Veit-Simon Eckle

Inhibition of T-type calcium current in rat thalamocortical neurons by isoflurane

Thalamocortical (TC) neurons provide the major sensory input to the mammalian somatosensory cortex. Decreased activity of these cells may be pivotal in the ability of general anesthetics to induce loss of consciousness and promote sleep (hypnosis). T-type voltage-gated calcium currents (T-currents) have a key function regulating the cellular excitability of TC neurons and previous studies have indicated that volatile general anesthetics may alter the excitability of these neurons. Using a patch-clamp technique, we investigated the mechanisms whereby isoflurane, a common volatile anesthetic, modulates isolated T-currents and T-current-dependent excitability of native TC neurons in acute brain slices of the rat. In voltage-clamp experiments, we found that isoflurane strongly inhibited peak amplitude of T-current, yielding an IC(50) of 1.1 vol-% at physiological membrane potentials. Ensuing biophysical studies demonstrated that inhibition was more prominent at depolarized membrane potentials as evidenced by hyperpolarizing shifts in channel availability curves. In current-clamp experiments we found that isoflurane decreased the rate of depolarization of low-threshold-calcium spikes (LTCSs) and consequently increased the latency of rebound spike firing at the same concentrations that inhibited isolated T-currents. This effect was mimicked by a novel selective T-channel blocker 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2). In contrast, isoflurane and TTA-P2 had minimal effect on resting membrane potential and cell input resistance. We propose that the clinical properties of isoflurane may at least partly be provided by depression of thalamic T-currents.

Propofol modulates phasic and tonic GABAergic currents in spinal ventral horn interneurones

BACKGROUND Surgical interventions like skin incisions trigger withdrawal reflexes which require motor neurones and local circuit interneurones in the spinal ventral horn. This region plays a key role in mediating immobilizing properties of the GABAergic anaesthetic propofol. However, it is unclear how propofol modulates GABA(A) receptors in the spinal ventral horn and whether tonic or phasic inhibition is involved. METHODS Organotypic spinal cord tissue slices were prepared from mice. Whole-cell recordings were performed for quantifying effects of propofol on GABA(A) receptor-mediated phasic transmission and tonic conductance. RESULTS Propofol increased GABAergic phasic transmission by a prolongation of the decay time constant in a concentration-dependent manner. The amount of the charge transferred per inhibitory post-synaptic current, described by the area under the curve, was significantly augmented by 1 µM propofol (P<0.01). A GABA(A) receptor-mediated tonic current was not induced by 1 µM propofol but at a concentration of 5 µM (P<0.05). CONCLUSIONS Propofol depresses ventral horn interneurones predominantly by phasic rather than by tonic GABA(A) receptor-mediated inhibition. However, the present results suggest that the involvement of a tonic inhibition might contribute to the efficacy of propofol to depress nociceptive reflexes at high concentrations of the anaesthetic.

Opposing Actions of Sevoflurane on GABAergic and Glycinergic Synaptic Inhibition in the Spinal Ventral Horn

Background The ventral horn is a major substrate in mediating the immobilizing properties of the volatile anesthetic sevoflurane in the spinal cord. In this neuronal network, action potential firing is controlled by GABAA and glycine receptors. Both types of ion channels are sensitive to volatile anesthetics, but their role in mediating anesthetic-induced inhibition of spinal locomotor networks is not fully understood. Methodology/Principal Findings To compare the effects of sevoflurane on GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs) whole-cell voltage-clamp recordings from ventral horn interneurons were carried out in organotypic spinal cultures. At concentrations close to MAC (minimum alveolar concentration), decay times of both types of IPSCs were significantly prolonged. However, at 1.5 MAC equivalents, GABAergic IPSCs were decreased in amplitude and reduced in frequency. These effects counteracted the prolongation of the decay time, thereby decreasing the time-averaged GABAergic inhibition. In contrast, amplitudes and frequency of glycinergic IPSCs were not significantly altered by sevoflurane. Furthermore, selective GABAA and glycine receptor antagonists were tested for their potency to reverse sevoflurane-induced inhibition of spontaneous action potential firing in the ventral horn. These experiments confirmed a weak impact of GABAA receptors and a prominent role of glycine receptors at a high sevoflurane concentration. Conclusions At high concentrations, sevoflurane mediates neuronal inhibition in the spinal ventral horn primarily via glycine receptors, and less via GABAA receptors. Our results support the hypothesis that the impact of GABAA receptors in mediating the immobilizing properties of volatile anesthetics is less essential in comparison to glycine receptors.

ALX 1393 inhibits spontaneous network activity by inducing glycinergic tonic currents in the spinal ventral horn.

