Thorsten M. Smul

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.

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.

Desflurane-induced Postconditioning Is Mediated by β-Adrenergic SignalingRole of β1- and β2-Adrenergic Receptors, Protein Kinase A, and Calcium/Calmodulin-dependent Protein Kinase II

Background:Anesthetic preconditioning is mediated by β- adrenergic signaling. This study was designed to elucidate the role of β-adrenergic signaling in desflurane-induced postconditioning. Methods:Pentobarbital-anesthetized New Zealand White rabbits were subjected to 30 min of coronary artery occlusion followed by 3 h of reperfusion and were randomly assigned to receive vehicle (control), 1.0 minimum alveolar concentration of desflurane, esmolol (30 mg · kg−1 · h−1) for the initial 30 min of reperfusion or throughout reperfusion, the β2-adrenergic receptor blocker ICI 118,551 (0.2 mg/kg), the protein kinase A inhibitor H-89 (250 μg/kg), or the calcium/calmodulin-dependent protein kinase II inhibitor KN-93 (300 μg/kg) in the presence or absence of desflurane. Protein expression of protein kinase B, calcium/calmodulin-dependent protein kinase II, and phospholamban was measured by Western immunoblotting. Myocardial infarct size was assessed by triphenyltetrazolium staining. Results:Infarct size was 57 ± 5% in control. Desflurane postconditioning reduced infarct size to 36 ± 5%. Esmolol given during the initial 30 min of reperfusion had no effect on infarct size (54 ± 4%) but blocked desflurane-induced postconditioning (58 ± 5%), whereas esmolol administered throughout reperfusion reduced infarct size in the absence or presence of desflurane to 42 ± 6% and 41 ± 7%, respectively. ICI 118,551 and KN-93 did not affect infarct size (62 ± 4% and 62 ± 6%, respectively) but abolished desflurane-induced postconditioning (57 ± 5% and 64 ± 3%, respectively). H-89 decreased infarct size in the absence (36 ± 5%) or presence (33 ± 5%) of desflurane. Conclusions:Desflurane-induced postconditioning is mediated by β-adrenergic signaling. However, β-adrenergic signaling displays a differential role in cardioprotection during reperfusion.

Desflurane-induced Preconditioning against Myocardial Infarction Is Mediated by Nitric Oxide

Background: Volatile anesthetics induce myocardial preconditioning through a signal transduction pathway that is remarkably similar to that observed during ischemic preconditioning. Nitric oxide–dependent signaling plays an important role in anesthetic and ischemic preconditioning. Therefore, the authors tested the hypothesis that desflurane-induced preconditioning is mediated by nitric oxide. Methods: Barbiturate-anesthetized rabbits were instrumented for measurement of hemodynamics. All rabbits were subjected to 30-min coronary artery occlusion followed by 3 h of reperfusion. Myocardial infarct size was assessed with triphenyltetrazolium chloride staining. Myocardial nitric oxide synthase activity was assessed with a [3H]l-arginine–conversion assay. Rabbits were randomized to five separate experimental groups. They received 0.0 or 1.0 minimum alveolar concentration desflurane for 30 min, which was discontinued 30 min before ischemia in the absence or presence of the nitric oxide synthase inhibitor N&ohgr;-nitro-l-arginine (l-NA). l-NA was given either 20 min before or 10 min after desflurane administration, respectively. Data are mean ± SEM. Results: Infarct size was 56 ± 8% in control experiments. Desflurane significantly (P < 0.05) reduced infarct size to 35 ± 4%. Preconditioning by desflurane was totally blocked by administration of l-NA either during or after desflurane inhalation (58 ± 4 and 59 ± 9%, respectively). l-NA alone had no effect on infarct size (56 ± 7%). Nitric oxide synthase activity was significantly (P < 0.05) increased by desflurane. Conclusion: The results demonstrate that desflurane-induced preconditioning markedly reduced myocardial infarct size. This beneficial effect was blocked by the nitric oxide synthase inhibitor l-NA either during or after desflurane-administration. These data suggest that early desflurane-induced preconditioning is mediated by nitric oxide.

