Lipid Emulsion Restoration of Myocardial Contractions After Bupivacaine-Induced Asystole In Vitro: A Benefit of Long- and Medium-Chain Triglyceride Over Long-Chain Triglyceride.

BACKGROUND: The relative efficacies of a long- and medium-chain triglyceride (LCT/MCT) emulsion and an LCT emulsion for treatment of bupivacaine (BPV)-induced cardiac toxicity are poorly defined. METHODS: After inducing asystole by BPV, varied concentrations (1%-12%) of either LCT/MCT (Lipofundin; B. Braun, Melsungen, Germany) or LCT emulsion (Intralipid; Fresenius Kabi, Upsala, Sweden) were applied to observe the recovery of stimulated contractile responses and contractile forces in either a recirculating or washout condition for 60 minutes, using guinea pig papillary muscles. The recirculation condition was used to demonstrate BPV binding by lipid emulsion. The washout condition was used to determine whether the time-dependent recovery of contraction is due to their metabolic enhancement. Oxfenicine, an inhibitor of carnitine palmitoyltransferase I in heart mitochondria, was used to evaluate the effect of each lipid emulsion on mitochondrial metabolic inhibition by BPV. To examine the effect of the lipid emulsion alone on contractility, either lipid emulsion was examined. BPV concentrations in solution and myocardial tissues were measured. RESULTS: In the recirculating condition, LCT/MCT emulsions (2%-12%) restored regular stimulated contractile responses in all muscles. Eight percent and 12% LCT/MCT emulsions led to complete recovery of contractile forces after 30 minutes. Meanwhile, LCT emulsions (4%-12%) did not restore regular stimulated contractile responses in some muscles (6, 3, and 2 in 9 muscles each in 4%, 8%, and 12% emulsions, respectively). Partial recovery, approximately 60%, of contractile forces was observed with 8% and 12% LCT emulsions. In the washout experiments, after asystole, LCT/MCT emulsions (1%-12%) restored contractility to baseline levels earlier and greater than LCT emulsion. Partial recovery, approximately 60%, was observed with a high concentration of LCT emulsion (12%). In the oxfenicine-pretreated group, the contractile recovery was enhanced with LCT/MCT emulsion but showed no change with LCT emulsion. Contractile depression by 40% was observed with high concentrations of LCT emulsion alone (8% and 12%), whereas no depression or enhanced contraction was observed with LCT/MCT emulsion (1%-12%) alone. Both types of lipid emulsions (2%-12%) caused concentration-related reductions of tissue BPV levels; LCT/MCT emulsions reduced tissue BPV levels slightly greater than LCT emulsion in a recirculating condition. CONCLUSIONS: An LCT/MCT emulsion was more beneficial than an LCT emulsion in terms of local anesthetic-binding and metabolic enhancement for treating acute BPV toxicity. The metabolic benefit of MCT, combined with the local anesthetic-binding effect of LCT, in an LCT/MCT emulsion may improve contractile function better than an LCT emulsion in an isolated in vitro animal myocardium model.

Intralipid Restoration of Myocardial Contractions Following Bupivacaine-Induced Asystole: Concentration- and Time-Dependence In Vitro.

BACKGROUND: The concentration- and time-response relationships of lipid emulsion (LE; Intralipid) on the recovery of myocardial contractility following bupivacaine (BPV)-induced asystole are poorly defined. METHODS: After achieving asystole by 500-µM BPV, varied concentrations of LE were applied to determine the recovery of stimulated contractile responses and contractions in the cardiac tissues of guinea pigs at a 1.2-Hz stimulation rate. These experiments were performed with LE in either a recirculating (2%-16%) or washout (nonrecirculating) condition (0.05%-12%) for 60 minutes. The effect of LE itself (0.05%-12%) was examined. Oxfenicine was used to evaluate the metabolic action of LE to reverse asystole. BPV concentrations in solution and myocardial tissues were measured. RESULTS: In the recirculation condition, partial recovery of contractile forces was observed for 60 minutes at 4%, 8%, and 12% LE. A contracture followed after exposure to 16% LE in some asystolic muscles. In the washout experiments, following asystole, LE (0.05%-12%) had no effect on the recovery time of the first and regular contractile responses. LE (0.1%-8%) restored contractility to baseline levels after 45 minutes; partial recovery was shown with lower (0.05%) and higher (12%) concentrations. Oxfenicine did not alter the recovery of contractile forces. Contractile depression was observed with 12% LE alone. Concentration-related reduction of tissue BPV concentration by LE was observed in both circulating conditions. CONCLUSIONS: LE induced time- and concentration-dependent recovery of stimulated myocardial contractions from BPV-induced asystole. The lipid uptake effect, along with other undefined mechanisms of LE, seems to contribute to the recovery of contractile function; however, the LE effect on myocardial metabolism is less likely involved at this concentration (500 µM) of BPV.