BACKGROUND Strychnine-sensitive glycine receptors are activated by glycine and facilitate chloride influx into neurons. Glycinergic transmission might be either mediated by synaptic or extrasynaptic glycine receptors. While phasic neurotransmission is provided by a synaptic pathway, activation of extrasynaptic glycine receptors induces tonic inhibition. The glycine transporter 2 (GlyT2) regulates the uptake of glycine into presynaptic boutons. It is not determined yet, whether inhibition of GlyT2 by ALX 1393 can produce inhibition of spinal motoric networks and, whether phasic or tonic glycinergic inhibition is mostly enhanced. METHODS We investigated the effect of ALX 1393 on spontaneous action potential firing activity by extracellular recordings in the ventral horn area of organotypic spinal cultures. Additionally, using the whole-cell patch-clamp technique, we defined the influence of GlyT2 inhibition on tonic and phasic glycinergic transmission in commissural interneurons of the ventral horn. RESULTS GlyT2 inhibition by ALX 1393 potently reduced neuronal action potential activity in a concentration-dependent manner (n=211). The half maximal effect of ALX 1393 was observed at 100 ± 31 nM. Moreover, 88.3 ± 2.6% of the action potential activity was suppressed at 1 μM. Whole-cell patch-clamp recordings unveiled that ALX 1393 (200 nM) induced a tonic current (-45.7 ± 11.6 pA, n=5) that was significantly reversed by application of the competitive glycine receptor antagonist strychnine. Contrastingly, phasic glycinergic transmission was not augmented by GlyT2 inhibition (charge transferred per time period for control conditions: 1.1 ± 0.1 pC, n=7, for ALX 1393: 0.9 ± 0.2 pC, n=7, p>0.05). CONCLUSION GlyT2 inhibition induced glycinergic tonic currents, which might be the underlying mechanism for the observed suppression of spontaneous action potential activity by ALX 1393 in the spinal ventral horn. Silencing neuronal action potential activity by blocking GlyT2 might be a novel principle to inhibit locomotor circuits in the ventral horn area and to induce muscle relaxation.

Spinal cord--skeletal muscle cocultures detect muscle-relaxant action of botulinum neurotoxin A.

The mouse LD50 assay is routinely used for potency testing of botulinum toxins. Unfortunately, this test is associated with severe pain and distress in animals and requires large quantities of mice. Here we used cocultures of spinal cord and muscle tissue as an alternative for probing botulinum toxins. Cocultures were prepared from mouse embryonic tissue (C57/BL6J) and cultured for 24-27 days. In these cultures spontaneous muscle activity was quantified in sham- and botulinum toxin-treated cultures for up to 3 days by video microscopy. At a concentration of 58 fmol/L or higher, incobotulinumtoxin A significantly reduced the frequency of muscle contractions within 24 hours after incubation. Hence, nerve-muscle-cultures are similar sensitive as the mouse LD50 assay. The limit of detection, as observed in our study, is close to the most sensitive cell-based bioassays, capable to detect concentrations of botulinum neurotoxin A between 30 and 50 fmol/L. However, spontaneous muscle activity of individual cultures displayed considerable fluctuations when evaluated on a day-to-day basis. Generally, the authors would like to emphasize, that in its present form, this in vitro assay might be too laborious for botulinum toxin potency testing. Thus, methodical improvements to decrease data variability are the next milestone to be passed towards developing this model into an assay that can be utilized for reducing animal experimentation.

Botulinum toxin B increases intrinsic muscle activity in organotypic spinal cord–skeletal muscle co-cultures

In organotypic spinal cord-skeletal muscle co-cultures, motoneurons are driven by locomotor commands and induce contractions in surrounding muscle fibres. Using these co-cultures, it has been shown that effects of organophosphorus compounds on neuromuscular synapses can be determined in vitro. In the present study we aimed to extend this in vitro tool for pharmacologic testing of botulinum toxin B. This neurotoxin is widely used for the treatment of dystonia. Besides its effects on the neuromuscular junction, botulinum toxins may also act at centrally located synapses. Incubation with botulinum toxin B (Neurobloc(®)) induced a significant increase in muscular activity after 24, 48 and 72h. Application of the NMDA- and AMPA-receptor antagonists AP5 (20μM) and CNQX (15μM) induced a similar augmentation of muscle activity after 48 and 72h, respectively. Administration of the glycine- and GABA(A)-receptor antagonists strychnine (1μM) and bicuculline (100μM) did not alter intrinsic muscle activity. In contrast, application of a non-depolarizing muscle relaxant rocuronium bromide reduced the muscle activity in a dose-dependent manner. Our findings suggest that glutamatergic synapses in the spinal cord are more sensitive to botulinum toxin B than synaptic contacts between spinal motoneurons and muscle fibres.

