Claudia Benkwitz

Determination of the EC50 amnesic concentration of etomidate and its diffusion profile in brain tissue: implications for in vitro studies.

Background:Etomidate is a widely used general anesthetic that has become a useful tool to investigate mechanisms of anesthetic action in vivo and in brain slices. However, the free aqueous concentration of etomidate that corresponds to amnesia in vivo and the diffusion profile of etomidate in brain slices are not known. Methods:The authors assessed the effect of intraperitoneally injected etomidate on contextual fear conditioning in mice. Etomidate concentrations in brain tissue were obtained by high-performance liquid chromatography. Uptake studies in 400-&mgr;m-thick brain slices were used to calculate the diffusion and partition coefficients of etomidate. A diffusion model was used to calculate the expected concentration profile within a brain slice as a function of time and depth. The predicted rate of drug equilibration was compared with the onset of electrophysiologic effects on inhibitory circuit function in recordings from hippocampal brain slices. Results:Etomidate impaired contextual fear conditioning with an ED50 dose of 11.0 ± 0.1 mg after intraperitoneal injection, which corresponded to an EC50 brain concentration of 208 ± 9 ng/g. The brain:artificial cerebrospinal fluid partition coefficient was 3.35, yielding an EC50,amnesia aqueous concentration of 0.25 &mgr;m. The diffusion coefficient was approximately 0.2 × 10−6 cm2/s. The development of etomidate action in hippocampal brain slices was compatible with the concentration profile predicted by this diffusion coefficient. Conclusions:The free aqueous concentration of etomidate corresponding to amnesia, as defined by impaired contextual fear conditioning in mice, is 0.25 &mgr;m. Diffusion of etomidate into brain slices requires approximately an hour to reach 80% equilibration at a typical recording depth of 100 &mgr;m. This information will be useful in designing and interpreting in vitro studies using etomidate.

Influence of GABAA receptor γ2 splice variants on receptor kinetics and isoflurane modulation

Background:γ-Aminobutyric acid type A (GABAA) receptors, the major inhibitory receptors in the brain, are important targets of many drugs, including general anesthetics. These compounds exert multiple effects on GABAA receptors, including direct activation, prolongation of deactivation kinetics, and reduction of inhibitory postsynaptic current amplitudes. However, the degree to which these actions occur differs for different agents and synapses, possibly because of subunit-specific effects on postsynaptic receptors. In contrast to benzodiazepines and intravenous anesthetics, there is little information available about the subunit dependency of actions of volatile anesthetics. Therefore, the authors studied in detail the effects of isoflurane on recombinant GABAA receptors composed of several different subunit combinations. Methods:Human embryonic kidney 293 cells were transiently transfected with rat complementary DNAs of α1β2, α1β2γ2L, α1β2γ2S, α5β3, or α5β3γ2S subunits. Using rapid application and whole cell patch clamp techniques, cells were exposed to 10- and 2,000-ms pulses of γ-aminobutyric acid (1 mm) in the presence or absence of isoflurane (0.25, 0.5, 1.0 mm). Anesthetic effects on decay kinetics, peak amplitude, net charge transfer and rise time were measured. Statistical significance was assessed using the Student t test or one-way analysis of variance followed by the Tukey post hoc test. Results:Under control conditions, incorporation of a γ2 subunit conferred faster deactivation kinetics and reduced desensitization. Isoflurane slowed deactivation, enhanced desensitization, and reduced peak current amplitude in αβ receptors. Coexpression with a γ2 subunit caused these effects of isoflurane to be substantially reduced or abolished. Although the two γ2 splice variants imparted qualitatively similar macroscopic kinetic properties, there were significant quantitative differences between effects of isoflurane on deactivation and peak current amplitude in γ2S- versus γ2L-containing receptors. The net charge transfer resulting from brief pulses of γ-aminobutyric acid was decreased by isoflurane in αβ but increased in αβγ receptors. Conclusions:The results indicate that subunit composition does substantially influence modulation of GABAA receptors by isoflurane. Specifically, the presence of a γ2 subunit and the identity of its splice variant are important factors in determining physiologic and pharmacologic properties. These results may have functional implications in understanding how anesthetic effects on specific types of GABAA receptors in the brain contribute to changes in brain function and behavior.

Lidocaine Enhances Gαi Protein Function

Background Local anesthetics inhibit several G protein–coupled receptors by interaction with the G&agr;q protein subunit. It is not known whether this effect on G protein function can be extrapolated to other classes of G proteins. The authors investigated interactions of lidocaine with the human adenosine 1 receptor (hA1R)–coupled signaling pathway. Activated A1Rs couple to adenylate cyclase via the pertussis toxin sensitive G&agr;i protein, thereby decreasing cyclic adenosine monophosphate formation. A1Rs are widely expressed and abundant in the spinal cord, brain, and heart. Interactions of LAs with the hA1R-coupled transduction cascade therefore might produce a broad range of clinically relevant effects. Methods The function of hA1Rs stably expressed in Chinese hamster ovary cells was determined with assays of cyclic adenosine monophosphate, receptor binding, and guanosine diphosphate/guanosine triphosphate &ggr;35S exchange by using reconstituted defined G protein subunits. Involvement of phosphodiesterase and G&agr;i was characterized by using the phosphodiesterase inhibitor rolipram and pertussis toxin, respectively. Results Lidocaine (10−9–10−1 M) had no significant effects on agonist or antagonist binding to the hA1R or on receptor–G protein interactions. However, cyclic adenosine monophosphate levels were reduced significantly to 50% by the LAs, even in the absence of an A1R agonist or presence of an A1R antagonist. This effect was unaffected by rolipram (10 &mgr;m), but abolished completely by pretreatment with pertussis toxin, which inactivates the G&agr;i protein. Therefore, the main target site for LAs in this pathway is located upstream from adenylate cyclase. Conclusions Lidocaine potentiates G&agr;i-coupled A1R signaling by reducing cyclic adenosine monophosphate production. The study suggests an interaction site for LAs in a G&agr;i-coupled signaling pathway, with the G&agr;i protein representing the prime candidate. Taken together with previous results showing inhibitory LA interactions on the G&agr;q protein subunit, the data in the current study support the hypothesis that specific G protein subunits represent alternative sites of LA action.

