Octavian Toma

The noble gas xenon induces pharmacological preconditioning in the rat heart in vivo via induction of PKC-ɛ and p38 MAPK

1 Xenon is an anesthetic with minimal hemodynamic side effects, making it an ideal agent for cardiocompromised patients. We investigated if xenon induces pharmacological preconditioning (PC) of the rat heart and elucidated the underlying molecular mechanisms. 2 For infarct size measurements, anesthetized rats were subjected to 25 min of coronary artery occlusion followed by 120 min of reperfusion. Rats received either the anesthetic gas xenon, the volatile anesthetic isoflurane or as positive control ischemic preconditioning (IPC) during three 5‐min periods before 25‐min ischemia. Control animals remained untreated for 45 min. To investigate the involvement of protein kinase C (PKC) and p38 mitogen‐activated protein kinase (MAPK), rats were pretreated with the PKC inhibitor calphostin C (0.1 mg kg−1) or the p38 MAPK inhibitor SB203580 (1 mg kg−1). Additional hearts were excised for Western blot and immunohistochemistry. 3 Infarct size was reduced from 50.9±16.7% in controls to 28.1±10.3% in xenon, 28.6±9.9% in isoflurane and to 28.5±5.4% in IPC hearts. Both, calphostin C and SB203580, abolished the observed cardioprotection after xenon and isoflurane administration but not after IPC. Immunofluorescence staining and Western blot assay revealed an increased phosphorylation and translocation of PKC‐ɛ in xenon treated hearts. This effect could be blocked by calphostin C but not by SB203580. Moreover, the phosphorylation of p38 MAPK was induced by xenon and this effect was blocked by calphostin C. 4 In summary, we demonstrate that xenon induces cardioprotection by PC and that activation of PKC‐ɛ and its downstream target p38 MAPK are central molecular mechanisms involved. Thus, the results of the present study may contribute to elucidate the beneficial cardioprotective effects of this anesthetic gas.

Desflurane Preconditioning Induces Time-dependent Activation of Protein Kinase C Epsilon and Extracellular Signal-regulated Kinase 1 and 2 in the Rat Heart In Vivo

Background:Activation of protein kinase C epsilon (PKC-&egr;) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) are important for cardioprotection by preconditioning. The present study investigated the time dependency of PKC-&egr; and ERK1/2 activation during desflurane-induced preconditioning in the rat heart. Methods:Anesthetized rats were subjected to regional myocardial ischemia and reperfusion, and infarct size was measured by triphenyltetrazoliumchloride staining (percentage of area at risk). In three groups, desflurane-induced preconditioning was induced by two 5-min periods of desflurane inhalation (1 minimal alveolar concentration), interspersed with two 10-min periods of washout. Three groups did not undergo desflurane-induced preconditioning. The rats received 0.9% saline, the PKC blocker calphostin C, or the ERK1/2 inhibitor PD98059 with or without desflurane preconditioning (each group, n = 7). Additional hearts were excised at four different time points with or without PKC or ERK1/2 blockade: without further treatment, after the first or the second period of desflurane-induced preconditioning, or at the end of the last washout phase (each time point, n = 4). Phosphorylated cytosolic PKC-&egr; and ERK1/2, and membrane translocation of PKC-&egr; were determined by Western blot analysis (average light intensity). Results:Desflurane significantly reduced infarct size from 57.2 ± 4.7% in controls to 35.2 ± 16.7% (desflurane-induced preconditioning, mean ± SD, P < 0.05). Both calphostin C and PD98059 abolished this effect (58.8 ± 13.2% and 64.2 ± 15.4% respectively, both P < 0.05 versus desflurane-induced preconditioning). Cytosolic phosphorylated PKC-&egr; reached its maximum after the second desflurane-induced preconditioning and returned to baseline after the last washout period. Both calphostin C and PD98059 inhibited PKC-&egr; activation. ERK1/2 phosphorylation reached its maximum after the first desflurane-induced preconditioning and returned to baseline after the last washout period. Calphostin C had no effect on ERK1/2 phosphorylation. Conclusions:Both, PKC and ERK1/2 mediate desflurane-induced preconditioning. PKC-&egr; and ERK1/2 are both activated in a time dependent manner during desflurane-induced preconditioning, but ERK1/2 activation during desflurane-induced preconditioning is not PKC dependent. Moreover, ERK1/2 blockade abolished PKC-&egr; activation, suggesting ERK-dependent activation of PKC-&egr; during desflurane-induced preconditioning.

