Grace L. Chien

Met5-enkephalin protects isolated adult rabbit cardiomyocytes via δ-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 β-endorphin did not. ME-induced protection was blocked by the δ-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 δ-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, 1µmol/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 1µmol/L naloxone hydrochloride+IP group (19.0 ± 6.5 vs. 48.9 ± 8.4, P = ns) but IP was blocked by 100 µmol/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.

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.

Remifentanil limits infarct size but attenuates preconditioning-induced infarct limitation

ObjectivesWe investigated the influence of the narcotic anesthetic remifentanil on irreversible myocardial ischemic injury. MethodsNew Zealand White rabbits were anesthetized with propofol (0.7–1.8 mg·kg−1·min−1) and then subjected to 30 min regional myocardial ischemia and 3 h reperfusion (CON). Some animals also underwent ischemic preconditioning, elicited by either one (IP1) or two (IP2) cycles of 5 min ischemia and 5 min reperfusion, and/or remifentanil, administered either as a transient infusion mimicking the preconditioning protocol (RP2, 10 μg·kg−1·min−1) or as a continuous infusion (R, 3–10 μg·kg−1·min−1). Rabbits were randomly assigned to experimental groups. Infarct size was assessed with tetrazolium. Results are reported as mean±SD. ResultsNon-preconditioned infarct size was ∼50% of the area-at-risk (49.6±20.1% CON). Both one and two cycles of ischemic preconditioning markedly reduced infarct size (49.6±20.1% CON versus 18.6±8.6% IP and versus 7.5±7.6% IP2; both p<0.001). Preconditioning with remifentanil modestly reduced infarct size (49.6±20.1% CON versus 29.3±8.5% RP2; p<0.01). However, sustained administration of remifentanil did not provide protection (49.6±20.1% CON versus 43.9±16.2% R), and it attenuated the protection offered by preconditioning (49.6±20.1% CON versus 35.6±20.7% R+IP1, p=NS; and versus 14.5±14.5% R+IP2; p<0.05). ConclusionTransient pre-ischemic administration of remifentanil modestly reduces infarct size in propofol-anesthetized rabbits, but continuous administration of remifentanil increases the threshold for ischemic preconditioning-induced infarct limitation.

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.