Assessing the Risks of Noncardiac Surgery for Children With Congenital Heart Disease

Children with congenital heart disease (CHD) who were once considered “inoperable” are increasingly living into adulthood with improved quality of life. By some accounts, there are now more adults than children living with CHD [(1)][1]. This turning point reflects decades of improvement across

Wandering Epidural Catheter

A MALE patient, 73 yr old, had a Flexitip Plus (Arrow International, Reading, PA) 19gauge end-hole, wire-reinforced thoracic epidural catheter inserted for postthoracotomy pain management. He underwent highresolution spiral computed tomography scan with the catheter in situ before its use to evaluate previous bronchoscopic findings. A composite set of sagittal computed tomography images (figs. A and B) show twoand three-dimensional thoracic spine reconstructions that highlight the track of the epidural catheter in this patient. Figure A shows a three-dimensional reconstruction of the spine sliced down midline, looking toward the right with the catheter looped away from the viewer, and resting adjacent to the pedicle (bold arrow). Figure B is a two-dimensional cross-sectional computed tomography image demonstrating the course of the epidural catheter from outside the skin to within the epidural space, with anterolateral placement of the catheter tip terminating adjacent to the pedicle (circles). The catheter first loops into the anterior and then back into the lateral epidural space terminating adjacent to the pedicle. This is a commonly underappreciated track of an epidural catheter. It is generally assumed that epidural catheters travel cephalad in the posterior epidural space. However, this image shows definitive evidence of the circuitous route often taken by an epidural catheter. Conventional teaching is to insert an epidural catheter 5 cm into the epidural space. Several studies contradict this and show that a low number of catheters are successfully advanced into the epidural space without coiling or deviating from their intended track. Loose areolar tissue, fat, and blood vessels in the epidural space can contribute to deviations in final placement of the catheter tip. Lim et al. showed that approximately 65% of epidural catheter tips deviate laterally in the epidural space immediately after leaving the Tuohy needle tip. The catheter tip followed a straight direction in the posterior epidural space for more than 2 cm in only 35% of placements. Further, only 13% of catheter tips could be advanced 4 cm without coiling. Choi et al. observed 17.6% of epidural catheters curling in the epidural space. They also reported that 63.5% of cephalad-directed thoracic epidural catheters (Touhy bevel up) were actually advanced as such, and 78% of caudally directed epidural catheters (Touhy bevel down) were advanced that direction. A negative consequence of lateral epidural catheter tip deviation is misplacement into the paravertebral area via an intervertebral foramen. Misplacement into the anterior epidural space is one of the most common causes of unilateral epidural block.

Double-Blind, Randomized, Placebo-Controlled Trial Comparing the Effects of Antithrombin Versus Placebo on the Coagulation System in Infants with Low Antithrombin Undergoing Congenital Cardiac Surgery.

OBJECTIVES To determine whether precardiopulmonary bypass (CPB) normalization of antithrombin levels in infants to 100% improves heparin sensitivity and anticoagulation during CPB and has beneficial effects into the postoperative period. DESIGN Randomized, double-blinded, placebo-controlled prospective study. SETTING Multicenter study performed in 2 academic hospitals. PARTICIPANTS The study comprised 40 infants younger than 7 months with preoperative antithrombin levels <70% undergoing CPB surgery. INTERVENTIONS Antithrombin levels were increased with exogenous antithrombin to 100% functional level intraoperatively before surgical incision. MEASUREMENTS AND MAIN RESULTS Demographics, clinical variables, and blood samples were collected up to postoperative day 4. Higher first post-heparin activated clotting times (sec) were observed in the antithrombin group despite similar initial heparin dosing. There was an increase in heparin sensitivity in the antithrombin group. There was significantly lower 24-hour chest tube output (mL/kg) in the antithrombin group and lower overall blood product unit exposures in the antithrombin group as a whole. Functional antithrombin levels (%) were significantly higher in the treatment group versus placebo group until postoperative day 2. D-dimer was significantly lower in the antithrombin group than in the placebo group on postoperative day 4. CONCLUSION Supplementation of antithrombin in infants with low antithrombin levels improves heparin sensitivity and anticoagulation during CPB without increased rates of bleeding or adverse events. Beneficial effects may be seen into the postoperative period, reflected by significantly less postoperative bleeding and exposure to blood products and reduced generation of D-dimers.

Thrombotic Endotracheal Tube Occlusion After Administration of Recombinant Factor VIIa

c RECOMBINANT HUMAN FACTOR VIIa (rFVIIa) promotes hemostasis by interacting with both the extrinsic nd intrinsic pathways of the coagulation cascade.1 Although FVIIa may be very effective in the treatment of severe bleedng after cardiac surgery, its cost and potential to cause major hrombotic complications have limited its use. Data describing hrombotic events associated with rFVIIa are scarce, and there s a debate as to the role of rFVIIa after cardiac surgery.2-4 A case in which the administration of rFVIIa for persistent bleeding after cardiopulmonary bypass (CPB) in a child was followed by repeated, complete thrombotic occlusion of the endotracheal tube (ETT) is presented.