Barbara Sinner

The acute myotoxic effects of bupivacaine and ropivacaine after continuous peripheral nerve blockades.

Compared with bupivacaine, acute myotoxicity of ropivacaine is less severe. Thus, in this study we compared the long term myotoxic effects of both drugs in a clinically relevant setting. Femoral nerve catheters were inserted in anesthetized pigs, and either 20 mL of bupivacaine (5 mg/mL) or ropivacaine (7.5 mg/mL) was injected. Subsequently, bupivacaine (2.5 mg/mL) and ropivacaine (3.75 mg/mL) were continuously infused (8 mL/h) over 6 h. Control animals were treated with corresponding volumes of normal saline. After 7 and 28 days, respectively, muscle samples were dissected at the former injection sites, and histological patterns of muscle damage were blindly scored (0 = no damage to 3 = marked lesions/myonecrosis) and compared. No morphological tissue changes were detected in control animals. In the observed period, both local anesthetics induced morphologically identical patterns of calcific myonecrosis, formation of scar tissue, and a marked rate of fiber regeneration. However, bupivacaine’s effects were constantly more pronounced than those of ropivacaine. These data show that both drugs induce irreversible skeletal muscle damage in a clinically relevant model, and confirm the exceeding rate of myotoxicity of bupivacaine. However, the clinical impact of these long term myotoxic effects still has to be assessed.

Differential effects of bupivacaine on intracellular Ca2+: Regulation potential mechanisms of its myotoxicity

BACKGROUND Bupivacaine produces skeletal muscle damage in clinical concentrations. It has been suggested that this may be caused by an increased intracellular level of [Ca2+]. Therefore, the aim of this study was to investigate direct intracellular effects of bupivacaine on Ca2+ release from the sarcoplasmic reticulum (SR), on Ca2+ uptake into the SR, and on Ca2+ sensitivity of the contractile proteins. METHODS Saponin skinned muscle fibers from the extensor digitorum longus muscle of BALB/c mice were examined according to a standardized procedure described previously. For the assessment of effects on Ca2+ uptake and release from the SR, bupivacaine was added to the loading solution and the release solution, respectively. Force transients and force decays were monitored, and the position of the curve relating relative isometric force free [Ca2+] was evaluated in the presence or absence of bupivacaine. RESULTS Bupivacaine induces Ca2+ release from the SR. In addition, the Ca2+ loading procedure is suppressed, resulting in smaller caffeine-induced force transients after loading in the presence of bupivacaine. The decay of caffeine-induced force transients is reduced by bupivacaine, and it also shifts [Ca2+]-force relation toward lower [Ca2+]. CONCLUSIONS These data reveal that bupivacaine does not only induce Ca2+ release from the SR, but also inhibits Ca2+ uptake by the SR, which is mainly regulated by SR Ca2+ adenosine triphosphatase activity. It also has a Ca2+ -sensitizing effect on the contractile proteins. These mechanisms result in increased intracellular [Ca2+] concentrations and may thus contribute to its pronounced skeletal muscle toxicity.

Lipid emulsion improves recovery from bupivacaine-induced cardiac arrest, but not from ropivacaine- or mepivacaine-induced cardiac arrest.

BACKGROUND: Cardiac toxicity significantly correlates with the lipophilicity of local anesthetics (LAs). Recently, the infusion of lipid emulsions has been shown to be a promising approach to treat LA-induced cardiac arrest. As the postulated mechanism of action, the so-called “lipid sink” effect may depend on the lipophilicity of LAs. In this study, we investigated whether lipid effects differ with regard to the administered LAs. METHODS: In the isolated rat heart, cardiac arrest was induced by administration of equipotent doses of bupivacaine, ropivacaine, and mepivacaine, respectively, followed by cardiac perfusion with or without lipid emulsion (0.25 mL · kg−1 · min−1). Subsequently, the times from the start of perfusion to return of first heart activity and to recovery of heart rate and rate-pressure product (to 90% of baseline values) were assessed. RESULTS: In all groups, lipid infusion had no effects on the time to the return of any cardiac activity. However, recovery times of heart rate and rate-pressure product (to 90% of baseline values) were significantly shorter with the administration of lipids in bupivacaine-induced cardiac toxicity, but not in ropivacaine- or mepivacaine-induced cardiac toxicity. CONCLUSIONS: These data show that the effects of lipid infusion on LA-induced cardiac arrest are strongly dependent on the administered LAs itself. We conclude that lipophilicity of LAs has a marked impact on the efficacy of lipid infusions to treat cardiac arrest induced by these drugs.

