segment shortening by sonomicrometry. Crystals were placed in the mid-myocardium (approximately 4-6 mm deep) 5 to 15 mm apart and aligned in a manner such that the intercrystal axis was parallel to the direction of myocardial fiber shortening.
Experimental protocol
Animals in both groups were allowed a 30-minute stabilization period after all instrumentation was complete. A total of 19 pigs were randomized into 2 groups. The control group (n = 11) underwent a 15-minute LAD occlusion and 3-hour reperfusion. The ischemic preconditioning group (n = 8) underwent 2 cycles of 5-minute LAD occlusion and 10-minute reperfusion before the 15-minute occlusion. All animals received heparin (100 U/kg body weight, intravenously) before the coronary occlusion and lidocaine (2% solution; 2 mg/kg, intravenous bolus) immediately before reperfusion. After 3 hours of reperfusion, the ischemic area at risk was determined by reoccluding the LAD and infusing a 0.5% Evans blue solution into the left ventricle while occluding the aorta. The area at risk was devoid of the Evans blue stain. The animals were killed while under deep anesthesia with an intracardiac bolus of KCl, and the hearts were excised. Crystal placement in the ischemic and nonischemic beds was verified after excision of the heart.
Area at risk and infarct size measurement
The isolated left ventricles were cut in 4 slices of equal thickness in a plane parallel to the atrioventricular groove. Each slice was weighed and compressed between 2 transparent Plexiglas acrylic plates (Rohm & Hass Company, Philadelphia, Pa) separated by a distance of 8 mm to achieve uniform thickness. The cross-sectional surface and ischemic areas of each slice were traced onto a transparency sheet. The slices were then incubated in a 1% triphenyltetrazolium chloride solution in phosphate-buffered saline solution at 37°C for 15 minutes. The presence of a brick-red stain indicated viable tissue although nonviable tissue (infarcted) remained pale. If any infarct was present, the tissue slices were again compressed between the Plexiglas acrylic plates and retraced. The areas on the tracings were quantified with a digitizer (Mustek 1200 III, parallel port scanner at 200 dpi; Mustek Incorporated, Irvine, Calif) and graphic analysis software (Sigma Scan Pro Automated Image Analysis Software; Jandel Scientific, SPSS Inc, San Rafael, Calif). The percent area at risk was calculated for each slice by dividing the area at risk by the total slice area. The sum of the areas at risk of all slices was divided by the sum of the areas of all slices to obtain the percentage of the left ventricle that was ischemic.
Data and statistical analysis
All hemodynamic and sonomicrometry signals were fed through a 32-bit analog digital converter into an online data acquisition computer with customized software (Augury; Coyote Bay Instruments, Manchester, NH). End-diastole was defined as the onset of positive rate of pressure rise (+dP/dt) and end-systole defined as 20 ms before peak –dP/dt. Segment shortening was defined as end-diastolic length (EDL)–end-systolic length (ESL), and percent segment shortening (% SS) was calculated as (EDL – ESL/EDL) x 100%. All hemodynamic data were continuously displayed on a computer monitor. Stroke work (SW) was calculated by quantifying the area within the pressure-segment–length loops generated during each cardiac cycle.
ESPLR, PRSW, and PRSWA were generated from the segment length, and LVP data collected during brief (7-second) vena cava occlusions. The inferior vena cava was occluded by gradual tightening of a snare formed of umbilical tape around its supradiaphragmatic portion. During data acquisition ventilation was held at end expiration to avoid effects of varying venous return on preload. Slope of the ESPLR (Ees) was calculated according to the methods of Aversano and colleagues, 23 where end-systole during each cardiac cycle was defined as the point on the LVP-segment length loop that maximized the value of Ees. The segment length (x-axis) intercept at an LVP of 70 mm Hg (L70) was derived with the slope and x- and y-intercept data from the ESPLR. PRSW and PRSWA were calculated according to the methods of Glower and colleagues. 24 PRSW was based on linear regression of the relationship between SW and end-diastolic segment length calculated by the equation SW = Msw (EDL–Lw), where Msw is the slope of PRSW and Lw is the x-axis intercept. 24PRSWA was determined by the formula: PRSWA = Msw/2 (1.2 Lw max – Lw)2, where Lw max was the maximum x-axis intercept during the entire experiment. Baseline and caval occlusion data were saved at specific time points in the protocol for off-line analysis. An average of 9 to 11 beats was used in each calculation.