The physiologic implications of isolated alpha(1) adrenergic stimulation.

Phenylephrine and methoxamine are direct-acting, predominantly α(1) adrenergic receptor (AR) agonists. To better understand their physiologic effects, we screened 463 articles on the basis of PubMed searches of "methoxamine" and "phenylephrine" (limited to human, randomized studies published in English), as well as citations found therein. Relevant articles, as well as those discovered in the peer-review process, were incorporated into this review. Both methoxamine and phenylephrine increase cardiac afterload via several mechanisms, including increased vascular resistance, decreased vascular compliance, and disadvantageous alterations in the pressure waveforms produced by the pulsatile heart. Although pure α(1) agonists increase arterial blood pressure, neither animal nor human studies have ever shown pure α(1)-agonism to produce a favorable change in myocardial energetics because of the resultant increase in myocardial workload. Furthermore, the cost of increased blood pressure after pure α(1)-agonism is almost invariably decreased cardiac output, likely due to increases in venous resistance. The venous system contains α(1) ARs, and though stimulation of α(1) ARs decreases capacitance and may transiently increase venous return, this gain may be offset by changes in afterload, venous compliance, and venous resistance. Data on the effects of α(1) stimulation in the central nervous system show conflicting changes, while experimental animal data suggest that renal blood flow is reduced by α(1)-agonists, and both animal and human data suggest that gastrointestinal perfusion may be reduced by α(1) tone.

The clinical implications of isolated alpha(1) adrenergic stimulation.

Phenylephrine is a direct-acting, predominantly α(1) adrenergic receptor agonist used by anesthesiologists and intensivists to treat hypotension. A variety of physiologic studies suggest that α-agonists increase cardiac afterload, reduce venous compliance, and reduce renal bloodflow. The effects on gastrointestinal and cerebral perfusion are controversial. To better understand the effects of phenylephrine in a variety of clinical settings, we screened 463 articles on the basis of PubMed searches of "methoxamine," a long-acting α agonist, and "phenylephrine" (limited to human, randomized studies published in English), as well as citations found therein. Relevant articles, as well as those discovered in the peer-review process, were incorporated into this review. Phenylephrine has been studied as an antihypotensive drug in patients with severe aortic stenosis, as a treatment for decompensated tetralogy of Fallot and hypoxemia during 1-lung ventilation, as well as for the treatment of septic shock, traumatic brain injury, vasospasm status-postsubarachnoid hemorrhage, and hypotension during cesarean delivery. In specific instances (critical aortic stenosis, tetralogy of Fallot, hypotension during cesarean delivery) in which the regional effects of phenylephrine (e.g., decreased heart rate, favorable alterations in Q(p):Q(s) ratio, improved fetal oxygen supply:demand ratio) outweigh its global effects (e.g., decreased cardiac output), phenylephrine may be a rational pharmacologic choice. In pathophysiologic states in which no regional advantages are gained by using an α(1) agonist, alternative vasopressors should be sought.

Implementation of an HDR brachytherapy-based breast IORT program: Initial experiences.

