Propidium iodide compares favorably with histology and triphenyl tetrazolium chloride in the assessment of experimentally-induced infarct size.

Quantitative assessment of infarct size after histology processing or by tetrazolium staining requires long reperfusion times to facilitate the delineation of the injured tissue. We evaluated the nucleic acid stain propidium iodide as an alternate technique for assessment of infarct size because it does not require extended reperfusion and is accomplished with more simple tissue processing. Eight mice underwent 45 min of coronary artery occlusion and 24 h of reperfusion, after which propidium iodide was administered and allowed to circulate for 15 min. Hearts were excised, sliced into transverse slices approximately 620 micro m thick, and photographed for determination of infarction with propidium iodide. Slices were processed by standard histology techniques, and infarction was evaluated using classical criteria of necrosis. In four other mice, infarct size was assessed by both triphenyl tetrazolium chloride and propidium iodide staining. A total of 46 slices were analysed and infarction was described as a percentage of the cross-sectional area of the slice. The correlation (r=0.94), orthogonal regression line (y=0.9x+1.8), and the percentage of the cross-section infarcted (histology 17.5+/-3.0%v propidium iodide 18.5+/-3.0%), demonstrates that infarct size assessment after propidium iodide staining or histology processing yields the same results. Similarly, propidium iodide measurement of infarct size was comparable to that obtained with TCC staining (22.5+/-4.1%v 25.4+/-5.4%, respectively, r=0.84). We conclude that propidium iodide staining yields accurate infarct size assessment and is a simpler alternative to tetrazolium staining or histologic processing.

Met5-enkephalin protects isolated adult rabbit cardiomyocytes via delta-opioid receptors.

In rats and rabbits, endogenous opioid peptides participate in ischemic preconditioning. However, it is not known which endogenous opioid(s) can trigger cardioprotection. We examined preconditioning-induced and opioid-induced limitation of cell death in isolated, calcium-tolerant, adult rabbit cardiomyocytes. Cells were subjected to simulated ischemia by pelleting and normothermic hypoxic incubation. Preconditioning was elicited with 15 min of simulated ischemia followed by 15 min of resuspension and reoxygenation. All cells underwent 180 min of simulated ischemia. Cell death was assessed by trypan blue permeability. Morphine protected cells, as did preconditioning; naloxone blocked the preconditioning-induced protection. Exogenous Met5-enkephalin (ME) induced protection, but exogenous beta-endorphin did not. ME-induced protection was blocked by the delta-selective antagonist naltrindole. Additionally, two other proenkephalin products, Leu5-enkephalin and Met5-enkephalin-Arg-Phe, provided protection equipotent to ME. These data suggest that one or more proenkephalin products interact with delta-opioid receptors to endogenously trigger opioid-mediated protection.

Naloxone blockade of myocardial ischemic preconditioning does not require central nervous system participation.

