Isoflurane-induced Preconditioning Research Articles

Involvement of Cyclophilin D and Calcium in Isoflurane-induced Preconditioning.

The mitochondrial permeability transition pore (PTP) has been established as an important mediator of ischemia-reperfusion-induced cell death. The matrix protein cyclophilin D (CypD) is the best known regulator of PTP opening. Therefore, the authors hypothesized that isoflurane, by inhibiting the respiratory chain complex I, another regulator of PTP, might reinforce the myocardial protection afforded by CypD inhibition. Adult mouse or isolated cardiomyocytes from wild-type or CypD knockout (CypD-KO) mice were subjected to ischemia or hypoxia followed by reperfusion or reoxygenation. Infarct size was assessed in vivo. Mitochondrial membrane potential and PTP opening were assessed using tetramethylrhodamine methyl ester perchlorate and calcein-cobalt fluorescence, respectively. Fluo-4 AM and rhod-2 AM staining allowed the measurement, by confocal microscopy, of Ca transient and Ca transfer from sarcoplasmic reticulum (SR) to mitochondria after caffeine stimulation. Both inhibition of CypD and isoflurane significantly reduced infarct size (-50 and -37%, respectively) and delayed PTP opening (+63% each). Their combination had no additive effect (n = 6/group). CypD-KO mice displayed endogenous protection against ischemia-reperfusion. Isoflurane depolarized the mitochondrial membrane (-28%, n = 5), decreased oxidative phosphorylation (-59%, n = 5), and blunted the caffeine-induced Ca transfer from SR to mitochondria (-22%, n = 7) in the cardiomyocytes of wild-type mice. Importantly, this transfer was spontaneously decreased in the cardiomyocytes of CypD-KO mice (-25%, n = 4 to 5). The results suggest that the partial inhibitory effect of isoflurane on respiratory complex I is insufficient to afford a synergy to CypD-induced protection. Isoflurane attenuates the Ca transfer from SR to mitochondria, which is also the prominent role of CypD, and finally prevents PTP opening.

The involvement of protein kinase C-ε in isoflurane induced preconditioning of human embryonic stem cell - derived Nkx2.5+ cardiac progenitor cells

BackgroundAnesthetic preconditioning can improve survival of cardiac progenitor cells exposed to oxidative stress. We investigated the role of protein kinase C and isoform protein kinase C-ε in isoflurane-induced preconditioning of cardiac progenitor cells exposed to oxidative stress.MethodsCardiac progenitor cells were obtained from undifferentiated human embryonic stem cells. Immunostaining with anti-Nkx2.5 was used to confirm the differentiated cardiac progenitor cells. Oxidative stress was induced by H2O2 and FeSO4. For anesthetic preconditioning, cardiac progenitor cells were exposed to 0.25, 0.5, and 1.0 mM of isoflurane. PMA and chelerythrine were used for protein kinase C activation and inhibition, while εψRACK and εV1-2 were used for protein kinase C -ε activation and inhibition, respectively.ResultsIsoflurane-preconditioning decreased the death rate of Cardiac progenitor cells exposed to oxidative stress (death rates isoflurane 0.5 mM 12.7 ± 9.3 %, 1.0 mM 12.0 ± 7.7 % vs. control 31.4 ± 10.2 %). Inhibitors of both protein kinase C and protein kinase C -ε abolished the preconditioning effect of isoflurane 0.5 mM (death rates 27.6 ± 13.5 % and 25.9 ± 8.7 % respectively), and activators of both protein kinase C and protein kinase C - ε had protective effects from oxidative stress (death rates 16.0 ± 3.2 % and 10.6 ± 3.8 % respectively).ConclusionsBoth PKC and PKC-ε are involved in isoflurane-induced preconditioning of human embryonic stem cells -derived Nkx2.5+ Cardiac progenitor cells under oxidative stress.