PURPOSE: A multidisciplinary team at our institution developed a novel method of intraoperative breast radiation therapy (precision breast intraoperative radiation therapy [PB-IORT]) that uses high-dose-rate brachytherapy with CT on-rails imaging to deliver high-dose, customized radiotherapy to patients with early-stage breast cancer. This report summarizes our program's experience developing and implementing PB-IORT. METHODS AND MATERIALS: Literature on PB-IORT was reviewed including published articles and abstracts. To evaluate case volume, all patients with a breast cancer diagnosis who underwent breast surgery or breast radiation (2010-2017) at our academic institution were identified. Patients were stratified into pre-IORT and post-IORT eras with initiation of our PB-IORT program in October 2013. Overall trends in surgical and radiation therapy volume in each era were analyzed by linear regression. Travel distance for all surgical patients was calculated using Google Maps (Alphabet Inc.) and then compared between IORT and non-IORT patients. RESULTS: Data from a PB-IORT Phase 1 trial found that the primary endpoints were met and that PB-IORT is feasible and safe. The direct health system's delivery costs for PB-IORT exceed those of 16-fraction whole-breast irradiation when accounting for consumable supplies (multilumen balloon applicator = $2,750 per patient). There was a significant increase in yearly growth of breast cancer surgical volume with PB-IORT. CONCLUSIONS: Accrual rates for the ongoing Phase II trial have been quicker than expected in an area where more research is needed. The rapid accrual indicates patient interest and demand for this treatment and that it is very feasible to get more data from randomized trials.

TASK channels contribute to neuroprotective action of inhalational anesthetics.

Postconditioning with inhalational anesthetics can reduce ischemia-reperfusion brain injury, although the cellular mechanisms for this effect have not been determined. The current study was designed to test if TASK channels contribute to their neuroprotective actions. Whole cell recordings were used to examine effects of volatile anesthetic on TASK currents in cortical neurons and to verify loss of anesthetic-activated TASK currents from TASK-/- mice. A transient middle cerebral artery occlusion (tMCAO) model was used to establish brain ischemia-reperfusion injury. Quantitative RT-PCR analysis revealed that TASK mRNA was reduced by >90% in cortex and hippocampus of TASK-/- mice. The TASK-/- mice showed a much larger region of infarction than C57BL/6 J mice after tMCAO challenge. Isoflurane or sevoflurane administered after the ischemic insult reduced brain infarct percentage and neurological deficit scores in C57BL/6 J mice, these effect were reduced in TASK-/- mice. Whole cell recordings revealed that the isoflurane-activated background potassium current observed in cortical pyramidal neurons from wild type mice was conspicuously reduced in TASK-/- mice. Our studies demonstrate that TASK channels can limit ischemia-reperfusion damage in the cortex, and postconditioning with volatile anesthetics provides neuroprotective actions that depend, in part, on activation of TASK currents in cortical neurons.

HCN subunit-specific and cAMP-modulated effects of anesthetics on neuronal pacemaker currents.

General anesthetics have been a mainstay of surgical practice for more than 150 years, but the mechanisms by which they mediate their important clinical actions remain unclear. Ion channels represent important anesthetic targets, and, although GABA(A) receptors have emerged as major contributors to sedative, immobilizing, and hypnotic effects of intravenous anesthetics, a role for those receptors is less certain in the case of inhalational anesthetics. The neuronal hyperpolarization-activated pacemaker current (Ih) is essential for oscillatory and integrative properties in numerous cell types. Here, we show that clinically relevant concentrations of inhalational anesthetics modulate neuronal Ih and the corresponding HCN channels in a subunit-specific and cAMP-dependent manner. Anesthetic inhibition of Ih involves a hyperpolarizing shift in voltage dependence of activation and a decrease in maximal current amplitude; these effects can be ascribed to HCN1 and HCN2 subunits, respectively, and both actions are recapitulated in heteromeric HCN1-HCN2 channels. Mutagenesis and simulations suggest that apparently distinct actions of anesthetics on V(1/2) and amplitude represent different manifestations of a single underlying mechanism (i.e., stabilization of channel closed state), with the predominant action determined by basal inhibition imposed by individual subunit C-terminal domains and relieved by cAMP. These data reveal a molecular basis for multiple actions of anesthetics on neuronal HCN channels, highlight the importance of proximal C terminus in modulation of HCN channel gating by diverse agents, and advance neuronal pacemaker channels as potentially relevant targets for clinical actions of inhaled anesthetics.