OBJECTIVE: The hypothesis that naloxone blockade of ischemic preconditioning (IP)-induced infarct limitation does not require central nervous system participation was evaluated using quaternary naloxone in anesthetized rabbits (Study I) and naloxone hydrochloride in isolated rabbit hearts (Study II). METHODS: In Study I, rabbits underwent 30 min coronary artery occlusion and 180 min reperfusion. IP was elicited with a 5 min coronary artery occlusion beginning 15 min before the 30 min occlusion. Intravenous naloxone methiodide, 12.9 mg/kg, was bolused 10 or 1 min before IP. In Study II, rabbit hearts underwent 45 min coronary artery occlusion and 120 min reperfusion. IP was elicited with 2 cycles of 5 min coronary artery occlusion plus 5 min reperfusion, beginning 20 min before the 45 min occlusion. Naloxone hydrochloride, 1 mumol/L or 100 mumol/L, was added to the buffer perfusate for 25 min preceding the long coronary artery occlusion. In both studies, infarct size was assessed with tetrazolium, normalized to risk volume, and analyzed using ANOVA. RESULTS: In both studies, IP reduced infarct size compared to control (6.3 +/- 2.3 vs. 29.5 +/- 4.4, P = 0.007, Study I; 11.8 +/- 4.7 vs. 47.7 +/- 6.7, P = 0.03, Study II). In Study I, IP was not blocked when naloxone methiodide was given 10 min before IP (13.8 +/- 4.8 vs. 42.3 +/- 5.4, P = 0.004) but was blocked when given 1 min before IP (25.3 +/- 7.2 vs. 28.4 +/- 5.0, P = ns). In Study II, infarct size was intermediate in the 1 mumol/L naloxone hydrochloride + IP group (19.0 +/- 6.5 vs. 48.9 +/- 8.4, P = ns) but IP was blocked by 100 mumol/L naloxone hydrochloride (62.6 +/- 4.5 vs. 56.2 +/- 6.7, P = ns). CONCLUSION: Naloxone blockade of IP-induced infarct limitation involves a cardiac mechanism.

High spinal anesthesia does not alter experimental myocardial infarction size or ischemic preconditioning.

OBJECTIVE: The role of the central nervous system in the development of myocardial infarction and ventricular fibrillation in virgin and ischemically preconditioned myocardium was investigated. DESIGN: Infarct size and ventricular arrhythmias were assessed after regional ischemia-reperfusion. Animals were randomly assigned to four groups: (1) preconditioned, central nervous system intact; (2) nonpreconditioned, nervous system intact; (3) preconditioned, nervous system blocked; and (4) nonpreconditioned, nervous system blocked. Differences in hemodynamics and infarct size were assessed with analysis of variance, and differences in ventricular fibrillation were assessed with the Kruskal-Wallis test. SETTING: Experiments were performed in the Anesthesiology Research Laboratory at a medical center. PARTICIPANTS: Anesthetized open-chest New Zealand white rabbits were used for these studies. INTERVENTIONS: Rabbits underwent 30 minutes of coronary artery occlusion and 3 hours of reperfusion. The central nervous system was blocked with total spinal anesthesia. Ischemic preconditioning was elicited with 5 minutes of coronary artery occlusion and 10 minutes of reperfusion. Infarction was assessed with tetrazolium and expressed as a percentage of the risk zone (mean +/- SEM). MEASUREMENTS AND MAIN RESULTS: Preconditioning resulted in infarct size limitation compared with the control (8% +/- 4% v 43% +/- 5%; p < 0.001) and delayed the onset of fibrillation (15.5 minutes v 11 minutes; p = 0.001). Spinal blockade neither altered nonpreconditioned infarct size nor attenuated preconditioning (32% +/- 7% v 8% +/- 3%; p = 0.04), but it was associated with ventricular fibrillation in 24/25 rabbits as compared with 6/14 rabbits without blockade. In blocked animals, preconditioning resulted in a decreased duration of fibrillation (2.5 minutes v 12.5 minutes; p = 0.0004). However, spinal blockade eliminated the preconditioning-induced delay in fibrillation (10 minutes v 12 minutes; p = NS). CONCLUSIONS: It is concluded that (1) activation of efferent sympathetic nerves is not necessary for ischemic preconditioning; (2) preconditioning delays the onset of ventricular arrhythmias; and (3) spinal blockade exacerbates ischemia-induced ventricular arrhythmias.

Naloxone blockade of myocardial ischemic preconditioning is stereoselective.

