Franz Kehl

Mechanism of Preconditioning by Isoflurane in Rabbits: A Direct Role for Reactive Oxygen Species

Background Reactive oxygen species (ROS) contribute to myocardial protection during ischemic preconditioning, but the role of the ROS in protection against ischemic injury produced by volatile anesthetics has only recently been explored. We tested the hypothesis that ROS mediate isoflurane-induced preconditioning in vivo. Methods Pentobarbital-anesthetized rabbits were instrumented for measurement of hemodynamics and were subjected to a 30 min coronary artery occlusion followed by 3 h reperfusion. Rabbits were randomly assigned to receive vehicle (0.9% saline), or the ROS scavengers N-acetylcysteine (NAC; 150 mg/kg) or N-2-mercaptopropionyl glycine (2-MPG; 1 mg · kg−1· min−1), in the presence or absence of 1.0 minimum alveolar concentration (MAC) isoflurane. Isoflurane was administered for 30 min and then discontinued 15 min before coronary artery occlusion. A fluorescent probe for superoxide anion production (dihydroethidium, 2 mg) was administered in the absence of the volatile anesthetic or 5 min before exposure to isoflurane in 2 additional groups (n = 8). Myocardial infarct size and superoxide anion production were assessed using triphenyltetrazolium staining and confocal fluorescence microscopy, respectively. Results Isoflurane (P < 0.05) decreased infarct size to 24 ± 4% (mean ± SEM; n = 10) of the left ventricular area at risk compared with control experiments (43 ± 3%; n = 8). NAC (43 ± 3%; n = 7) and 2-MPG (42 ± 5%; n = 8) abolished this beneficial effect, but had no effect on myocardial infarct size (47 ± 3%; n = 8 and 46 ± 3; n = 7, respectively) when administered alone. Isoflurane increased superoxide anion production as compared with control experiments (28 ± 12 vs. −6 ± 9 fluorescence units;P < 0.05). Conclusions The results indicate that ROS produced following administration of isoflurane contribute to protection against myocardial infarction in vivo.

Mechanical function of the left atrium: New insights based on analysis of pressure-volume relations and doppler echocardiography

THE left atrium (LA) serves three major roles that exert a profound effect on left ventricular (LV) filling and overall cardiovascular performance. The LA is a contractile chamber that actively empties immediately before the onset of LV systole and establishes final LV enddiastolic volume. The LA is a reservoir that stores pulmonary venous return during LV contraction and isovolumic relaxation after the closure and before the opening of the mitral valve. Lastly, the LA is a conduit that empties its contents into the LV down a pressure gradient after the mitral valve opens and continues to passively transfer pulmonary venous blood flow during LV diastasis. These contraction, reservoir, and conduit functions of the LA mechanically facilitate the transition between the almost continuous flow through the pulmonary venous circulation and the intermittent filling of the LV. The contractile activity of the LA was initially described by William Harvey in 1628. This “booster pump” contribution to cardiac output normally accounts for approximately 20% of LV stroke volume but becomes increasingly important to the preservation of cardiovascular performance in patients with reduced LV compliance. The enhanced significance of atrial systole to LV filling in patients with LV dysfunction is emphasized by the frequently observed development of clinical signs and symptoms of heart failure when LA contraction is improperly timed or eliminated with the onset of atrial tachyarrhythmias. These adverse effects are reversed with the subsequent restoration of normal sinus rhythm and LA contraction. The relative impact of LA reservoir function on early LV filling was initially recognized by Henderson et al., and the dependence of reservoir function on LA compliance was later identified by Suga. While these and other early studies provided seminal information about LA function, comprehensive evaluation of LA performance in the normal and diseased heart was limited by lack of effective techniques for reproducibly measuring continuous LA volume and pulmonary venous blood flow until the 1980s. This objective has subsequently been facilitated by the application of pressure–volume theory adapted from LV function analysis and by the widespread use of two-dimensional and Doppler echocardiography. This article critically reviews recent advances in the understanding of LA physiology derived from pressure–volume relations and echocardiography, discusses the mechanical consequences of primary LA dysfunction, examines LA mechanical adaptation to LV dysfunction, and describes current knowledge about the actions of volatile and intravenous anesthetics on LA function in vivo.