Variability in Mechanical Ventilation: What's All the Noise About?

Controlled mechanical ventilation is characterized by a fixed breathing frequency and tidal volume. Physiological and mathematical models have demonstrated the beneficial effects of varying tidal volume and/or inspiratory pressure during positive-pressure ventilation. The addition of noise (random changes) to a monotonous nonlinear biological system, such as the lung, induces stochastic resonance that contributes to the recruitment of collapsed alveoli and atelectatic lung segments. In this article, we review the mechanism of physiological pulmonary variability, the principles of noise and stochastic resonance, and the emerging understanding that there are beneficial effects of variability during mechanical ventilation.

Distinct regulation of expressed calcium channels 2.3 in Xenopus oocytes by direct or indirect activation of protein kinase C.

Protein kinase C (PKC)-dependent regulation of voltage-gated Ca (Ca(v); with alpha(1)beta1Balpha2/delta subunits) channel 2.3 was investigated using phorbol 12-myristate 13-acetate (PMA), or by M(1) muscarinic receptor activation in Xenopus oocytes. The inward Ca(2+)-current with Ba(2+) (I(Ba)) as the charge carrier was potentiated by PMA or acetyl-beta-methylcholine (MCh). The inactivating [I(inact)] and non-inactivating [I(noninact)] components of I(Ba) and the time constant of inactivation tau(inact) were all increased by MCh or PMA. This may be a PKC-dependent action since the effect of MCh and PMA was blocked by Ro-31-8425 or beta-pseudosubstrate. MCh effect was blocked by atropine, guanosine-5'-O-(2-thiodiphosphate) trilithium (GDPbetaS) or U-73122. The effect of MCh but not PMA was blocked by the inhibition of inositol-1,4,5-trisphosphate (IP3) receptors, intracellular Ca(2+) ([Ca(2+)](i)) or the translocation of conventional PKC (cPKC) with heparin, BAPTA and betaC2.4, respectively. While a lower concentration (25 nM) of Ro-31-8425 blocked MCh, a higher concentration (500 nM) of Ro-31-8425 was required to block PMA action. This differential susceptibility of MCh and PMA to heparin, BAPTA, betaC2.4 or Ro-31-8425 is suggestive of the involvement of Ca(2+)-dependent cPKC in MCh action, whereas cPKC and Ca(2+)-independent novel PKC (nPKC) in PMA action. PMA led to additional increase in I(Ba) that was already potentiated by preadministered MCh (1 or 10 microM), leading to the suggestion that differential phosphorylation sites for cPKC and nPKC may be present in the alpha(1)2.3 subunit of Ca(v) 2.3 channels.

Formalin-induced short- and long-term modulation of Cav currents expressed in Xenopus oocytes: an in vitro cellular model for formalin-induced pain.

Xenopus oocytes expressing high voltage-gated calcium channels (Ca(v)) were exposed to formalin (0.5%, v/v, 5 min.) and the oocyte death and Ca(v) currents were studied for up to 10 days. Ca(v) channels were expressed with alpha(1)beta(1)b and alpha(2)delta sub-units and the currents (I(Ba)) were studied by voltage clamp. None of the oocytes was dead during the exposure to formalin. Oocyte death was significant between day 1 and day 5 after the exposure to formalin and was uniform among the oocytes expressing various Ca(v) channels. Peak I(Ba) of all Ca(v) and A(1), the inactivating current component was decreased whereas the non-inactivated R current was not affected by 5 min. exposure to formalin. On day 1 after the exposure to formalin, Ca(v)1.2c currents were increased, 2.1 and 2.2 currents were decreased and 2.3 currents were unaltered. On day 5, both peak I(Ba) and A(1) currents were increased. Ca(v)1.2c, 2.2 and 2.3 currents were increased and Ca(v)2.1 was unaltered on day 10 after the exposure to formalin. Protein kinase C (PKC) may be involved in formalin-induced increase in Ca(v) currents due to the (i) requirement for Ca(v)beta(1)b sub-units; (ii) decreased phorbol-12-myristate,13-acetate potentiation of Ca(v)2.3 currents; (iii) absence of potentiation of Ca(v)2.3 currents following down-regulation of PKC; and (iv) absence of potentiation of Ca(v)2.2 or 2.3 currents with Ser-->Ala mutation of Ca(v)alpha(1)2.2 or 2.3 sub-units. Increased Ca(v) currents and PKC activation may coincide with changes observed in in vivo pain investigations, and oocytes incubated with formalin may serve as an in vitro model for some cellular mechanisms of pain.