Differential Effects of Bupivacaine and Ropivacaine Enantiomers on Intracellular Ca2+Regulation in Murine Skeletal Muscle Fibers

Background: Increased intracellular Ca2+ concentrations are considered to be a major pathomechanism in local anesthetic myotoxicity. Racemic bupivacaine and S-ropivacaine cause Ca2+ release from the sarcoplasmic reticulum of skeletal muscle fibers and simultaneously inhibit Ca2+ reuptake. Examining the optical isomers of both agents, the authors investigated stereoselective effects on muscular Ca2+ regulation to get a closer insight in subcellular mechanisms of local anesthetic myotoxicity. Methods: R- and S-enantiomers as well as racemic mixtures of both agents were tested in concentrations of 1, 5, 10, and 15 mm. Saponin-skinned muscle fibers from the extensor digitorum longus muscle of BALB/c mice were examined according to a standardized procedure. For the assessment of effects on Ca2+ uptake and release from the sarcoplasmic reticulum, agents were added to the loading solution and the release solution, respectively, and force and Ca2+ transients were monitored. Results: The effects of S-enantiomers on both Ca2+ release and reuptake were significantly more pronounced than those of racemic mixtures and R-enantiomers, respectively. In addition, the effects of racemates were markedly stronger than those of R-enantiomers. With regard to Ca2+ release, the effects of bupivacaine isomers were more pronounced than the isomers of ropivacaine. Conclusions: These data show that stereoselectivity is involved in alterations of intracellular Ca2+ regulation by bupivacaine and ropivacaine. S-enantiomers seem to be more potent than R-enantiomers, with intermediate effects of racemic mixtures. In addition, lipophilicity also seems to determine the extent of Ca2+ release by local anesthetics.

The toxic effects of s(+)-ketamine on differentiating neurons in vitro as a consequence of suppressed neuronal Ca2+ oscillations.

BACKGROUND: In the immature brain, neuronal Ca2+ oscillations are present during a time period of high plasticity and regulate neuronal differentiation and synaptogenesis. In this study we examined the long-term blockade of hippocampal Ca2+ oscillations, the role of the N-methyl-D-aspartate (NMDA) receptors and the effects of S(+)-ketamine on neuronal synapsin expression. METHODS: Hippocampal neurons were incubated at day 15 in culture with the specific NMDA receptor antagonists dizocilpine (MK 801, 100 &mgr;M) or S(+)-ketamine (3 &mgr;M to 25 &mgr;M) for 24 hours. Terminal-deoxynucleotidyl-transferase (TUNEL) and activated caspase3 were used to detect apoptotic neurons. Ca2+ oscillations were detected after loading the neurons with the Ca2+-sensitive dye fura-2AM, and dual wavelength excitation fluorescence microscopy was performed. Ca2+/calmodulin kinase II (CaMKII) was measured using Western blots. Synapsin was identified with confocal antisynapsin immunofluorescence. RESULTS: Blocking the NMDA receptor with MK 801 or 25 &mgr;M S(+)-ketamine resulted in a significant increase in apoptotic neurons. MK 801 led to a significant increase in cytosolic Ca2+ concentration and reduction of the amplitude and frequency of the Ca2+ oscillations. Similar to MK 801, the long-term application of S(+)-ketamine resulted in a significant increase in cytosolic Ca2+ concentration 24 hours after washout. This was associated with a down-regulation of the CaMKII and a reduction of the synapsin 24 hours after washout. CONCLUSION: Neuronal Ca2+ oscillations mediate neuronal differentiation and synaptogenesis via activating CaMKII. By acting via the NMDA receptor, S(+)-ketamine exerts its toxic effect through the suppression of neuronal Ca2+ oscillations, down-regulation of the CaMKII, and consecutively reduced synaptic integrity.