Hyperglycemia Prevents Isoflurane-induced Preconditioning against Myocardial Infarction

Background Volatile anesthetics stimulate but hyperglycemia attenuates activity of mitochondrial adenosine triphosphate–regulated potassium channels. The authors tested the hypothesis that acute hyperglycemia interferes with isoflurane-induced preconditioning in vivo. Methods Barbiturate-anesthetized dogs (n = 79) were instrumented for measurement of hemodynamics. Myocardial infarct size and collateral blood flow were assessed with triphenyltetrazolium chloride staining and radioactive microspheres, respectively. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3 h of reperfusion. Dogs were randomly assigned to receive an infusion of normal saline (normoglycemic controls) or 15% dextrose in water to increase blood glucose concentrations to 300 or 600 mg/dl in the absence or presence of isoflurane (0.5 or 1.0 minimum alveolar concentration [MAC]) in separate experimental groups. Isoflurane was discontinued, and blood glucose concentrations were allowed to return to baseline values before left anterior descending coronary artery occlusion. Results Myocardial infarct size was 26 ± 1% of the left ventricular area at risk in control experiments. Isoflurane reduced infarct size (15 ± 2 and 13 ± 1% during 0.5 and 1.0 MAC, respectively). Hyperglycemia alone did not alter infarct size (26 ± 2 and 33 ± 4% during 300 and 600 mg/dl, respectively). Moderate hyperglycemia blocked the protective effects of 0.5 MAC (25 ± 2%) but not 1.0 MAC isoflurane (13 ± 2%). In contrast, severe hyperglycemia prevented reductions of infarct size during both 0.5 MAC (29 ± 3%) and 1.0 MAC isoflurane (28 ± 4%). Conclusions Acute hyperglycemia attenuates reductions in myocardial infarct size produced by isoflurane in dogs.

Is isoflurane-induced preconditioning dose related?

Background Volatile anesthetics precondition against myocardial infarction, but it is unknown whether this beneficial action is threshold- or dose-dependent. The authors tested the hypothesis that isoflurane decreases myocardial infarct size in a dose-dependent fashion in vivo. Methods Barbiturate-anesthetized dogs (n = 40) were instrumented for measurement of systemic hemodynamics including aortic and left ventricular pressures and rate of increase of left ventricular pressure. Dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3 h of reperfusion and were randomly assigned to receive either 0.0, 0.25, 0.5, 1.0, or 1.25 minimum alveolar concentration (MAC) isoflurane in separate groups. Isoflurane was administered for 30 min and discontinued 30 min before left anterior descending coronary artery occlusion. Results Infarct size (triphenyltetrazolium staining) was 29 ± 2% of the area at risk in control experiments (0.0 MAC). Isoflurane produced significant (P < 0.05) reductions of infarct size (17 ± 3, 13 ± 1, 14 ± 2, and 11 ± 1% of the area at risk during 0.25, 0.5, 1.0, and 1.25 MAC, respectively). Infarct size was inversely related to coronary collateral blood flow (radioactive microspheres) in control experiments and during low (0.25 or 0.5 MAC) but not higher concentrations of isoflurane. Isoflurane shifted the linear regression relation between infarct size and collateral perfusion downward (indicating cardioprotection) in a dose-dependent fashion. Conclusions Concentrations of isoflurane as low as 0.25 MAC are sufficient to precondition myocardium against infarction. High concentrations of isoflurane may have greater efficacy to protect myocardium during conditions of low coronary collateral blood flow.

Sevoflurane-induced preconditioning of rat brain in vitro and the role of KATP channels

In the present study we tested the ability of the inhalation anesthetic sevoflurane to induce preconditioning against hypoxia in vitro. Rat hippocampal slices were prepared using established procedures. After 90 min of incubation, slices were exposed for 30 min to 0, 1, 2 or 3 minimum alveolar concentration (MAC) of sevoflurane under normoxic conditions (95% O2/5% CO2). Fifteen minutes later, slices were exposed to 13-min hypoxia (95% N2/5% CO2) followed by 30-min reoxygenation. The amplitude of extracellularly recorded, orthodromically evoked, CA1 population spikes (neuronal function) at the end of the reoxygenation period was measured and used to quantify the degree of recovery of neuronal function posthypoxia. To assess the role that the mitochondrial KATP channel plays in preconditioning, its blocker, 5-hydroxydecanoic acid (5-HD), was added during sevoflurane exposure. Sevoflurane-preconditioning with 1, 2 and 3 MAC increased the degree of recovery of neuronal function after 13-min hypoxia and 30-min reoxygenation from 51 +/- 1% (0 MAC), to 55 +/- 3%, 63 +/- 3%, and 72 +/- 2%, respectively. The effect of 3 MAC sevoflurane was blocked by 5-HD (53 +/- 3%), whereas 5-HD alone had no effect (48 +/- 3%) on the recovery of neuronal function from hypoxia. It is concluded that sevoflurane is capable of inducing preconditioning in vitro in a dose-dependent fashion and involves activation of mitochondrial KATP channels.