The Role of Emulsified Isoflurane in Multi-Organ Protection

Aim of review: This review describes the multi-organ protective effects of emulsified isoflurane.Method: This review examines the recent experimental researches involving emulsified isoflurane-induced preconditioning and postconditioning in multiple organs.Recent findings: Emulsified isoflurane is a new formulation of isoflurane in lipid emulsion. It enables an intravenous, intraperitoneal injection or oral (rather than the traditional inhalation) route of administration for this anesthetic. Pharmacological preconditioning and postconditioning have been demonstrated to be the protective strategies in clinical settings for the high risk patients with various organ dysfunctions. In recent years, there has been increased interest in exploring the protective effect of emulsified isoflurane in multiple organs including heart, brain, kidney, liver and lung, providing potential approaches translating these convincing experimental conditioning effects to clinical practice.Summary: Emulsified isoflurane could exert strong organ-protective effects. With an understanding of its potential mechanisms, therapeutic approaches may be applied to the future organ-protective properties, transferring convincing experimental evidences into clinical therapies. Citation: Zhao-Yang Hu, Jin Liu. The role of emulsified isoflurane in multiorgan protection. J Anesth Perioper Med 2015; 2: 29-35. doi: 10.24015/JAPM.2015.0005This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Isoflurane decreases death of human embryonic stem cell-derived, transcriptional marker Nkx2.5+ cardiac progenitor cells

Cardiac progenitor cells (CPCs) derived from human embryonic stem cells (hESCs) can multiply and generate cardiomyocytes, offering their tremendous potential for cardiac regenerative therapy. However, poor survival under stressful conditions is a major hurdle in the regeneration. We investigated whether isoflurane-induced preconditioning can increase hESC-derived CPC survival under oxidative stress. Undifferentiated hESCs were cultured in suspension with 20% FBS (fetal bovine serum) and 20 ng/ml of BMP-4 (bone morphogenetic protein-4) to form embryoid bodies and grown onto Matrigel-coated plates for 2-3 weeks. To characterise the differentiated CPCs, immunostaining for Nkx2.5 (nonspecific transcriptional marker) and Isl-1 was performed. hESC-derived CPCs were exposed to oxidative stress induced by H(2) O(2) and FeSO(4) . For anaesthetic preconditioning, CPCs were exposed to isoflurane (0.25, 0.5, 1.0 mM). CPC survival was determined by trypan blue exclusion. A mitoK(ATP) channels inhibitor, 5-hydroxydecanoic acid (200 μM) and an opener, diazoxide (100 μM), were used to investigate the involvement of mitoK(ATP) channels. hESC-derived CPCs stained with Nkx2.5 were 95 ± 3% of total cell number. Isoflurane (0.5 and 1.0 mM)-preconditioned CPCs showed a significantly lower death rate compared with control (0.5 mM: 30.6 ± 10.7% and 1.0 mM: 28.5 ± 6.2% vs. control: 43.2 ± 9.9%). Inhibition of mitoK(ATP) channels with 5-HD completely abolished the protective effects of isoflurane. Diazoxide significantly decreased CPC death (29.5 ± 12.4%). However, when diazoxide was applied to CPC preconditioned with isoflurane, CPC death did not decrease further (28.7 ± 10.9%). Isoflurane increased hESC-derived Nkx2.5(+) CPC survival under oxidative stress, and mitoK(ATP) channels may be involved in the protective effect.

Letter by Lotz et al Regarding Article, “Reactive Oxygen Species–Induced Stimulation of 5′ AMP-Activated Protein Kinase Mediates Sevoflurane-Induced Cardioprotection”

To the Editor: AMP kinase (AMPK) activation represents a unique molecular event in cardioprotection against ischemia/reperfusion injury, potentially linking myocardial metabolism to a number of cardioprotective signaling pathways such as nitric oxide and reactive oxygen species signaling. In a recently published article in Circulation , Lamberts et al1 demonstrated that AMPK activation contributed to the signaling cascade in the powerful cardioprotection conferred by sevoflurane-induced preconditioning. This finding is intriguing because AMPK activation has the potential to overcome the loss of anesthetic-induced preconditioning during diabetes mellitus.2 The published report provides important information and useful insights, particularly regarding the triangle of AMPK signaling, reactive oxygen species generation, …

Attenuation of Isoflurane-Induced Preconditioning and Reactive Oxygen Species Production in the Senescent Rat Heart