Alterations in intracellular Ca2+-homeostasis of skeletal muscle fibers during sepsis.

Objective:To investigate changes in intracellular Ca2+-regulation and Ca2+-sensitivity of the contractile apparatus in murine skeletal muscle fibers during sepsis. Design and Setting:Animal study in a university-based research laboratory. Subjects:Isolated muscle fibers (M. extensor digitorum longus) of septic mice. Interventions:In one group, sepsis was induced in “black six” mice using cecal ligation and puncture (CLP). In a second group, laparotomy (SHAM), and in a third group, general anesthesia (GA) was performed. Saponin-skinned skeletal muscle fibers were examined 2, 3, 5, and 7 days after treatment, and caffeine-induced Ca2+-release from the sarcoplasmic reticulum (SR) as well as Ca2+-sensitivity of the contractile apparatus were assessed. Measurements and Results:In the CLP group, Ca2+-release significantly decreased over 5 days and increased again after 7 days. In the SHAM group, Ca2+-release decreased at days 2 and 3, whereas no changes were observed in the GA group. Ca2+-sensitivity significantly increased over 5 days in the CLP group and decreased again at day 7. In the SHAM group, Ca2+-sensitivity increased at days 2 and 3, and no changes were seen in the GA group. Conclusions:In murine skeletal muscle fibers, Ca2+-release from the SR decreases during sepsis, with effects being most pronounced 2–3 days after CLP. In parallel, Ca2+-sensitivity of the contractile apparatus is increased, and all changes are reversible. Thus, these effects might be involved in skeletal muscle dysfunction during sepsis as corresponding changes are less pronounced or absent in control groups.

Cardiac effects of induction agents in the septic rat heart

IntroductionThe current debate about the side effects of induction agents, e.g. possible adrenal suppression through etomidate, emphasizes the relevance of choosing the correct induction agent in septic patients. However, cardiovascular depression is still the most prominent adverse effect of these agents, and might be especially hazardous in septic patients presenting with a biventricular cardiac dysfunction - or so-called septic cardiomyopathy. Therefore, we tested the dose-response direct cardiac effects of clinically available induction agents in an isolated septic rat heart model.MethodsA polymicrobial sepsis was induced via cecal ligation and single puncture. Hearts (n = 50) were isolated and randomly assigned to five groups, each receiving etomidate, s(+)-ketamine, midazolam, propofol, or methohexitone at concentrations of 1 × 10-8 to 1 × 10-4 M. Left ventricular pressure, contractility and lusitropy, and coronary flow were measured. Cardiac work, myocardial oxygen delivery, oxygen consumption, and percentage of oxygen extraction were calculated.ResultsAll of the induction agents tested showed a dose-dependent depression of cardiac work. Maximal cardiac work dysfunction occurred in the rank order of s(+)-ketamine (-6%) <etomidate (-17%) <methohexitone (-31%) <midazolam (-38%) <propofol (-50%). In addition, propofol showed a maximum decrease in contractility of -38%, a reduction in lusitropy of -44%, and a direct vasodilator effect by increasing coronary flow by +29%.ConclusionsOverall, this study demonstrates that these tested drugs indeed have differential direct cardiac effects in the isolated septic heart. Propofol showed the most pronounced adverse direct cardiac effects. In contrast, S(+)ketamine showed cardiovascular stability over a wide range of concentrations, and might therefore be a beneficial alternative to etomidate.

Addition of Acetylsalicylic Acid to Heparin for Anticoagulation Management During Pumpless Extracorporeal Lung Assist