Mechanisms of xenon- and isoflurane-induced preconditioning – a potential link to the cytoskeleton via the MAPKAPK-2/HSP27 pathway

We previously demonstrated that the anesthetic gas xenon exerts cardioprotection by preconditioning in vivo via activation of protein kinase C (PKC)‐ɛ and p38 mitogen‐activated protein kinase (MAPK). P38 MAPK interacts with the actin cytoskeleton via the MAPK‐activated protein kinase‐2 (MAPKAPK‐2) and heat‐shock protein 27 (HSP27). The present study further elucidated the underlying molecular mechanism of xenon‐induced preconditioning (Xe‐PC) by focusing on a potential link of xenon to the cytoskeleton. Anesthetized rats received either xenon (Xe‐PC, n=6) or the volatile anesthetic isoflurane (Iso‐PC, n=6) during three 5‐min periods interspersed with two 5‐min and one final 10‐min washout period. Control rats (n=6) remained untreated for 45 min. Additional rats were either pretreated with the PKC inhibitor Calphostin C (0.1 mg kg−1) or with the p38 MAPK inhibitor SB203580 (1 mg kg−1) with and without anesthetic preconditioning (each, n=6). Hearts were excised for immunohistochemistry of F‐actin fibers and phosphorylated HSP27. Phosphorylation of MAPKAPK‐2 and HSP27 were assessed by Western blot. HSP27 and actin colocalization were investigated by co‐immunoprecipitation. Xe‐PC induced phosphorylation of MAPKAPK‐2 (control 1.0±0.2 vs Xe‐PC 1.6±0.1, P<0.05) and HSP27 (control 5.0±0.5 vs Xe‐PC 9.8±1.0, P<0.001). Both effects were blocked by Calphostin C and SB203580. Xe‐PC enhanced translocation of HSP27 to the particulate fraction and increased F‐actin polymerization. F‐actin and pHSP27 were colocalized after Xe‐PC. Xe‐PC activates MAPKAPK‐2 and HSP27 downstream of PKC and p38 MAPK. These data link Xe‐PC to the cytoskeleton, revealing new insights into the mechanisms of Xe‐PC in vivo.

Morphine induces late cardioprotection in rat hearts in vivo: the involvement of opioid receptors and nuclear transcription factor kappaB.