Although attenuation of anesthetic preconditioning in aged ex vivo heart models has been studied extensively, there are no comparable in vivo studies. To extend previous work and to address a possible mechanism underlying age-related differences, we investigated isoflurane-induced preconditioning and reactive oxygen species (ROS) production in the aged rat heart in vivo. Male Fisher 344 rats were assigned from their respective age groups (young, 3-5 mo; old, 20-24 mo) to either receive 30 min of 1.0 minimum alveolar concentration isoflurane or to a control group. Rats were subjected to coronary artery occlusion for 30 min followed by 2 h of reperfusion. A fluorescent probe for superoxide anion production (dihydroethidium, 1 mg) was administered in the absence of the isoflurane or just before isoflurane exposure in four additional groups. Myocardial infarct size and superoxide anion production were assessed using triphenyltetrazolium staining and epifluorescence microscopy, respectively. Isoflurane decreased myocardial infarct size of young rats (26.7% +/- 3.0%) compared with young controls (50.9% +/- 1.9%; P < 0.001), whereas isoflurane did not significantly affect myocardial infarct size of old rats (39.1% +/- 0.9%) compared with old controls (46.5% +/- 2.4%; P > 0.05). Isoflurane increased ROS levels in young rats (430.5 +/- 95.9 arbitrary units [AU]) compared with young controls (162.7 +/- 25.5 AU; P < 0.01). In contrast, no significant changes in ROS levels were observed in old animals (316.4 +/- 56.3 AU isoflurane versus 233.8 +/- 59.2 AU control). Reduction in the cardioprotective effects of isoflurane and attenuation of isoflurane-stimulated ROS production were observed in the senescent myocardium in vivo.

ANESTHETIC PRECONDITIONING CONFERS ACUTE CARDIOPROTECTION VIA UP-REGULATION OF MANGANESE SUPEROXIDE DISMUTASE AND PRESERVATION OF MITOCHONDRIAL RESPIRATORY ENZYME ACTIVITY

The cellular and molecular mechanisms that underlie cardioprotection against I/R by anesthetic-induced preconditioning (APC) require further elucidation. Using isoflurane as a representative anesthetic, we evaluated the hypothesis that APC induces myocardial protection against I/R by attenuation of excessive reactive oxygen species and restoration of mitochondrial bioenergetics through postischemic up-regulation of manganese superoxide dismutase (MnSOD) expression and preservation of respiratory enzyme activity. Pentobarbital anesthetized open-chest Sprague-Dawley rats were subject to 30-min left coronary artery occlusion, followed by 120-min reperfusion. Before ischemia, rats were randomly assigned to receive 0.9% saline, two cycles of brief coronary artery occlusion and reperfusion, or a 30-min exposure to 1.0 minimum alveolar concentration isoflurane in the absence or presence of a specific mitochondrial adenosine triphosphate-sensitive potassium (KATP) channel blocker, 5-hydroxydecanoate; a membrane-permeable superoxide scavenger, 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl; or a NOS inhibitor, N(G)-nitro-L-arginine methyl ester. Isoflurane exposure induced an initial increase in myocardial superoxide (O2-), but not NO level. It also significantly decreased infarct size and restored mitochondrial respiratory enzyme activity or ATP production in I/R rat hearts, along with suppression of the O2- surge at reperfusion and increase in MnSOD expression or enzyme activity. These protective effects were abrogated by 5-hydroxydecanoate or 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl, but not by N(G)-nitro-L-arginine methyl ester pretreatment. These results suggest that opening of mitochondrial KATP channel, followed by O2- signaling, induces postischemic augmentation of MnSOD and preservation of mitochondrial respiratory enzyme activities, leading to attenuated cardiac O2- surge and restored ATP production during reperfusion, and underlie APC-induced cardioprotection.

Isoflurane Activates Human Cardiac Mitochondrial Adenosine Triphosphate-Sensitive K+ Channels Reconstituted in Lipid Bilayers

Activation of the mitochondrial adenosine triphosphate (ATP)-sensitive K+ channel (mitoK(ATP)) has been proposed as a critical step in myocardial protection by isoflurane-induced preconditioning in humans and animals. Recent evidence suggests that reactive oxygen species (ROS) may mediate isoflurane-mediated myocardial protection. In this study, we examined the direct effect of isoflurane and ROS on human cardiac mitoK(ATP) channels reconstituted into the lipid bilayers. Inner mitochondrial membranes were isolated from explanted human left ventricles not suitable for heart transplantation and fused into lipid bilayers in symmetrical potassium glutamate solution (150 mM). ATP-sensitive K+ currents were recorded before and after exposure to isoflurane and H2O2 under voltage clamp. The human mitoK(ATP) was identified by its sensitivity to inhibition by ATP and 5-hydroxydecanoate. Addition of isoflurane (0.8 mM) increased the open probability of the mitoK(ATP) channels, either in the presence or absence of ATP inhibition (0.5 mM). The isoflurane-mediated increase in K+ currents was completely inhibited by 5-hydroxydecanoate. Similarly, H2O2 (200 microM) was able to activate the mitoK(ATP) previously inhibited by ATP. These data confirm that isoflurane, as well as ROS, directly activates reconstituted human cardiac mitoK(ATP) channel in vitro, without apparent involvement of cytosolic protein kinases, as commonly proposed. Activation of the mitoK(ATP) channel may contribute to the myocardial protective effect of isoflurane in the human heart.