Role of the β1-Adrenergic Pathway in Anesthetic and Ischemic Preconditioning against Myocardial Infarction in the Rabbit Heart In Vivo

Background:Anesthetic and ischemic preconditioning share similar signal transduction pathways. The authors tested the hypothesis that the β1-adrenergic signal transduction pathway mediates anesthetic and ischemic preconditioning in vivo. Methods:Pentobarbital-anesthetized (30 mg/kg) rabbits (n = 96) were instrumented for measurement of systemic hemodynamics and subjected to 30 min of coronary artery occlusion and 3 h of reperfusion. Sixty minutes before occlusion, vehicle (control), 1.0 minimum alveolar concentration desflurane, or sevoflurane, and esmolol (30.0 mg · kg−1 · h−1) were administered for 30 min, respectively. Administration of a single 5-min cycle of ischemic preconditioning was instituted 35 min before coronary artery occlusion. In separate groups, the selective blocker esmolol or the protein kinase A inhibitor H-89 (250 μg/kg) was given alone and in combination with desflurane, sevoflurane, and ischemic preconditioning. Results:Baseline hemodynamics and area at risk were not significantly different between groups. Myocardial infarct size (triphenyltetrazolium staining) as a percentage of area at risk was 61 ± 4% in control. Desflurane, sevoflurane, and ischemic preconditioning reduced infarct size to 34 ± 2, 36 ± 5, and 23 ± 3%, respectively. Esmolol did not alter myocardial infarct size (65 ± 5%) but abolished the protective effects of desflurane and sevoflurane (57 ± 4 and 52 ± 4%, respectively) and attenuated ischemic preconditioning (40 ± 4%). H-89 did not alter infarct size (60 ± 4%) but abolished preconditioning by desflurane (57 ± 5%) and sevoflurane (61 ± 1%). Ischemic preconditioning (24 ± 7%) was not affected by H-89. Conclusions:The results demonstrate that anesthetic preconditioning is mediated by the β1-adrenergic pathway, whereas this pathway is not essential for ischemic preconditioning. These results indicate important differences in the mechanisms of anesthetic and ischemic preconditioning.

Activation of mitochondrial large-conductance calcium-activated K+ channels via protein kinase A mediates desflurane-induced preconditioning.

BACKGROUND:ATP-regulated K+ channels are involved in anesthetic-induced preconditioning (APC). The role of other K+ channels in APC is unclear. We tested the hypothesis that APC is mediated by large-conductance calcium-activated K+ channels (KCa). METHODS:Pentobarbital-anesthetized male C57BL/6 mice were subjected to 45 min of coronary artery occlusion and 3 h reperfusion. Thirty minutes before coronary artery occlusion, 1.0 MAC desflurane was administered for 15 min alone or in combination with the large-conductance KCa channel activator NS1619 (1 &mgr;g/g i.p.), its respective vehicle dimethylsulfoxide (10 &mgr;L/g i.p.), the large-conductance KCa channel blocker iberiotoxin (0.05 &mgr;g/g i.p.), or the protein kinase A (PKA) inhibitor H-89 (0.5 &mgr;g/g intraventricular). Infarct size was determined with triphenyltetrazolium chloride and area at risk with Evans blue. Mitochondrial and sarcolemmal localization of large-conductance KCa channels in cardiac myocytes was investigated with immunocytochemical staining of isolated cardiac myocytes. RESULTS:Desflurane significantly reduced infarct size compared with control animals (7.4% ± 0.8% vs 51.3% ± 6.1%; P < 0.05). Activation of large-conductance KCa channels by NS1619 (7.5% ± 1.8%; P < 0.05) mimicked and blockade of large-conductance KCa channels by iberiotoxin (49.1% ± 7.5%) abrogated desflurane-induced preconditioning. PKA blockade by H-89 abolished desflurane-induced (45.1% ± 4.0%) but not NS1619-induced (9.0% ± 2.4%, P < 0.05) preconditioning. Immunocytochemical staining revealed that large-conductance KCa channels were localized in the mitochondria but not in the sarcolemma of cardiac myocytes. CONCLUSION:These data suggest that desflurane-induced APC is mediated in part by activation of mitochondrial large-conductance KCa channels, and that activation of these channels by desflurane is mediated by PKA.

Isoflurane does not produce a second window of preconditioning against myocardial infarction in vivo.