Electrophysiologic mechanism underlying action potential prolongation by sevoflurane in rat ventricular myocytes.

BACKGROUND: Despite prolongation of the QTc interval in humans during sevoflurane anesthesia, little is known about the mechanisms that underlie these actions. In rat ventricular myocytes, the effect of sevoflurane on action potential duration and underlying electrophysiologic mechanisms were investigated. METHODS: The action potential was measured by using a current clamp technique. The transient outward K current was recorded during depolarizing steps from -80 mV, followed by brief depolarization to -40 mV and then depolarization up to +60 mV. The voltage dependence of steady state inactivation was determined by using a standard double-pulse protocol. The sustained outward current was obtained by addition of 5 mm 4-aminopyridine. The inward rectifier K current was recorded from a holding potential of -40 mV before their membrane potential was changed from -130 to 0 mV. Sevoflurane actions on L-type Ca current were also obtained. RESULTS: Sevoflurane prolonged action potential duration, whereas the amplitude and resting membrane potential remained unchanged. The peak transient outward K current at +60 mV was reduced by 18 +/- 2% (P < 0.05) and 24 +/- 2% (P < 0.05) by 0.35 and 0.7 mm sevoflurane, respectively. Sevoflurane had no effect on the sustained outward current. Whereas 0.7 mm sevoflurane did not shift the steady state inactivation curve, it accelerated the current inactivation (P < 0.05). The inward rectifier K current at -130 mV was little altered by 0.7 mm sevoflurane. L-type Ca current was reduced by 28 +/- 3% (P < 0.05) and 33 +/- 1% (P < 0.05) by 0.35 and 0.7 mm sevoflurane, respectively. CONCLUSIONS: Action potential prolongation by clinically relevant concentrations of sevoflurane is due to the suppression of transient outward K current in rat ventricular myocytes.

Myocardial depressant effects of desflurane: mechanical and electrophysiologic actions in vitro.

BACKGROUND: The authors determined whether desflurane altered myocardial excitation-contraction coupling and electrophysiologic behavior in the same manner as isoflurane and sevoflurane. METHODS: The effects of desflurane on isometric force in guinea pig ventricular papillary muscles were studied in modified standard and in 26 mM K(+) Tyrode solution with 0.1 microm isoproterenol. Desflurane effects on sarcoplasmic reticulum Ca(2+) release were also determined by examining its actions on rat papillary muscles, guinea pig papillary muscles in low-Na(+) Tyrode solution, and rapid cooling contractures. Normal and slow action potentials were recorded using a conventional microelectrode technique. Ca(2+) and K(+) currents of guinea pig ventricular myocytes were examined. RESULTS: Desflurane (5.3% and 11.6%) decreased peak force to approximately 70% and 40% of the baseline, respectively, similar to the effects of equianesthetic isoflurane concentrations. With isoproterenol in 26 mM K(+) Tyrode solution, desflurane markedly depressed late peaking force and modestly depressed early peak force. The rested state contractions of rat myocardium or guinea pig myocardium in low-Na(+) Tyrode solution were modestly depressed, whereas rapid cooling contractures were virtually abolished after desflurane administration. Desflurane significantly prolonged the action potential duration. Desflurane reduced L-type Ca(2+) current and the delayed outward K(+) current but did not alter the inward rectifier K(+) current. CONCLUSIONS: Myocardial depression by desflurane is due to decreased Ca(2+) influx, whereas depolarization-activated sarcoplasmic reticulum Ca(2+) release is modestly depressed, similar to the actions of isoflurane and sevoflurane. Desflurane depressed the delayed outward K(+) current associated with significant lengthening of cardiac action potentials.