Effectiveness of Isoflurane in Inducing Delayed Preconditioning against Myocardial Infarction In Vivo

We thank Xia et al. for their gracious comments about our recent work characterizing the role of endothelial nitric oxide synthase in delayed preconditioning against myocardial infarction produced by isoflurane.1The authors mention that isoflurane-induced preconditioning requires the generation of reactive oxygen species. In fact, we have previously demonstrated that isoflurane produces small quantities of reactive oxygen species independent of ischemia and reperfusion as detected using dihydroethidium staining and confocal laser microscopy.2These data provided direct evidence that exposure to isoflurane produces a small burst of reactive oxygen species via opening of mitochondrial adenosine triphosphate–sensitive potassium channels that triggers preconditioning.3Xia et al. suggest that the duration of coronary artery occlusion may contribute to the relative efficacy of volatile anesthetics during acute or delayed preconditioning. Previous data indicated that isoflurane did not produce delayed preconditioning in dogs exposed to a 60-min left anterior descending coronary artery occlusion,4in contrast to the findings in rabbits when a 30-min coronary occlusion was used.1Although these results may have been related to the duration of coronary occlusion, it is more likely the findings were related to differences in systemic hemodynamics and coronary collateral blood flow between species. Coronary artery occlusions of 30 or 60 min in duration typically produce myocardial infarct sizes of approximately 40 and 33% in rabbits and dogs, respectively. Heart rates in barbiturate-anesthetized rabbits and dogs are approximately 240 and 130 beats/min, respectively. As a result, myocardial oxygen consumption before and during coronary occlusion is substantially higher in rabbits as compared with dogs. In addition to this more pronounced ischemic burden, rabbits have little if any coronary collateral blood flow.5In contrast, the canine model of ischemia and reperfusion used in our previous investigation4is complicated by variable degrees of coronary collateral perfusion, which must be considered when interpreting the results. We believe that it would also be premature to extrapolate our findings in barbiturate-anesthetized, acutely instrumented rabbits1to patients with coronary artery disease undergoing cardiac surgery using cardiopulmonary bypass, as suggested by Xia et al. In contrast to the arguments of Xia et al. , there is little experimental evidence supporting the hypothesis that propofol produces substantial cardioprotective effects against ischemia–reperfusion injury in vivo . However, a large body of experimental evidence supports the contention that volatile anesthetics exert important protective effects against reversible and irreversible ischemic injury.6To date, several clinical trials have provided preliminary data to corroborate these experimental findings. In particular, De Hert et al. 7demonstrated that sevoflurane but not propofol preserved myocardial function and attenuated increases in troponin I release in patients undergoing coronary artery bypass graft surgery. These data suggested that sevoflurane but not propofol produces myocardial protection in humans at risk for ischemic injury.7Further large-scale, multicenter clinical trials should be performed to define the utility of volatile anesthetics as cardioprotective agents in humans.*Medical College of Wisconsin and the Clement J. Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin. pspagel@mcw.edu

Effects of Nitrous Oxide on the Rat Heart In Vivo

Background. For nitrous oxide, a preconditioning effect on the heart has yet not been investigated. This is important because nitrous oxide is commonly used in combination with volatile anesthetics, which are known to precondition the heart. The authors aimed to clarify (1) whether nitrous oxide preconditions the heart, (2) how it affects protein kinase C (PKC) and tyrosine kinases (such as Src) as central mediators of preconditioning, and (3) whether isoflurane-induced preconditioning is influenced by nitrous oxide.Methods. For infarct size measurements, anesthetized rats were subjected to 25 min of coronary artery occlusion followed by 120 min of reperfusion. Rats received nitrous oxide (60%), isoflurane (1.4%) or isoflurane-nitrous oxide (1.4%/60%) during three 5-min periods before index ischemia (each group, n = 7). Control animals remained untreated for 45 min. Additional hearts (control, 60% nitrous oxide alone%, and isoflurane-nitrous oxide [0.6%/60%, in equianesthetic doses]) were excised for Western blot of PKC-epsilon and Src kinase (each group, n = 4).Results. Nitrous oxide had no effect on infarct size (59.1 +/- 15.2% of the area at risk vs. 51.1 +/- 10.9% in controls). Isoflurane (1.4%) and isoflurane-nitrous oxide (1.4%/60%) reduced infarct size to 30.9 +/- 10.6 and 28.7 +/- 11.8% (both P &lt; 0.01). Nitrous oxide (60%) had no effect on phosphorylation (2.3 +/- 1.8 vs. 2.5 +/- 1.7 in controls, average light intensity, arbitrary units) and translocation (7.0 +/- 4.3 vs. 7.4 +/- 5.2 in controls) of PKC-epsilon. Src kinase phosphorylation was not influenced by nitrous oxide (4.6 +/- 3.9 vs. 5.0 +/- 3.8; 3.2 +/- 2.2 vs. 3.5 +/- 3.0). Isoflurane-nitrous oxide (0.6%/60%, in equianesthetic doses) induced PKC-epsilon phosphorylation (5.4 +/- 1.9 vs. 2.8 +/- 1.5; P &lt; 0.001) and translocation to membrane regions (13.8 +/- 13.0 vs. 6.7 +/- 2.0 in controls; P &lt; 0.05).Conclusions. Nitrous oxide is the first inhalational anesthetic without preconditioning effect on the heart. However, isoflurane-induced preconditioning and PKC-epsilon activation are not influenced by nitrous oxide.