Desflurane-Induced Preconditioning Has a Threshold That Is Lowered by Repetitive Application and Is Mediated by β2-Adrenergic Receptors

OBJECTIVE An optimal administration protocol to induce a maximal effect of anesthetic preconditioning has not been evaluated to date. In this study, desflurane preconditioning was characterized with respect to its threshold, dose dependency, and continuous versus repetitive application. Furthermore, the role of beta(2)-adrenergic receptors in anesthetic preconditioning was tested. DESIGN A randomized controlled study. SETTING Laboratory study in a University hospital. SUBJECTS New Zealand white rabbits in vivo. INTERVENTIONS Systemic hemodynamics were continuously measured. Rabbits were subjected to 30 minutes of coronary artery occlusion and 3 hours of reperfusion. Animals received desflurane continuously for 30 minutes at 0.5, 1.0, or 1.5; desflurane for 90 minutes at 0.5 or 1.5 MAC; or repetitively for three 10-minute periods at 0.5, 1.0, or 1.5 MAC before coronary occlusion. The beta(2)-adrenergic receptor blocker ICI 118,551 (0.2 mg/kg) or saline placebo was given in the absence or presence of 1.0 MAC desflurane. Myocardial infarct size was measured with triphenyltetrazolium staining. MEASUREMENTS AND MAIN RESULTS Myocardial infarct size was 61% +/- 5% in control experiments. Desflurane, administered continuously at 0.5 MAC for 30 minutes (52% +/- 4%) or 90 minutes (56% +/- 8%) had no effect, whereas 0.5 MAC of desflurane given repetitively reduced infarct size to 36% +/- 7%. Desflurane administered continuously for 30 minutes at 1.0 or 1.5 MAC reduced infarct size to 35% +/- 5% and 39% +/- 4%, respectively. Repetitive application at 1.0 MAC (37% +/- 6%) or 1.5 MAC (29% +/- 4%) and continuous administration of 1.5 MAC for 90 minutes (32% +/- 6%) did not result in further infarct size reduction. ICI 118,551 did not affect infarct size (53% +/- 2%) but abolished desflurane preconditioning (51% +/- 5%). CONCLUSION beta(2)-Adrenergic receptors mediate desflurane-induced preconditioning. Desflurane-induced preconditioning has a threshold that can be lowered by repetitive administration.

The effectiveness and patient comfort of the novel streamlined pharynx airway liner (SLIPA®) compared with the conventional laryngeal mask airway in ophthalmic surgery

BACKGROUND: The novel, disposable streamlined pharynx airway liner (SLIPA®) has recently been introduced into clinical practice. It has no inflatable cuff, because the shape of the SLIPA® closely resembles the anatomy of the pharynx. METHODS: We compared the SLIPA® with the conventional laryngeal mask airway (LMA®) regarding handling, safety, sealing of the pharynx, and patient comfort in 124 adult patients (ASA I–III) undergoing ophthalmic surgery under general anesthesia. RESULTS: Insertion of the SLIPA® was straightforward in 88%, slightly difficult in 10%, and obviously difficult in 0% of cases. The SLIPA® could not be inserted in 2% of patients. In the LMA® group, insertion was straightforward in 90%, slightly difficult in 8%, obviously difficult in 2%, and a failure in 0% of patients. Maximum seal pressure was 24 ± 6 mm H2O with the SLIPA® and 24 ± 4 mm H2O with the LMA®. Gastric air insufflation was noticed in 19% of patients in the SLIPA® group and 3% in the LMA® group (P < 0.05). No regurgitation of gastric contents was observed. Removal of the airway was uneventful in all cases. Blood traces were noted on the surface of the device in 20% in the SLIPA® versus 11% (n.s.) in the LMA® group. Complaints of a sore throat were recorded in 2% vs. 14% in the SLIPA® and the LMA® group, respectively. CONCLUSION: The SLIPA® is a useful alternative to the conventional LMA® in patients undergoing minor surgery. However, it is associated with a higher incidence of gastric air insufflation, which may increase the risk of aspiration.

