Jan Stumpner

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.

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 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.

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.

Aromatase inhibition attenuates desflurane-induced preconditioning against acute myocardial infarction in male mouse heart in vivo.

The volatile anesthetic desflurane (DES) effectively reduces cardiac infarct size following experimental ischemia/reperfusion injury in the mouse heart. We hypothesized that endogenous estrogens play a role as mediators of desflurane-induced preconditioning against myocardial infarction. In this study, we tested the hypothesis that desflurane effects local estrogen synthesis by modulating enzyme aromatase expression and activity in the mouse heart. Aromatase metabolizes testosterone to 17β- estradiol (E2) and thereby significantly contributes to local estrogen synthesis. We tested aromatase effects in acute myocardial infarction model in male mice. The animals were randomized and subjected to four groups which were pre-treated with the selective aromatase inhibitor anastrozole (A group) and DES alone (DES group) or in combination (A+DES group) for 15 minutes prior to surgical intervention whereas the control group received 0.9% NaCl (CON group). All animals were subjected to 45 minutes ischemia following 180 minutes reperfusion. Anastrozole blocked DES induced preconditioning and increased infarct size compared to DES alone (37.94±15.5% vs. 17.1±3.62%) without affecting area at risk and systemic hemodynamic parameters following ischemia/reperfusion. Protein localization studies revealed that aromatase was abundant in the murine cardiovascular system with the highest expression levels in endothelial and smooth muscle cells. Desflurane application at pharmacological concentrations efficiently upregulated aromatase expression in vivo and in vitro. We conclude that desflurane efficiently regulates aromatase expression and activity which might lead to increased local estrogen synthesis and thus preserve cellular integrity and reduce cardiac damage in an acute myocardial infarction model.

Effects of crystalloids and colloids on liver and intestine microcirculation and function in cecal ligation and puncture induced septic rodents

BackgroundSeptic acute liver and intestinal failure is associated with a high mortality. We therefore investigated the influence of volume resuscitation with different crystalloid or colloid solutions on liver and intestine injury and microcirculation in septic rodents.MethodsSepsis was induced by cecal ligation and puncture (CLP) in 77 male rats. Animals were treated with different crystalloids (NaCl 0.9% (NaCl), Ringer’s acetate (RA)) or colloids (Gelafundin 4% (Gel), 6% HES 130/0.4 (HES)). After 24 h animals were re-anesthetized and intestinal (n = 6/group) and liver microcirculation (n = 6/group) were obtained using intravital microscopy, as well as macrohemodynamic parameters were measured. Blood assays and organs were harvested to determine organ function and injury.ResultsHES improved liver microcirculation, cardiac index and DO2-I, but significantly increased IL-1β, IL-6 and TNF-α levels and resulted in a mortality rate of 33%. Gel infused animals revealed significant reduction of liver and intestine microcirculation with severe side effects on coagulation (significantly increased PTT and INR, decreased haemoglobin and platelet count). Furthermore Gel showed severe hypoglycemia, acidosis and significantly increased ALT and IL-6 with a lethality of 29%. RA exhibited no derangements in liver microcirculation when compared to sham and HES. RA showed no intestinal microcirculation disturbance compared to sham, but significantly improved the number of intestinal capillaries with flow compared to HES. All RA treated animals survided and showed no severe side effects on coagulation, liver, macrohemodynamic or metabolic state.ConclusionsGelatine 4% revealed devastated hepatic and intestinal microcirculation and severe side effects in CLP induced septic rats, whereas the balanced crystalloid solution showed stabilization of macro- and microhemodynamics with improved survival. HES improved liver microcirculation, but exhibited significantly increased pro-inflammatory cytokine levels. Crystalloid infusion revealed best results in mortality and microcirculation, when compared with colloid infusion.

Desflurane-induced post-conditioning against myocardial infarction is mediated by calcium-activated potassium channels: role of the mitochondrial permeability transition pore