Protein kinase C translocation and Src protein tyrosine kinase activation mediate isoflurane-induced preconditioning in vivo: potential downstream targets of mitochondrial adenosine triphosphate-sensitive potassium channels and reactive oxygen species.

The authors tested the hypotheses that protein kinase C (PKC)-specific isoform translocation and Src protein tyrosine kinase (PTK) activation play important roles in isoflurane-induced preconditioning in vivo. Rats (n = 125) instrumented for measurement of hemodynamics underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion and received 0.9% saline (control); PKC inhibitors chelerythrine (5 mg/kg), rottlerin (0.3 mg/kg), or PKC-epsilonV1-2 peptide (1 mg/kg); PTK inhibitors lavendustin A (1 mg/kg) or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1; 1 mg/kg); mitochondrial adenosine triphosphate-sensitive potassium channel antagonist 5-hydroxydecanote (10 mg/kg); or reactive oxygen species scavenger N-acetylcysteine (150 mg/kg) in the absence and presence of a 30-min exposure to isoflurane (1.0 minimum alveolar concentration) in separate groups. Isoflurane was discontinued 15 min before coronary occlusion (memory period). Infarct size was determined using triphenyltetrazolium staining. Immunohistochemistry and confocal microscopic imaging were performed to examine PKC translocation in separate groups of rats. Isoflurane significantly (P < 0.05) reduced infarct size (40 +/- 3% [n = 13]) as compared with control experiments (58 +/- 2% [n = 12]). Chelerythrine, rottlerin, PKC-epsilonV1-2 peptide, lavendustin A, PP1, 5-hydroxydecanote, and N-acetylcysteine abolished the anti-ischemic actions of isoflurane (58 +/- 2% [n = 8], 50 +/- 3% [n = 9], 53 +/- 2% [n = 9], 59 +/- 3% [n = 6], 57 +/- 3% [n = 7], 60 +/- 3% [n = 7], and 53 +/- 3% [n = 6], respectively). Isoflurane stimulated translocation of the delta and epsilon isoforms of PKC to sarcolemmal and mitochondrial membranes, respectively. Protein kinase C-delta, PKC-epsilon, and Src PTK mediate isoflurane-induced preconditioning in the intact rat heart. Opening of mitochondrial adenosine triphosphate-sensitive potassium channels and generation of reactive oxygen species are upstream events of PKC activation in this signal transduction process.

Intracellular mechanism of mitochondrial adenosine triphosphate-sensitive potassium channel activation with isoflurane.