The administration of a volatile anesthetic shortly before a prolonged ischemic episode exerts protective effects against myocardial infarction similar to those of ischemic preconditioning. A second window of preconditioning (SWOP) against myocardial infarction can also be elicited by brief episodes of ischemia when this occurs 24 h before prolonged coronary artery occlusion. Whether remote exposure to a volatile anesthetic also causes delayed myocardial protection is unknown. We tested the hypothesis that the administration of isoflurane 24 h before ischemia produces a SWOP against infarction. Barbiturate-anesthetized dogs (n = 25) were instrumented for measurement of hemodynamics, including aortic and left ventricular (LV) pressures and LV +dP/dtmax, and subjected to a 60-min left anterior descending coronary artery occlusion followed by 3 h of reperfusion. Myocardial infarct size and coronary collateral blood flow were assessed with triphenyltetrazolium chloride staining and radioactive microspheres, respectively. Two groups of dogs received 1.0 minimum alveolar anesthetic concentration isoflurane for 30 min or 6 h that was discontinued 30 min (acute) or 24 h (delayed) before ischemia and reperfusion, respectively. A control group of dogs did not receive isoflurane. Infarct size was 27% ± 3% of the LV area at risk in the absence of pretreatment with isoflurane. Acute, but not remote, administration of isoflurane reduced infarct size (12% ± 1% and 31% ± 3%, respectively). No differences in hemodynamics or transmural myocardial perfusion during or after occlusion were observed between groups. The results indicate that isoflurane does not produce a SWOP when administered 24 h before prolonged myocardial ischemia in vivo.

Comparison of isoflurane-, sevoflurane-, and desflurane-induced pre- and postconditioning against myocardial infarction in mice in vivo.

The murine in vivo model of acute myocardial infarction is increasingly used to investigate anesthetic-induced preconditioning (APC) and postconditioning (APOST). However, in mice the potency of different volatile anesthetics to reduce myocardial infarct size (IS) has never been investigated systematically nor in a head to head comparison with regard to ischemic preconditioning (IPC) and postconditioning (IPOST). Male C57BL/6 mice were subjected to 45 min of coronary artery occlusion (CAO) and 180 min of reperfusion. To induce APC, 1.0 MAC isoflurane (ISO), sevoflurane (SEVO) or desflurane (DES) was administered 30 min prior to CAO for 15 min. In an additional group, ISO was administered 45 min prior to CAO for 30 min. To induce APOST, 1.0 MAC ISO, SEVO or DES was administered for 18 min starting 3 min prior to the end of CAO. IPC was induced by 3 or 6 cycles of 5 min ischemia/reperfusion, 40 or 60 min prior to CAO, respectively. IPOST was induced by 3 cycles of 30 sec reperfusion/ischemia at the beginning of reperfusion. Area at risk (AAR) and IS were determined with Evans Blue and TTC staining, respectively. IS (IS/AAR) was 50 ± 4% (mean ± SEM) in the control group and was significantly (*P < 0.05) reduced by 3×5 IPC (26 ± 3%*), 6×5 IPC (26 ± 4%*), IPOST (20 ± 2%*), ISO APOST (19 ± 1%*), SEVO APOST (15 ± 1%*), DES APOST (14 ± 2%*) and SEVO APC (27 ± 6%*). ISO APC significantly reduced IS compared to control when administered 30 min (33 ± 4%*), but not when administered 15 min (48 ± 6%). DES APC significantly reduced IS compared to control and to SEVO APC (7 ± 1%*). Within the paradigm of preconditioning, the potency of volatile anesthetics to reduce myocardial infarct size in mice significantly increases from ISO over SEVO to DES, whereas within the paradigm of postconditioning the potency of these volatile anesthetics to reduce myocardial infarct size in mice is similar.

Impact of Ischemia and Reperfusion Times on Myocardial Infarct Size in Mice In Vivo

The murine in vivo model of acute myocardial infarction is increasingly used to study signal transduction pathways. However, methodological details of this model are rarely published, and durations of ischemia and reperfusion (REP) time vary considerably among different laboratories. In this study, we tested the hypothesis that infarct size (IS) is dependent on both duration of ischemia and REP time. Pentobarbital-anesthetized male C57BL/6 mice were intubated, mechanically ventilated, and instrumented for continuous monitoring of mean arterial blood pressure and heart rate. After left fourth thoracotomy, the left anterior descending coronary artery was ligated. Mice were randomly assigned to receive 30, 45, or 60 mins of coronary artery occlusion (CAO) and 120, 180, or 240 mins of REP, respectively. IS was determined with triphenyltetrazolium chloride and area at risk (AAR) with Evans blue, respectively. Arterial blood gas analysis and hemodynamics were not different among groups. Prolongation of CAO from 30 to 60 mins significantly (* P < 0.05) increased IS from 18% ± 5% to 69% ± 3%*, from 20% ± 2% to 69% ± 6%* and from 42% ± 10% to 75% ± 2%* after 120, 180, and 240 mins REP, respectively. Moreover, IS was increased from 18% ± 5% to 42% ± 10%* (30 mins CAO) and from 40% ± 3% to 72% ± 6%* (45 mins CAO) when REP time was prolonged from 120 to 240 mins. IS was not increased when REP was prolonged from 120 to 240 mins at 60 mins CAO (69% ± 3% vs. 75% ± 2%). In the present study, we describe important methodological aspects of the murine in vivo model of acute myocardial infarction and provide evidence that, in this model, IS depends both on duration of ischemia and on REP time.