&dgr;1-opioid receptor agonists can induce cardioprotection by early and late preconditioning (LPC). Morphine (MO) is commonly used for pain treatment during acute coronary syndromes. We investigated whether MO can induce myocardial protection by LPC and whether a nuclear transcription factor &kgr;B (NF-&kgr;B)-dependent intracellular signaling pathway is involved. Rats were subjected to 25 min of regional ischemia and 2 h of reperfusion 24 h after treatment with saline (NaCl; 0.9% 5 mL), lipopolysaccharide of Escherichia coli (LPS; 1 mg/kg), or MO (3 mg/kg). LPS is a trigger of LPC and served as positive control. Naloxone (NAL) was used to investigate the role of opioid receptors in LPC and was given before NaCl, LPS, or MO application (trigger phase) or before ischemia-reperfusion (mediator phase). Infarct size (percentage area at risk) was 59% ± 9%, 51% ± 6%, or 53% ± 10% in the NaCl, NAL-NaCl, and NaCl-NAL groups, respectively. Pretreatment with MO reduced infarct size to 20% ± 6% after 24 h (MO-24h), and this effect was abolished by NAL in the trigger (NAL-MO, 53% ± 14%) and in the mediator (MO-NAL, 60% ± 8%) phases. Pretreatment with LPS reduced infarct size to 23% ± 8%. NAL administration in the trigger phase had no effect on infarct size (NAL-LPS 30% ± 16%), whereas NAL during the mediator phase of LPC abolished the LPS-induced cardioprotection (LPS-NAL 54% ± 8%). The role of NF-&kgr;B in morphine-induced LPC was investigated by Western blot and electrophoretic mobility shift assay. Morphine and LPS treatment increased phosphorylation of the inhibitory protein &kgr;B, leading to an increased activity of NF-&kgr;B. Thus, MO induces LPC similarly to LPS and it is likely that this cardioprotection is mediated at least in part by activation of NF-&kgr;B. Opioid receptors are involved as mediators in both MO- and LPS-induced LPC but as triggers only in MO-induced LPC.

Pharmacokinetics and tissue penetration of cefoxitin in obesity: implications for risk of surgical site infection.

BACKGROUND:Obesity is a significant risk factor for surgical site infections (SSIs), for poorly understood reasons. SSIs are a major cause of morbidity, prolonged hospitalization, and increased health care cost. Drug disposition in general is frequently altered in the obese. Preoperative antibiotic administration, achieving adequate tissue concentrations at the time of incision, is an essential strategy to prevent SSIs. Nonetheless, there is little information regarding antibiotic concentrations in obese surgical patients. This investigation tested the hypothesis that the prophylactic antibiotic cefoxitin may have delayed and/or diminished tissue penetration in the obese. METHODS:Plasma and tissue concentrations of cefoxitin were determined in obese patients undergoing abdominal and pelvic surgery (body mass index 43 ± 10 kg/m2, n = 14, 2 g cefoxitin) and in normal-weight patients and healthy volunteers (body mass index 20 ± 2 kg/m2, n = 13, 1 g cefoxitin). Tissue concentrations were measured using a microdialysis probe in the subcutaneous layer of the abdomen, and in adipose tissue excised at the time of incision and wound closure. RESULTS:Plasma concentrations and area under the concentration-time curve (AUC) were approximately 2-fold higher in the obese patients because of the 2-fold-higher dose. Dose-normalized concentrations were higher, although AUCs were not significantly different. Measured and dose-normalized subcutaneous cefoxitin concentrations and AUCs in the obese patients were significantly lower than in the normal-weight subjects. There was an inverse relationship between cefoxitin tissue penetration (AUCtissue/AUCplasma ratio) and body mass index. Tissue penetration was substantially lower in the obese patients (0.08 ± 0.07 vs 0.37 ± 0.26, P < 0.05). Adipose tissue cefoxitin concentrations in obese patients were only 7.8 ± 7.3 and 2.7 ± 1.4 &mgr;g/g, respectively, at incision and closure, below the minimum inhibitory concentration of 8 and 16 &mgr;g/mL, respectively, for aerobic and anaerobic microorganisms. CONCLUSION:Obese surgical patients have impaired tissue penetration of the prophylactic antibiotic cefoxitin, and inadequate tissue concentrations despite increased clinical dose (2 g). Inadequate tissue antibiotic concentrations may be a factor in the increased risk of SSIs in obese surgical patients. Additional studies are needed to define doses achieving adequate tissue concentrations.

Effects of nitrous oxide on the rat heart in vivo : Another inhalational anesthetic that preconditions the heart?

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-ϵ 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 < 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-ϵ. 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-ϵ phosphorylation (5.4 ± 1.9 vs. 2.8 ± 1.5; P < 0.001) and translocation to membrane regions (13.8 ± 13.0 vs. 6.7 ± 2.0 in controls; P < 0.05). Conclusions:Nitrous oxide is the first inhalational anesthetic without preconditioning effect on the heart. However, isoflurane-induced preconditioning and PKC-ϵ activation are not influenced by nitrous oxide.