The precise mechanism of isoflurane and mitochondrial adenosine triphosphate-sensitive potassium channel (mitoK(ATP)) interaction is still unclear, although the mitoK(ATP) is involved in isoflurane-induced preconditioning. We examined the role of various intracellular signaling systems in mitoK(ATP) activation with isoflurane. Mitochondrial flavoprotein fluorescence (MFF) was measured to quantify mitoK(ATP) activity in guinea pig cardiomyocytes. To confirm isoflurane-induced MFF, cells were exposed to Tyrode's solution containing either isoflurane (1.0 +/- 0.1 mM) or diazoxide and then both drugs together (n = 10 each). In other studies, the following drugs were each added during isoflurane administration: adenosine or the adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline (SPT); the protein kinase C (PKC) activators phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu); the PKC inhibitors polymyxin B and staurosporine; the tyrosine kinase inhibitor lavendustin A; or the mitogen-activated protein kinase inhibitor SB203580 (n = 10 each). Isoflurane potentiated MFF induced by diazoxide (100 micro M), and diazoxide also increased isoflurane-induced MFF. PMA (0.2 micro M), PDBu (1 micro M), and adenosine (100 micro M) induced MFF. However, SPT (100 micro M), polymyxin B (50 micro M), staurosporine (200 nM), lavendustin A (0.5 micro M), and SB203580 (10 micro M) all failed to inhibit the effect of isoflurane. Our results show that isoflurane, adenosine, and PKC activate mitoK(ATP). However, our data do not support an action of isoflurane through pathways involving adenosine, PKC, tyrosine kinase, or mitogen-activated protein kinase. These results suggest that isoflurane may directly activate mitoK(ATP). Our results show that isoflurane activates mitochondrial adenosine triphosphate-sensitive potassium (mitoK(ATP)) channels, but not through pathways involving adenosine, protein kinase C, tyrosine kinase, or p38 mitogen-activated protein kinase. Isoflurane may directly activate mitoK(ATP) channels.

N-acetylcysteine restores isoflurane-induced preconditioning against myocardial infarction during hyperglycemia.

Hyperglycemia generates reactive oxygen species and prevents isoflurane-induced preconditioning. The authors tested the hypothesis that scavenging reactive oxygen species with N-acetylcysteine will restore protection against myocardial infarction produced by isoflurane in vivo. Barbiturate-anesthetized dogs (n = 45) were instrumented for measurement of systemic hemodynamics. Myocardial infarct size and coronary collateral blood flow were measured with triphenyltetrazolium 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 0.9% saline or 15% dextrose in water to increase blood glucose concentrations to 600 mg/dl (hyperglycemia) in the absence or presence of isoflurane (1.0 minimum alveolar concentration) with or without pretreatment with N-acetylcysteine (150 mg/kg i.v.) in six experimental groups. Isoflurane was discontinued, and blood glucose concentrations were allowed to return to baseline values before left anterior descending coronary artery occlusion. Myocardial infarct size was 27 +/- 2% (n = 8) of the left ventricular area at risk in control experiments. Isoflurane significantly (P < 0.05) decreased infarct size (13 +/- 2%; n = 7). Hyperglycemia alone did not alter infarct size (29 +/- 3%; n = 7) but abolished the protective effect of isoflurane (25 +/- 2%; n = 8). N-Acetylcysteine alone did not affect infarct size (28 +/- 2%; n = 8) but restored isoflurane-induced cardioprotection during hyperglycemia (10 +/- 1%; n = 7). Acute hyperglycemia abolishes reductions in myocardial infarct size produced by isoflurane, but N-acetylcysteine restores these beneficial effects. The results suggest that excessive quantities of reactive oxygen species generated during hyperglycemia impair isoflurane-induced preconditioning in dogs.

Mitochondrial adenosine triphosphate-regulated potassium channel opening acts as a trigger for isoflurane-induced preconditioning by generating reactive oxygen species.

Whether the opening of mitochondrial adenosine triphosphate-regulated potassium (K(ATP)) channels is a trigger or an end effector of anesthetic-induced preconditioning is unknown. We tested the hypothesis that the opening of mitochondrial K(ATP) channels triggers isoflurane-induced preconditioning by generating reactive oxygen species (ROS) in vivo. Pentobarbital-anesthetized rabbits were subjected to a 30-min coronary artery occlusion followed by 3 h reperfusion. Rabbits were randomly assigned to receive a vehicle (0.9% saline) or the selective mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (5-HD) alone 10 min before or immediately after a 30-min exposure to 1.0 minimum alveolar concentration (MAC) isoflurane. In another series of experiments, the fluorescent probe dihydroethidium was used to assess superoxide anion production during administration of 5-HD or the ROS scavengers N-acetylcysteine or N-2-mercaptopropionyl glycine (2-MPG) in the presence or absence of 1.0 MAC isoflurane. Myocardial infarct size and superoxide anion production were measured using triphenyltetrazolium staining and confocal fluorescence microscopy, respectively. Isoflurane (P < 0.05) decreased infarct size to 19 +/- 3% (mean +/- SEM) of the left ventricular area at risk as compared to the control (38 +/- 4%). 5-HD administered before but not after isoflurane abolished this beneficial effect (37 +/- 4% as compared to 24 +/- 3%). 5-HD alone had no effect on infarct size (42 +/- 3%). Isoflurane increased fluorescence intensity. Pretreatment with N-acetylcysteine, 2-MPG, or 5-HD before isoflurane abolished increases in fluorescence, but administration of 5-HD after isoflurane only partially attenuated increases in fluorescence produced by the volatile anesthetic agent. The results indicate that mitochondrial K(ATP) channel opening acts as a trigger for isoflurane-induced preconditioning by generating ROS in vivo.