Convergent and reciprocal modulation of a leak K+ current and I(h) by an inhalational anaesthetic and neurotransmitters in rat brainstem motoneurones.

Neurotransmitters and volatile anaesthetics have opposing effects on motoneuronal excitability which appear to reflect contrasting modulation of two types of subthreshold currents. Neurotransmitters increase motoneuronal excitability by inhibiting TWIK-related acid-sensitive K+ channels (TASK) and shifting activation of a hyperpolarization-activated cationic current (I(h)) to more depolarized potentials; on the other hand, anaesthetics decrease excitability by activating a TASK-like current and inducing a hyperpolarizing shift in I(h) activation. Here, we used whole-cell recording from motoneurones in brainstem slices to test if neurotransmitters (serotonin (5-HT) and noradrenaline (NA)) and an anaesthetic (halothane) indeed compete for modulation of the same ion channels - and we determined which prevails. When applied together under current clamp conditions, 5-HT reversed anaesthetic-induced membrane hyperpolarization and increased motoneuronal excitability. Under voltage clamp conditions, 5-HT and NA overcame most, but not all, of the halothane-induced current. When I(h) was blocked with ZD 7288, the neurotransmitters completely inhibited the K+ current activated by halothane; the halothane-sensitive neurotransmitter current reversed at the equilibrium potential for potassium (E(K)) and displayed properties expected of acid-sensitive, open-rectifier TASK channels. To characterize modulation of I(h) in relative isolation, effects of 5-HT and halothane were examined in acidified bath solutions that blocked TASK channels. Under these conditions, 5-HT and halothane each caused their characteristic shift in voltage-dependent gating of I(h). When tested concurrently, however, halothane decreased the neurotransmitter-induced depolarizing shift in I(h) activation. Thus, halothane and neurotransmitters converge on TASK and I(h) channels with opposite effects; transmitter action prevailed over anaesthetic effects on TASK channels, but not over effects on I(h). These data suggest that anaesthetic actions resulting from effects on either TASK or hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in motoneurones, and perhaps at other CNS sites, can be modulated by prevailing neurotransmitter tone.

Identification of sites responsible for potentiation of type 2.3 calcium currents by acetyl-beta-methylcholine.

To address mechanisms for the differential sensitivity of voltage-gated Ca2+ channels (Cav) to agonists, channel activity was compared in Xenopus oocytes coexpressing muscarinic M(1) receptors and different Cav alpha1 subunits, all with beta1B,alpha2/delta subunits. Acetyl-beta-methylcholine (MCh) decreased Cav 1.2c currents, did not affect 2.1 or 2.2 currents, but potentiated Cav 2.3 currents. Phorbol 12-myristate 13-acetate (PMA) did not affect Cav 1.2c or 2.1 currents but potentiated 2.2 and 2.3 currents. Comparison of the amino acid sequences of the alpha1 subunits revealed a set of potential protein kinase C phosphorylation sites in common between the 2.2 and 2.3 channels that respond to PMA and a set of potential sites unique to the alpha1 2.3 subunits that respond to MCh. Quadruple Ser --> Ala mutation of the predicted MCh sites in the alpha1 2.3 subunit (Ser-888, Ser-892, and Ser-894 in the II-III linker and Ser-1987 in the C terminus) caused loss of the MCh response but not the PMA response. Triple Ser --> Ala mutation of just the II-III linker sites gave similar results. Ser-888 or Ser-892 was sufficient for the MCh responsiveness, whereas Ser-894 required the presence of Ser-1987. Ser --> Asp substitution of Ser-888, Ser-892, Ser-1987, and Ser-892/Ser-1987 increased the basal current and decreased the MCh response but did not alter the PMA response. These results reveal that sites unique to the II-III linker of alpha1 2.3 subunits mediate the responsiveness of Cav 2.3 channels to MCh. Because Cav 2.3 channels contribute to action potential-induced Ca2+ influx, these sites may account for M1 receptor-mediated regulation of neurotransmission at some synapses.