This study tested the hypothesis that endogenous opioids are involved in the infarct limitation of myocardial ischemic preconditioning (IP). Blockade of IP-induced infarct limitation by (-)naloxone hydrochloride (-NAL) or its receptor-inactive stereoisomer (+)naloxone (+NAL) was evaluated. Fifty-two pentobarbitone-anesthetized, open-chest rabbits underwent 30 min coronary artery occlusion and 180 min reperfusion. Treatment groups were: control (n = 9), i.p. (n = 8), -NAL (n = 9) and -NAL/i.p. (n = 12), or +NAL (n = 6) and +NAL/i.p. (n = 8). i.p. was elicited with 5 min regional ischemia, beginning 15 min before the 30 min coronary occlusion. -NAL or +NAL, 3 mg/kg i.v. bolus, was given 25 min before the 30 min coronary occlusion. Infarct size was assessed with tetrazolium and expressed as a percentage of area-at-risk. There were no significant intergroup differences of area-at-risk. IP resulted in marked infarct limitation compared to control (control, 32.9 +/- 7.6% v i.p., 5.8 +/- 4.5%; P = 0.04). Neither -NAL nor +NAL alone altered infarct size compared to control, but -NAL did block the infarct limitation of i.p. (-NAL, 31.4 +/- 6.7% v -NAL/i.p., 24.3 +/- 6.2%) whereas +NAL did not (+NAL, 40.5 +/- 5.0% v +NAL/i.p., 13.7 +/- 3.6%; P = 0.02). In conclusion, naloxone blockade of i.p.-induced cardioprotection is stereospecific and therefore likely to be opioid receptor-mediated.

Adrenergic activation confers cardioprotection mediated by adenosine, but is not required for ischemic preconditioning.

BACKGROUND: The aim of this study was to determine whether (1) adrenergic activation is cardioprotective, (2) adrenergic cardioprotection occurs via adenosine receptor activation, and (3) ischemic preconditioning requires alpha-adrenergic activation. METHODS: Anesthetised open chest rabbits underwent 30 min coronary occlusion and 3 h reperfusion. Ischemic preconditioning was elicited with 5 min coronary occlusion and 10 min reperfusion. Activation of adrenergic receptors with endogenous norepinephrine was achieved with tyramine (0.28 mg/kg/min intravenously for 5 min). Adenosine receptors were blocked with 8-p-sulfophenyl theophylline (10 mg/kg intravenously), alpha 1-adrenergic receptors were selectively blocked with prazosin (0.1 mg/kg intravenously), and alpha-adrenergic receptors were blocked with phentolamine (4 mg/kg intravenously). RESULTS: Ischemic preconditioning reduced risk-adjusted infarct volume by 79% (P < 0.0005). This protection was attenuated by adenosine receptor blockade. Tyramine infusion resulted in a 1305% change from baseline plasma norepinephrine concentration (P < or = 0.01), and reduced infarct volume by 55% (P = 0.01). Adenosine receptor blockade abolished this protection. Blockade of alpha 1-adrenergic receptors with prazosin failed to abolish ischemic preconditioning (79 versus 89% reduction in infarct volume, without and with prazosin, respectively). Similarly, non-selective blockade of alpha-adrenergic receptors also failed to abolish ischemic preconditioning (79 versus 57% reduction without and with phentolamine, respectively). CONCLUSIONS: We conclude that the cardioprotection of ischemic preconditioning and alpha-adrenergic activation both involve adenosine, but ischemic preconditioning does not require alpha-adrenergic activation.

Fluorescent vs. radioactive microsphere measurement of regional myocardial blood flow.