Results are expressed as mean ± SEM. Reperfusion segment shortening was expressed as percent recovery of preischemic values. Stroke work, Ees, L70, PRSW, and PRSWA were expressed as absolute numbers.
Differences between the groups were determined with 2-factor analysis of variance with repeated measures across the second factor. If significant differences between groups were encountered, further analysis of variance was performed with Student t tests. Differences within groups were analyzed with 1-way analysis of variance.
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Results |
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. There were no statistically significant differences in SW between the 2 groups either at baseline (P = .35) or at 2 hours (P = .08) and 3 hours (P = .14) of reperfusion. At 1 hour of reperfusion, SW was lower in the preconditioned group (P = .01). Percent recovery of baseline regional SW after 3 hours of reperfusion was 50% ± 6% in controls and 51% ± 7% in preconditioned animals.
The ESPLR data collected during the caval occlusions are shown in Fig. 4, A and B. The reperfusion Ees values were not significantly different between the groups at any time (1 hour, P = .39; 2 hours, P = .27; 3 hours, P = .92). Furthermore the reperfusion Ees values were not significantly different from preischemic values within the groups (for controls 1-, 2-, and 3-hour P values were .88, .57, and .44, respectively, whereas for the preconditioned group they were .27, .42, and .18, respectively). The control and preconditioned groups both exhibited significant rightward-shifts in ESPLR throughout the 3-hour reperfusion period (Fig. 4, B). Although reperfusion L70 values tended to be higher in preconditioned hearts, there were no statistically significant differences between the groups (1-, 2-, and 3-hour P values were .08, .14, and .14, respectively).
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, shows the preischemic and reperfusion PRSW x-intercept values in the 2 groups, indicating both groups exhibited significant rightward shifts throughout reperfusion compared with baseline values. But there was no statistically significant difference between the groups (1-, 2-, and 3-hour P values vs control were .24, .35, and .31, respectively).
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Discussion |
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Several experimental and clinical studies have suggested that ischemic preconditioning reduces myocardial infarct size and ventricular arrhythmias, but its effects on postischemic ventricular function are less clear. Numerous studies show that globally ischemic isolated perfused rat hearts can be preconditioned, 9-11 and there are similar studies in isolated rabbit hearts. 12-14 However, additional reports in isolated rabbit 15,16 and guinea pig hearts 4 show that ischemic preconditioning does not improve postischemic function. Furthermore, high creatine kinase and lactate dehydrogenase release during reperfusion and high postischemic end-diastolic pressures in the isolated rat heart studies indicate that there was significant infarct. Jenkins and colleagues 17 measured both function and infarct size in globally ischemic isolated rabbit hearts and reported that ischemic preconditioning improved postischemic LVDP when infarct size was reduced, but preconditioning did not attenuate pure stunning in the absence of infarct.
Although there are numerous studies of ischemic preconditioning effects on postischemic function in isolated hearts, there are only a few studies in in vivo pure myocardial stunning models. Cohen and colleagues 18 reported that preconditioning improved segment shortening in the rabbit after 20 minutes of regional ischemia; however, they attributed this improvement to the associated 25% decrease in infarct size. Two frequently cited studies reported that neither a single cycle (Ovize and colleagues 19) nor 2 cycles (Miyamae and colleagues 20) of 5-minute preconditioning occlusions improve segment shortening after 15 minutes of regional ischemia in the canine or porcine model, respectively. However, any protective effect of the preconditioning may have been masked by the fact that the 5-minute preconditioning cycles reduced segment shortening considerably (52% and 25%, respectively) before the prolonged occlusion.