Activation of peroxisome-proliferator-activated receptors α and γ mediates remote ischemic preconditioning against myocardial infarction in vivo

Remote ischemic preconditioning (remote IPC) elicits a protective cardiac phenotype against myocardial ischemic injury. The remote stimulus has been hypothesized to act on major signaling pathways; however, its molecular targets remain largely undefined. We hypothesized that remote IPC exerts its effects by activating the peroxisome-proliferator-activated receptors (PPARs) α and γ, which have been previously implicated in cardioprotective signaling. Male New Zealand white rabbits (n = 78) were subjected to a 30-min coronary artery occlusion followed by three hours of reperfusion. Three cycles of remote IPC consisting of 10-min renal ischemia/reperfusion were performed. The animals either received the PPARα-antagonist GW6471 or the PPARγ-antagonist GW9662 alone or combined with remote IPC. Infarct size was determined gravimetrically. Tissue levels of 15d-prostaglandin J2 (15d-PGJ2), as well as the PPAR DNA binding were measured using specific assays. Reverse transcriptase polymerase chain reaction was used to analyze changes in endothelial nitric oxide synthase or inducible nitric oxide synthase (iNOS) mRNA expression in relative quantity (RQ). Data are mean ± SD. As a result, remote IPC significantly reduced the myocardial infarct size (42.2 ± 4.9%* versus 61 ± 1.9%), accompanied by an increased PPAR DNA-binding (189.6 ± 19.8RLU* versus 44.4 ± 9RLU), increased iNOS expression (3.5 ± 1RQ* versus 1RQ), as well as 15d-PGJ2 levels (179.7 ± 7.9 pg/mL* versus 127.9 ± 7.6 pg/mL). The protective response elicited by remote IPC, as well as the accompanying molecular changes were abolished by inhibiting PPARα (56.8 ± 4.7%; 61.1 ± 14.2RLU; and 1.91 ± 0.96RQ, respectively) or PPARγ (57.4 ± 3.3%; 52.7 ± 16.9RLU; and 1.54 ± 0.25RQ, respectively). (*Significantly different from control P < 0.05). In conclusion, the obtained results indicate that both PPARα and PPARγ play an essential role in remote IPC against myocardial infarction, impinging on the transcriptional control of iNOS expression.

Differential Role of Pim-1 Kinase in Anesthetic-induced and Ischemic Preconditioning against Myocardial Infarction

Background:Ischemic preconditioning (IPC) and anesthetic-induced preconditioning against myocardial infarction are mediated via protein kinase B. Pim-1 kinase acts downstream of protein kinase B and was recently shown to regulate cardiomyocyte survival. The authors tested the hypothesis that IPC and anesthetic-induced preconditioning are mediated by Pim-1 kinase. Methods:Pentobarbital-anesthetized male C57Black/6 mice were subjected to 45 min of coronary artery occlusion and 3 h of reperfusion. Animals received no intervention, Pim-1 kinase inhibitor II (10 &mgr;g/g intraperitoneally), its vehicle dimethyl sulfoxide (10 &mgr;l/g intraperitoneally), or 1.0 minimum alveolar concentration desflurane alone or in combination with Pim-1 kinase inhibitor II (10 &mgr;g/g intraperitoneally). IPC was induced by three cycles of 5 min ischemia–reperfusion each, and animals received IPC either alone or in combination with Pim-1 kinase inhibitor II (10 &mgr;g/g intraperitoneally). Infarct size was determined with triphenyltetrazolium chloride, and area at risk was determined with Evans blue (Sigma-Aldrich, Taufkirchen, Germany). Protein expression of Pim-1 kinase, Bad, phospho-BadSer112, and cytosolic content of cytochrome c were measured using Western immunoblotting. Results:Infarct size in the control group was 47 ± 2%. Pim-1 kinase inhibitor II (44 ± 2%) had no effect on infarct size. Desflurane (17 ± 3%) and IPC (19 ± 2%) significantly reduced infarct size compared with control (both P < 0.05 vs. control). Blockade of Pim-1 kinase completely abrogated desflurane-induced preconditioning (43 ± 3%), whereas IPC (35 ± 3%) was blocked partially. Desflurane tended to reduce cytosolic content of cytochrome c, which was abrogated by Pim-1 kinase inhibitor II. Conclusion:These data suggest that Pim-1 kinase mediates at least in part desflurane-induced preconditioning and IPC against myocardial infarction in mice.