BACKGROUND Desflurane (DES)-induced preconditioning is mediated by large-conductance calcium-activated potassium channels (BK(Ca)). Whether BK(Ca) are involved in anaesthetic-induced post-conditioning is unknown. We tested the hypothesis that DES-induced post-conditioning is mediated by BK(Ca) upstream of the mitochondrial permeability transition pore (mPTP). METHODS Pentobarbital-anaesthetized male C57Black/6 mice were subjected to 45 min coronary artery occlusion (CAO) and 3 h reperfusion. Animals received either no intervention or dimethylsulphoxide (DMSO, 10 µl g(-1)). DES (1.0 MAC, 7.5 vol%) was administered for 18 min, starting 3 min before the end of CAO. The following agents were given either alone or in combination with DES: the BK(Ca) activator NS1619 (1 µg g(-1)), the BK(Ca) inhibitor iberiotoxin (IbTx, 0.05 µg g(-1)), the mPTP opener atractyloside (ATRA, 25 µg g(-1)), and the mPTP inhibitor cyclosporine A (CYC A, 10 µg g(-1)). Infarct size (IS) was determined with triphenyltetrazolium chloride and the area at risk with Evans Blue, respectively. RESULTS IS in control animals was 48(6)%. Neither DMSO, IbTx nor ATRA affected myocardial IS. DES alone or NS1619 alone or the combination reduced IS (P<0.05), CYC A alone or in combination with IbTx or DES also reduced IS (P<0.05). DES-induced reduction of myocardial IS was completely abolished by IbTx and was partially blocked by ATRA and ATRA partially blocked IS reduction by NS1619. CONCLUSIONS These data suggest that DES-induced post-conditioning against myocardial infarction is mediated by BK(Ca) and mPTP. Cardioprotection by BK(Ca) activator NS1619 might occur, at least in part, independently of mPTP.

Desflurane-Induced Cardioprotection Against Ischemia-Reperfusion Injury Depends On Timing

OBJECTIVES The authors tested the hypothesis that desflurane-induced cardioprotection depends on the timing of application and whether desflurane-induced postconditioning is mediated by nitric oxide. DESIGN A prospective randomized vehicle-controlled study. SETTING A university research laboratory. SUBJECTS New Zealand White rabbits (N = 56). INTERVENTIONS Rabbits were instrumented and subjected to a 30-minute coronary artery occlusion (CAO) and 3 hours of reperfusion. Animals were randomized to 8 groups (n = 7) and received 0.0 or 1.0 minimum alveolar concentration desflurane for 30 minutes before CAO (PRE), during CAO (ISCH), after CAO (POST), before and after CAO (PRE + POST), or continuously for 90 minutes starting 30 minutes before CAO (PRE + ISCH + POST). In 2 separate experimental groups, the nitric oxide synthase inhibitor N-omega-nitro-L-arginine (L-NA) was administered before reperfusion in the presence or absence of desflurane. Data are mean +/- standard deviation. RESULTS Infarct size was 68% +/- 14% in control experiments. Desflurane significantly (p < 0.05) reduced infarct size in the PRE (43% +/- 9%) and POST groups (49% +/- 12%) but not in the ISCH group (69% +/- 9%). The PRE + ISCH + POST and PRE + POST groups produced similar reductions in infarct size to 47% +/- 12% and 43% +/- 9%, respectively. L-NA alone had no effect on infarct size (61% +/- 9%) but blocked postconditioning completely (L-NA + POST, 68% +/- 10%). CONCLUSIONS Desflurane induces pre- and postconditioning but does not confer cardioprotection during ischemia in rabbits. The combination of pre- and postconditioning or continuous application does not provide additional cardioprotection. Furthermore, desflurane-induced postconditioning is mediated by nitric oxide.

Peroxisome‐proliferator‐activated receptor γ mediates the second window of anaesthetic‐induced preconditioning

The second window of anaesthetic‐induced preconditioning (APC) is afforded by the interplay of multiple signalling pathways, whereas a similar protective response is mediated by peroxisome‐proliferator‐activated receptor γ (PPARγ) agonists. However, a possible role of this nuclear receptor during APC has not been studied to date. We investigated the hypothesis that the second window of APC is mediated by the activation of PPARγ. New Zealand White rabbits (n= 48) were subjected to 30 min of coronary artery occlusion followed by 3 h of reperfusion. The animals received desflurane (1.0 minimal alveolar concentration), the PPARγ antagonist GW9662, as well as the combined application of both, respectively, 24 h prior to coronary artery occlusion. Infarct size was determined gravimetrically; tissue levels of 15‐deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2) and nitrite/nitrate (NOx), as well as PPAR DNA binding were measured using specific assays. Data are presented as means ±s.e.m. Desflurane led to a reduced myocardial infarct size (41.7 ± 2.5 versus 61.8 ± 2.8%, P < 0.05), accompanied by significantly increased PPAR DNA binding (289.9 ± 33 versus 102.9 ± 18 relative light units, P < 0.05), as well as elevated tissue levels of 15d‐PGJ2 (224.4 ± 10.2 versus 116.9 ± 14.2 pg ml−1, P < 0.05) and NOx (14.9 ± 0.7 versus 5.4 ± 0.7 μm, P < 0.05). Pharmacological inhibition of PPARγ abolished these protective effects, resetting the infarct size (56.5 ± 2.9%), as well as PPAR DNA‐binding activity (91.2 ± 31 relative light units) and NOx tissue levels (5.9 ± 0.9 μm) back to control levels. Desflurane governs a second window of APC by increasing the production of 15d‐PGJ2, subsequently activating PPARγ, resulting in a diminished myocardial infarct size by increasing the downstream availability of NO.