Our results are consistent with those of Ovize, 19 Miyamae, 20 and their colleagues in terms of the lack of beneficial effect of ischemic preconditioning on postischemic regional ventricular function in the absence of myocardial infarction. We also observed a significant decrease in segment shortening after the two 5-minute preconditioning occlusions. In all of these studies, the stunning produced by the preconditioning occlusions, which likely would have persisted for several hours, confounds the interpretation of the potential beneficial effects of preconditioning on postischemic function. The present results with regional stroke work are less problematic to interpret. The 2 preconditioning cycles did not significantly reduce regional SW before the long occlusion, nor did this protocol improve regional SW after the prolonged occlusion. Because segment shortening is both preload and afterload sensitive and SW is preload sensitive, the use of these measures as reliable assessments of cardiac contractility is limited.
The novel aspect to the present study was our analysis of the effects of ischemic preconditioning on stunning measured by ESPLR, PRSW, and PRSWA. All of these measures of cardiac contractility are relatively load insensitive. 23,24 Ees is the slope of the ESPLR generated during brief vena cava occlusions and is analogous to end systolic elastance (Ees) of ESPVR. 25 Aversano and colleagues 23 first reported the reliability of ESPLR for measurements of regional contractility in an open chest preparation. In the present study, although Ees was not significantly altered with stunning, the ESPLR in both groups was rightward shifted throughout reperfusion, as indicated by significantly greater L70 values; there were no statistical differences between the groups. Thus preconditioning did not attenuate stunning when assessed by ESPLR.
Another load-insensitive measure of myocardial contractility is the PRSW. This index of contractility is the relationship between regional stroke work and EDL and has been shown to be linear and a load-insensitive measure of regional inotropic state in both normal and reperfused myocardium. 24 A decrease in contractility based on PRSW is exhibited by a decrease in the slope and an increase in the length axis intercept. In our study, the PRSW slopes after 3-hour reperfusion in both groups returned to values that were not statistically different from preischemic values. However, the regression lines in both groups were significantly rightward shifted throughout reperfusion, indicating persistent stunning consistent with the results of Glower and colleagues. 24
PRSWA has been reported to be the most sensitive for quantifying regional ischemia-induced ventricular dysfunction. 24 In our study, this index of regional contractility remained significantly reduced in both groups throughout reperfusion, indicating persistent myocardial stunning. Furthermore, the lack of statistical difference in the recovery of PRSWA between the 2 groups indicates that ischemic preconditioning did not improve this load-insensitive measure of contractility.
There are several limitations to the present study, which must be recognized. These results pertain only to the first 3 hours of reperfusion and do not reflect the potential benefits during the reperfusion beyond 3 hours. Although we observed no significant differences between the groups, it appeared that there was a slight trend for improved recovery of PRSWA in the ischemic preconditioned group at the conclusion of the study. This trend may have translated into a statistically significant difference if the reperfusion period had been longer. Another limitation is the sample size. Although the sample size of each group was 7, the study had sufficient power to detect a difference between the groups. It is also possible that ischemic preconditioning may have exerted a beneficial effect after a longer ischemic period. We limited the ischemia to 15 minutes to avoid the development of infarction because a 15-minute occlusion has been repeatedly shown to result in no infarct in either porcine or canine myocardium. 19,20 We did not see any infarction using triphenyltetrazolium chloride staining, which is consistent with these studies. The use of an open chest, pentobarbital anesthetized animal preparation may have influenced both the degree of ischemic injury and the precision of the load-insensitive measurements. Finally, it remains to be determined whether these findings in a regional ischemia model can be extrapolated to the setting of global ischemia.
Our findings are consistent with Cremer and colleagues, 27 who compared a group of patients receiving a preconditioning protocol identical to ours before arrest with cold blood cardioplegia versus patients receiving intermittent cold blood cardioplegia alone and found no benefit from ischemic preconditioning. There are other studies that report no beneficial effects of ischemic preconditioning during clinical cardiac operation. 28-30
In summary, the results of this study indicate that ischemic preconditioning does not ameliorate porcine myocardial stunning in the acute reperfusion period, when contractility is assessed by load-insensitive measurements. Extrapolating these results to the clinical arena ischemic preconditioning may have a limited role in minimizing reversible postischemic dysfunction after cardiac operation.
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Acknowledgments |
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