OBJECTIVES: This study compared simultaneous regional myocardial blood flow (RMBF) measurements using fluorescent microspheres (FM) and radiolabeled microspheres (RM). The utility of an internal standard during processing was also examined. METHODS: Paired FM and RM were injected into the left atrium of 9 anaesthetised rabbits. RMBF was altered by use of either regional ischaemia or (-)-N6-(2-phenylisopropyl)-adenosine. Radioactivity of blood reference and tissue samples was quantitated using standard methods. Samples were then digested with potassium hydroxide and microspheres recovered by vacuum filtration, with an additional label of FM as the internal standard. FM labels were extracted using Carbitol acetate and quantitated using fluorescence spectroscopy. Agreement between the fluorescent and radioactive methods was assessed using both orthogonal regression and difference-against-mean analyses. RESULTS: Using recovery-uncorrected data, the slope of the orthogonal regression of RM and FM-determined RMBF was not statistically different from 1, but the intercept was statistically different from 0 [-0.03(0.01), P = 0.005] and the mean RMBF by each method differed from one another [1.24(0.08) vs. 1.17(0.08) ml.min-1.g-1, P = 0.0002]. The mean +/- 2 s.d. of the differences of RMBF (RM minus FM) was +0.07 +/- 0.30 ml.min-1.g-1. Although recovery of FM from tissue averaged 97.6(1.2)%, use of the internal standard to correct for losses substantially improved the agreement between RM and FM-determined RMBF: the orthogonal regression slope was not statistically different from 1, the intercept was not statistically different from 0, and the means of the flows were not different. The mean +/- 2 s.d. of the differences of RMBF was -0.01 +/- 0.22 ml.min-1.g-1. The internal standard also improved RMBF estimates from samples with simulated large spillage during processing. CONCLUSION: Fluorescent microspheres are an equivalent alternative to radiolabeled microspheres for the estimation of RMBF. Although the overall recovery of microspheres using this technique was high, use of an internal standard is recommended for correction of random losses.

Anaesthetics alter the magnitude of infarct limitation by ischaemic preconditioning.

OBJECTIVE: The aim was to determine whether three commonly used animal anaesthetics alter the magnitude of infarct limitation achieved with ischaemic preconditioning. METHODS: Eighty four anaesthetised non-preconditioned and preconditioned open chest rabbits underwent a 30 min coronary occlusion followed by 3 h reperfusion. Ischaemic preconditioning was achieved with 5 min coronary occlusion beginning 15 min before the 30 min coronary occlusion. The anaesthetics studied were: pentobarbitone (30 mg.kg-1 intravenously +30-50 mg.kg-1.h-1 intravenously), isoflurane (1.5-2.5% end expiratory), and ketamine/xylazine (cocktail of 67 mg ketamine and 6.7 mg xylazine.ml-1, 1 ml.kg-1 intramuscularly +0.3-1.3 ml.kg-1.h-1 intramuscularly). Area at risk was delineated with ZnCdS particles and infarction assessed with tetrazolium. RESULTS: There were no significant differences in area at risk, heart rate, arterial pressure, and temperature between non-preconditioned and preconditioned hearts. Although infarct size was not significantly different among non-preconditioned hearts for each anaesthetic regimen (p = NS), the magnitude of infarct limitation with preconditioning varied with the anaesthetic employed (decrease in infarct size from control values of 81%, 44%, and 33% for pentobarbitone, isoflurane and ketamine/xylazine, respectively, p = 0.0145 for comparison of the three magnitudes, two factor ANOVA). CONCLUSION: Anaesthetic regimens affect the degree of infarct size limitation seen with ischaemic preconditioning.

"Normothermic range" temperature affects myocardial infarct size.

OBJECTIVES: This study tested the hypothesis that small changes in temperature above the hypothermic range may alter myocardial infarct size after acute coronary occlusion-reperfusion. A secondary hypothesis, that a correlation between temperature and infarct size may be independent of an associated change of heart rate, was also evaluated. METHODS: Eighteen pentobarbitone-anaesthetised, open chest rabbits underwent 30 min coronary artery occlusion and 3 h reperfusion at blood temperatures ranging from 35-42 degrees C, achieved and maintained using surface methods (not paced, group NP). In a second group of 11 animals, heart rate was held constant across the same range of temperatures (paced, group P), before and throughout coronary artery occlusion-reperfusion. Infarct sizes were assessed by the tetrazolium method. RESULTS: Target temperature was effectively controlled over the duration of the experimental protocol to within +/- 0.25 degrees C. Area at risk did not vary with temperature. Infarct size, normalised to area at risk, was correlated with temperature in both groups (infarct size = 7.9 x temp-250.0, r = 0.75, p = 0.0003, group NP; infarct size = 11.7 x temp-404.5, r = 0.88, p = 0.0004, group P). There was no significant difference between the slopes of these two lines (p = 0.18), indicating that the positive correlation between infarct size and temperature is not related to changes of heart rate. CONCLUSION: Temperatures in the range of 35-42 degrees C affect myocardial infarct size significantly, independent of heart rate.