Sevoflurane-induced Preconditioning Impact of Protocol and Aprotinin Administration on Infarct Size and Endothelial Nitric-Oxide Synthase Phosphorylation in the Rat Heart In Vivo

Background:Sevoflurane induces preconditioning (SevoPC). The effect of aprotinin and the involvement of endothelial nitric-oxide synthase (NOS) on SevoPC are unknown. We investigated (1) whether SevoPC is strengthened by multiple preconditioning cycles, (2) whether SevoPC is blocked by aprotinin, and (3) whether endothelial NOS plays a crucial role in SevoPC. Methods:Anesthetized male Wistar rats were randomized to 15 groups (each n = 6) and underwent 25-min regional myocardial ischemia and 2-h reperfusion. Controls were not treated further. Preconditioning groups inhaled 1 minimum alveolar concentration of sevoflurane for 5 min (SEVO-I), twice for 5 min each (SEVO-II), three times for 5 min each (SEVO-III), or six times for 5 min each (SEVO-VI). Aprotinin was administered with and without sevoflurane. Involvement of endothelial NOS was determined with the nonspecific NOS blocker N-nitro-l-arginine-methyl-ester, the specific neuronal NOS blocker 7-nitroindazole, and the specific inducible NOS blocker aminoguanidine. Results:SevoPC reduced infarct size in all protocols (SEVO-I, 42 ± 6%; SEVO-II, 33 ± 4%; SEVO-III, 11 ± 5%; SEVO-VI, 16 ± 4%; all P < 0.001 vs. control, 67 ± 3%) and was least after three and six cycles of sevoflurane (P < 0.001 vs. SEVO-II and -I, respectively). Aprotinin alone had no effect on infarct size but blocked SevoPC. N-nitro-l-arginine-methyl-ester abolished SevoPC (67 ± 4%; P < 0.05 vs. SEVO-III). Aminoguanidine and 7-nitroindazole blocked SevoPC only partially (25 ± 6 and 31 ± 6%, respectively; P < 0.05 vs. SEVO-III and control). SevoPC induced endothelial NOS phosphorylation, which was abrogated by aprotinin. Conclusion:SevoPC is strengthened by multiple preconditioning cycles, and phosphorylation of endothelial NOS is a crucial step in mediating SevoPC. These effects are abolished by aprotinin.

In vivo desflurane preconditioning evokes dynamic alterations of metabolic proteins in the heart--proteomic insights strengthen the link between bioenergetics and cardioprotection

Background: The cardioprotective effect of anaesthetic preconditioning as measured by reduction of ischaemia-reperfusion (I/R) injury is a well described phenomenon. However little is known about the impact on the myocardial proteome. We therefore investigated proteome dynamics at different experimental time points of a preconditioning protocol. Methods: Using an in vivo rat model of desflurane-induced preconditioning (DES-PC) cardiac tissue proteomes were analysed by a gel-based comparative approach. Treatment-dependent protein alterations were assessed by intra-group comparisons. Proteins were identified by mass-spectrometry. Results: A total of 40 protein spots were altered during the 30-minutes lasting preconditioning protocol. None of the proteins was differentially regulated consistently at all experimental time points. Interestingly, 1) the repeated administration of desflurane mostly accounted for proteome alterations during DES-PC, 2) the majority of altered protein species showed a decrease in abundance, 3) these changes primarily affected metabolic proteins involved in NADH/NAD+ redox balance, calcium homeostasis and acidosis and 4) protein alterations were not exclusively due to expression changes but also represented modifications of specific protein isoforms. Conclusion: DES-PC evokes dynamic alterations in the cardiac proteome which substantiate a tight regulation of bioenergetic proteins. Unique protein modifications may play a more important role in the preconditioning response.