Pump-driven extracorporeal membrane oxygenation (ECMO) or pumpless arterio-venous interventional lung assist (iLA) is associated with possible complications, mainly consisting of bleeding or thrombosis/clotting by cellular deposits on the membrane or extracorporeal circuit surfaces, which may reduce gas-exchange capacity. In this study, we report our experiences with the addition of low-dose acetylsalicylic acid (ASA 1.5 mg/kg body weight/d) to heparin for anticoagulation of a pumpless low-resistance gas-exchange membrane (Novalung GmbH, Talheim, Germany). We assessed changes in coagulation parameters and the demand for transfusion of blood components. Furthermore, we compared the function of the artificial membranes (oxygen transfer and capacity of CO2 removal) of the ASA group (n = 15) with that of a matched-pair control group treated with heparin alone. The mean duration of iLA treatment was 6.6 ± 3.7 days. The addition of ASA did not increase bleeding activity or the demand for transfusion. Relative changes of CO2 removal on day 3 expressed as a percentage in the ASA group were (mean value) −11.8% in comparison with control (−3.0%, p = 0.266), but the relative amount of oxygen transfer tended to be increased in the ASA group (+3.9%) and to be decreased in the control group (−14.7%, p = 0.214). Pao2/Fio2 ratio was significantly improved in the ASA group compared with the control group at day 5. The use of membranes per patient (membrane/patient ratio) tended to be decreased in patients treated with ASA (1.12 ± 0.34) in comparison with control (1.33 ± 0.62, p = 0.157). In the ASA group, one patient died due to multiple organ failure, whereas in the control group, five patients died. We conclude that supplementation of low-dose ASA during pumpless extracorporeal lung support is safe and might preserve the function of oxygen transfer.

Toxic effects of midazolam on differentiating neurons in vitro as a consequence of suppressed neuronal Ca2+-oscillations

BACKGROUND In immature neurons anesthetics induce apoptosis and influence neuronal differentiation. Neuronal Ca(2+)-oscillations regulate differentiation and synaptogenesis. We examined the effects of the long-term blockade of hippocampal Ca(2+)-oscillations with midazolam on neuronal synapsin expression. MATERIAL AND METHODS Hippocampal neurons were incubated at day 15 in culture with the specific GABA(A) receptor agonist muscimol (50μM) or with midazolam (100 and 300nM), respectively, for 24h. TUNEL and activated-Caspase-3 staining were used to detect apoptotic neurons. Ca(2+)-oscillations were detected using the Ca(2+)-sensitive dye FURA-2 and dual wavelength excitation fluorescence microscopy. Synapsin was identified with confocal anti-synapsin immunofluorescence microscopy. RESULTS Muscimol, when applied for 24h, decreased the amplitude and frequency Ca(2+)-oscillations significantly. Midazolam concentration-dependently suppressed the amplitude and frequency of the Ca(2+)-oscillations. This was associated by a downregulation of the synapsin expression 24h after washout. CONCLUSION Neuronal Ca(2+)-oscillations mediate neuronal differentiation and are involved in synaptogenesis. By acting via the GABA(A) receptor, midazolam exerts its toxic effect through the suppression of neuronal Ca(2+)-oscillations, a reduction in synapsin expression and consecutively reduced synaptic integrity.

Direct cardiac effects of dobutamine, dopamine, epinephrine, and levosimendan in isolated septic rat hearts.

In septic patients, myocardial depression-the so-called septic cardiomyopathy-needing inotropic support is common. The aim of this study was to compare the dose-responsive electrophysiological and mechanical properties concerning metabolic effects of clinically available inotropic agents in an isolated septic rat heart model. After 20 h of incubation, both sham-operated and septic (cecal ligation and single puncture) hearts from male Wistar rats (n = 64) were isolated and received dobutamine, dopamine, epinephrine, or levosimendan at concentrations of 10−9 to 10−3 M. Electrophysiological, mechanical, and metabolic properties were measured, and the myocardial oxygen supply-demand ratio and cardiac efficiency were calculated. With the exception of levosimendan, all of the drugs tested showed dose-dependent, significantly positive changes in chronotropy, inotropy, and lusitropy in all hearts. Maximum increases in septic hearts were dose-dependent and were ordered as follows: epinephrine > dopamine > dobutamine ⋙ levosimendan. These increases in cardiac performance were accompanied by a decrease in the myocardial oxygen supply-demand ratio. However, cardiac efficiency was significantly improved in the epinephrine-treated septic hearts. With the drug-induced increase in cardiac performance, the myocardial oxygen supply-demand ratio decreased proportionally in the epinephrine-, dobutamine-, and dopamine-treated septic hearts. However, epinephrine showed the most favorable results with regard to cardiac efficiency, and levosimendan showed no beneficial effect in septic hearts with regard to efficiency in this study.