Mechanism of Isoflurane-Induced Preconditioning in Rabbits: A Role for Reactive Oxygen Species

Mechanism of Isoflurane-Induced Preconditioning in Rabbits: A Role for Reactive Oxygen Species

Isoflurane-induced preconditioning is attenuated by diabetes.

Volatile anesthetics stimulate, but hyperglycemia attenuates, the activity of mitochondrial ATP-regulated K(+) channels. We tested the hypothesis that diabetes mellitus interferes with isoflurane-induced preconditioning. Acutely instrumented, barbiturate-anesthetized dogs were randomly assigned to receive 0, 0.32, or 0.64% end-tidal concentrations of isoflurane in the absence or presence of diabetes (3 wk after administration of alloxan and streptozotocin) in six experimental groups. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3 h of reperfusion. Myocardial infarct size (triphenyltetrazolium staining) was 29 +/- 3% (n = 8) of the left ventricular area at risk in control experiments. Isoflurane reduced infarct size (15 +/- 2 and 13 +/- 1% during 0.32 and 0.64% concentrations; n = 8 and 7 dogs, respectively). Diabetes alone did not alter infarct size (30 +/- 3%; n = 8) but blocked the protective effects of 0.32% (27 +/- 2%; n = 7) and not 0.64% isoflurane (18 +/- 3%; n = 7). Infarct size was directly related to blood glucose concentrations in diabetic dogs, but this relationship was abolished by higher concentrations of isoflurane. The results indicate that blood glucose and end-tidal isoflurane concentrations are important determinants of infarct size during anesthetic-induced preconditioning.

Isoflurane preconditions myocardium against infarction via release of free radicals.

Isoflurane exerts cardioprotective effects that mimic the ischemic preconditioning phenomenon. Generation of free radicals is implicated in ischemic preconditioning. The authors investigated whether isoflurane-induced preconditioning may involve release of free radicals. Sixty-one alpha-chloralose-anesthetized rabbits were instrumented for measurement of left ventricular (LV) pressure (tip-manometer), cardiac output (ultrasonic flowprobe), and myocardial infarct size (triphenyltetrazolium staining). All rabbits were subjected to 30 min of occlusion of a major coronary artery and 2 h of subsequent reperfusion. Rabbits of all six groups underwent a treatment period consisting of either no intervention for 35 min (control group, n = 11) or 15 min of isoflurane inhalation (1 minimum alveolar concentration end-tidal concentration) followed by a 10-min washout period (isoflurane group, n = 12). Four additional groups received the radical scavenger N-(2-mercaptoproprionyl)glycine (MPG; 1 mg. kg-1.min-1) or Mn(III)tetrakis(4-benzoic acid)porphyrine chloride (MnTBAP; 100 microg.kg-1.min-1) during the treatment period with (isoflurane + MPG; n = 11; isoflurane + MnTBAP, n = 9) or without isoflurane inhalation (MPG, n = 11; MnTBAP, n = 7). Hemodynamic baseline values were not significantly different between groups (LV pressure, 97 +/- 17 mmHg [mean +/- SD]; cardiac output, 228 +/- 61 ml/min). During coronary artery occlusion, LV pressure was reduced to 91 +/- 17% of baseline and cardiac output to 94 +/- 21%. After 2 h of reperfusion, recovery of LV pressure and cardiac output was not significantly different between groups (LV pressure, 83 +/- 20%; cardiac output, 86 +/- 23% of baseline). Infarct size was reduced from 49 +/- 17% of the area at risk in controls to 29 +/- 19% in the isoflurane group (P = 0.04). MPG and MnTBAP themselves had no effect on infarct size (MPG, 50 +/- 14%; MnTBAP, 56 +/- 15%), but both abolished the preconditioning effect of isoflurane (isoflurane + MPG, 50 +/- 24%, P = 0.02; isoflurane + MnTBAP, 55 +/- 10%, P = 0.001). Isoflurane-induced preconditioning depends on the release of free radicals.

Hyperglycemia prevents isoflurane-induced preconditioning against myocardial infarction.

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. 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. 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%). Acute hyperglycemia attenuates reductions in myocardial infarct size produced by isoflurane in dogs.