Cardioprotection provided by adenosine receptor activation is abolished by blockade of the KATP channel.

Adenosine agonists and openers of the ATP-sensitive potassium (KATP) channel have been reported to limit infarct size (IS). We tested the hypothesis that these phenomena are interdependent. Anesthetized swine underwent 60 min of coronary artery occlusion and 90 min of reperfusion. Preconditioning was elicited by two cycles comprising 10 min of occlusion and 10 min of reperfusion (n = 7 swine). An intracoronary infusion of adenosine (Ado; n = 10) or (-)-N6-(2-phenylisopropyl)-adenosine (R-PIA; n = 7) replaced preconditioning ischemia. KATP channels were blocked with sodium 5-hydroxydecanoate (5-HD) in the absence (n = 6) or presence (n = 8) of R-PIA. Control pigs (n = 7) received saline vehicle. IS was assessed with tetrazolium and normalized as percentage of area at risk. Preconditioning resulted in a reduced IS compared with Control (3.9 +/- 1.8 vs. 43.5 +/- 6.9%, respectively; P < 0.0005). Ado and R-PIA also reduced IS [21.1 +/- 6.8 (P < 0.01) and 11.2 +/- 7.4% (P < 0.005), respectively]. 5-HD alone did not alter IS, but it abolished R-PIA-induced cardioprotection (IS 5-HD + R-PIA = 48.6 +/- 13.2%). Thus Ado A1-receptor agonists mimicked the cardioprotection of ischemic preconditioning. The Ado-induced limitation of IS was abolished by blockade of the KATP channel. We conclude that both Ado A1 receptors and KATP channels may be involved in ischemic preconditioning.

Electrocardiographic R-wave changes during cardiac surgery.

The diagnostic accuracy of exercise electrocardiography has been improved by incorporation of R-wave gain factor to correct the measured ST-segment changes. If marked changes in R-wave amplitude occur in individual patients during cardiac operations, a similar gain factor correction may improve the intraoperative diagnosis of myocardial ischemia. This investigation was designed to determine the frequency and magnitude of intraoperative V5 R-wave amplitude changes during cardiac operations. Electrocardiograms were recorded from 83 patients while patients were awake, anesthetized (baseline), after placement of the Favaloro and Canadian sternal retractors, and at end-operation. Compared with baseline values, placement of the Canadian sternal retractor was associated with a reduction in V5 R-wave amplitude from 15 +/- 1 to 10 +/- 1 mm (mean +/- SEM), in V5 S-wave amplitude from 3.5 +/- 0.4 to 1.7 +/- 0.3 mm, and in absolute ST-segment deviation from 0.50 +/- 0.04 to 0.39 +/- 0.05 mm. Changes in V5 R-wave amplitude were correlated with changes in ST-segment deviation in patients with baseline ST-segment deviations greater than or equal to 0.5 mm (r = 0.55, P = 0.0004, n = 37). Changes associated with the Favaloro retractor and the respiratory cycle were less marked. However, the V5 R-wave amplitude was decreased from 15 +/- 1 to 9 +/- 1 mm at end-operation. In conclusion, sternal spreading with the Canadian retractor was associated with marked reductions in V5 R- and S-wave amplitudes and ST-segment deviations. Marked changes in V5 R-wave amplitude persisted after sternal closure.(ABSTRACT TRUNCATED AT 250 WORDS)