Intrajugular balloon catheter reduces air embolism in vitro and in vivo

Background Neurosurgical procedures requiring a sitting position may put the patient at risk of a potentially life-threatening air embolism. Transient manual jugular venous compression limits further air entry in this situation. This study presents an alternative technique aimed at reducing the risk of air embolism. Methods In an in vitro model, an intrajugular balloon catheter was inserted to demonstrate that this device prevents air embolism. In an in vivo study, this device was bilaterally placed into jugular vessels in pigs. Using an ultrasound technique, blood flow was monitored and jugular venous pressure was recorded before and during cuff inflation. Air was applied proximally to the inflated cuffs to test the hypothesis that this novel device blocks air passage. Results In vitro, the intrajugular balloon catheter reliably prevented further air entry (n=10). Additionally, accumulated air could be aspirated from an orifice of the catheter (n=10). In vivo, inflation of the catheter balloon completely obstructed venous blood flow (n=8). Bilateral inflation of the cuff significantly increased the proximal jugular venous pressure from 9.8 (2.4) mm Hg to 14.5 (2.5) mm Hg (n=8, P<0.05). Under conditions mimicking an air embolism, air passage across the inflated cuffs was prevented and 78 (20%) (n=6) of the air dose could be aspirated by the proximal orifice of the catheter. Conclusions These findings may serve as a starting point for the development of intrajugular balloon catheters designed to reduce the risk of air embolism in patients undergoing neurosurgery in a sitting position.

Incompatibility of Piritramide with Cephalosporins

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Präzipitation von Piritramid und Cefazolin

BACKGROUND Drug incompatibility might lead to precipitation with subsequent serious complications, such as transient pulmonary embolism. Recently, incompatibility of the opioid piritramide with cephalosporin antibiotics was described. As both drugs are frequently administered in a perioperative setting, the present study addressed the question whether the precipitation effect depends on the piritramide concentration or on the pH of the solution. Moreover, it was tested whether the precipitate reversibly dissolves in a physiological saline solution. METHODS Piritramide was diluted to the final test concentrations in 0.9 % sodium chloride solution. Precipitation tests were performed by combining 1 ml of the respective piritramide dilution with 1 ml of cefazolin (20 mg/ml) in a syringe. Precipitation was detected by visual inspection as an opaque whitish appearance of the mixture. Each concentration was tested 5 times. The pH values of the tested piritramide concentrations were determined using a 3-point calibrated pH meter. The precipitate formed in 1 ml of cefazolin (20 mg/ml) and 1 ml of piritramide (5 mg/ml) was diluted in 3 ml physiological saline. RESULTS The piritramide concentrations 5 mg/ml, 3.75 mg/ml and 3 mg/ml precipitated in the presence of cefazolin (20 mg/ml), while the concentrations 1.875 mg/ml, 1 mg/ml and 0.5 mg/ml did not produce a precipitate. To exclude the possibility that changes in pH of the tested dilutions might be responsible for these findings, the pH values of the piritramide dilutions were measured. The mean pH values of the concentrations 5 mg/ml, 3.75 mg/ml, 3 mg/ml, 1.875 mg/ml and 1 mg/ml did not differ significantly (pH 3.89 ± 0.004, n = 26, tested by ANOVA). However, the mean pH of 0.5 mg/ml was significantly different from the other tested dilutions (pH 3.98 ± 0.02, n = 6; p < 0.01 by ANOVA). After diluting the precipitate of piritramide and cefazolin in physiological saline the whitish precipitate completely dissolved and the resulting solution became clear (n = 5). CONCLUSION The results imply a concentration dependence of the precipitation with cefazolin, while a correlation with pH changes could not be detected. In cases of co-administration of cephalosporins and piritramide, a piritramide concentration of 1 mg/ml seems to be safe and does not form a precipitate. As the precipitate could be reversed by diluting in saline solution it is most likely that a proton switch between the carboxylic acid moiety of cefazolin and the amino group of piritramide causes the precipitation.

Recovery of residual curarization after red blood cell transfusion

Summary Background The muscle-relaxing effects of succinylcholine are terminated via hydrolysis by plasma cholinesterase. There are multiple genetic variants of this enzyme and clinical circumstances that might influence the activity of plasma cholinesterase and eventually lead to prolonged neuromuscular blockade following succinylcholine application. Case Report Here, we report a parturient woman with atonic bleeding who suffered significant blood loss (hemoglobin 6.0 g·dL−1). For surgical curettage, general anesthesia was performed by using short-acting succinylcholine. By the end of the 105-minute procedure, the patient’s trachea was extubated. After extubation she showed signs of the prolonged neuromuscular blocking action of succinylcholine. At this time, the patient received an AB0-compatible red blood cell transfusion and recovered instantly from neuromuscular blockade. The plasma cholinesterase (3.200 U·L−1) was below the normal range (4.900–12.000 U·L−1). Patient’s blood DNA analysis revealed heterozygously the genetic K variant of plasma cholinesterase. After red blood cell transfusion, serum potassium was elevated (5.7 mmol·L−1; 4.4 mmol·L−1 prior to transfusion). Conclusions Pregnancy, blood loss and genetic variation contributed to impairment of plasma cholinesterase. Due to high-speed red blood cell transfusion, hemolytic release of erythrocyte cholinesterase might have terminated the neuromuscular blocking succinylcholine effect.