Prevention of isoflurane-induced preconditioning by 5-hydroxydecanoate and gadolinium: possible involvement of mitochondrial adenosine triphosphate-sensitive potassium and stretch-activated channels.

Both mitochondrial adenosine triphosphate-sensitive potassium (MKATP) channels (selectively blocked by 5-hydroxydecanoate) and stretch-activated channels (blocked by gadolinium) have been involved in the mechanism of ischemic preconditioning. Isoflurane can reproduce the protection afforded by ischemic preconditioning. We sought to determine whether isoflurane-induced preconditioning may involve MKATP and stretch-activated channels. Anesthetized open-chest rabbits underwent 30 min of coronary occlusion followed by 3 h of reperfusion. Before this, rabbits were randomized into one of six groups and underwent a treatment period consisting of either no intervention for 40 min (control group; n = 9) or 15 min of isoflurane inhalation (1.1% end tidal) followed by a 15-min washout period (isoflurane group; n = 9). The two groups received an intravenous bolus dose of either 5-hydroxydecanoate (5 mg/kg) or gadolinium (40 micromol/kg) before coronary occlusion and reperfusion (5-hydroxydecanoate, n = 9; gadolinium, n = 7). Two additional groups received 5-hydroxydecanoate or gadolinium before isoflurane exposure (isoflurane-5-hydroxydecanoate, n = 10; isoflurane-gadolinium, n = 8). Area at risk and infarct size were assessed by blue dye injection and tetrazolium chloride staining. Area at risk was comparable among the six groups (29 +/- 7, 30 +/- 5, 27 +/- 6, 35 +/- 7, 31 +/- 7, and 27 +/- 4% of the left ventricle in the control, isoflurane, isoflurane-5-hydroxydecanoate, 5-hydroxydecanoate, isoflurane-gadolinium, and gadolinium groups, respectively). Infarct size averaged 60 +/- 20% (SD) in untreated controls versus 54 +/- 27 and 65 +/- 15% of the risk zone in 5-hydroxydecanoate- and gadolinium-treated controls (P = nonsignificant). In contrast, infarct size in the isoflurane group was significantly reduced to 26 +/- 11% of the risk zone (P < 0.05 vs.control). Both 5-hydroxydecanoate and gadolinium prevented this attenuation: infarct size averaged 68 +/- 23 and 56 +/- 21% of risk zone in the isoflurane-5-hydroxydecanoate and isoflurane-gadolinium groups, respectively (P = nonsignificant vs.control). 5-Hydroxydecanoate and gadolinium inhibited pharmacologic preconditioning by isoflurane. This result suggests that MKATP channels and mechanogated channels are probably involved in this protective mechanism.

Colchicine inhibits isoflurane-induced preconditioning.

When administered before prolonged myocardial ischemia and reperfusion, isoflurane exerts potent cardioprotective effects similar to those inferred by ischemic preconditioning. To determine whether an intact cytoskeleton is critically important in isoflurane-induced preconditioning, the authors used a rabbit model in which isoflurane-induced myocardial preconditioning decreases myocardial infarct size (IS) substantially. In this model, the authors tested whether the microtubule depolymerizing agent, colchicine, would inhibit isoflurane-induced myocardial preconditioning. Myocardial IS was measured in four groups of propofol-anesthetized rabbits, each subjected to 30 min of anterolateral coronary occlusion followed by 3 h of reperfusion. Groups differed only in the pretreatments given, and only the control group received no pretreatment. An isoflurane-preconditioned group was pretreated with 15 min of end-tidal isoflurane, 1.1%, and then 15 min of washout. An isoflurane-plus-colchicine group was administered 2 mg/kg colchicine intravenously before isoflurane pretreatment. A colchicine-control group was administered 2 mg/kg colchicine but no isoflurane pretreatment. Myocardial IS and area at risk (AR) were defined by staining. Data were analyzed by analysis of variance or covariance. Infarct size, expressed as a percentage of AR (IS:AR) was 33.6%+/-8.8% (SD) in the control group. Isoflurane preexposure reduced myocardial IS:AR significantly, to 11.8%+/-9.1%. Colchicine pretreatment eliminated the preconditioning-like effect of isoflurane (IS:AR = 32.6%+/-8.7%). Colchicine alone did not alter IS (IS:AR = 27.6%+/-7.1%; P = not significant). Colchicine abolished the preconditioning effect of isoflurane but did not increase IS when administered alone. An intact microtubular cytoskeleton is critically important in the process of volatile anesthetic-induced preconditioning.