[Intern(euron)al affairs : The role of specific neocortical interneuron classes in the interaction between acetylcholine and GABAergic anesthetics]. / Intern(euron)al affairs : Die Rolle spezifischer neokortikaler Interneuronentypen bei der Interaktion Acetylcholins mit GABAergen Anästhetika.

Acetylcholine is a neuromodulator which is released throughout the central nervous system and plays an essential role in consciousness and cognitive processes including attention and learning. Due to its 'activating' effect on the neuronal and behavioral level its interaction with anesthetics has long been of interest to anesthesiologists. It is widely held that a reduction of the release of acetylcholine by general anesthetics constitutes part of the anesthetic effect. This notion is backed by numerous human and animal studies, but is also in seeming contradiction to findings that acetylcholine activates specific classes of inhibitory neurons: if acetylcholine excites elements within the neuronal network responsible for the release of the inhibitory neurotransmitter γ-aminobutyric acid (GABA), its withdrawal should diminish, not enhance, the effect of anesthetics.Focusing on cortical circuits, we present an overview of recent advances in cellular neurophysiology, particularly the interactions between inhibitory neuron classes, which provide insights on the interaction between acetylcholine and GABA.

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.

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.

How much oxidative stress exists without the liver?

BACKGROUND: Oxidative stress (OS) represents an important pathogenetic factor of acute liver failure and chronic liver diseases. To elucidate whether the liver itself is a major source of OS, the present study was performed to assess OS and antioxidant status in an anhepatic porcine model. METHODS: Six pigs underwent a total hepatectomy, five pigs were sham operated. OS and antioxidant status were evaluated by measuring plasma concentrations of malondialdehyde (MDA), xanthine oxidase (XO), superoxide dismutase (SOD) and the ferric reducing ability of plasma (FRAP). They were sampled at the start of the experiment, immediately after surgery, and then at 8 and 16 hours post hepatectomy. RESULTS: Increased concentrations of MDA were observed in anhepatic pigs postoperatively (p < 0.02) and 8 hours after hepatectomy (p < 0.003) compared to controls. XO activity increased soon after hepatectomy (22.6 ±â€Š5.4 mU/L versus 3.3 ±â€Š2.1 mU/L in sham animals, p < 0.03) but returned to normal values in the further course. SOD levels did not change during the observational period in both groups. FRAP values rose significantly in the anhepatic animals compared to control (p < 0.015). A significant positive correlation was observed between MDA levels and FRAP levels (Spearman's ρ = 0.62; p < 0.0001). CONCLUSIONS: These findings show that hepatectomy does not completely prevent the occurrence of OS because the production and regulation of OS are also located outside the liver.

[Precipitation of piritramide and cefazolin. Study of the dependence on concentration and pH]. / Präzipitation von Piritramid und Cefazolin. Untersuchungen zur Abhängigkeit von Konzentration und pH.

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.

[Is deep anesthesia dangerous?]. / Ist tiefe Narkose gefährlich?

Regarding the question of an adequate depth of anesthesia, over the past decade anesthesiologists have focused on the prevention of intraoperative consciousness in combination with explicit memory. Recent studies approached the topic from a different way postulating that deep anesthesia, quantified as time with a bispectral index (BIS)< 45, is associated with increased postoperative mortality and four out of the five published studies revealed such a correlation. However, the finding is limited by a suboptimal study design, e.g. none of the studies presented randomized data. Furthermore, it is ambiguous whether the correlation is causal as the administration of deep anesthesia determines higher postoperative mortality or the study results reveal an epiphenomenon. An epiphenomenon implies e.g. that patients with cancer respond to general anesthesia with deeper cortical depression. In summary, as long as there is a lack of adequately performed randomized trials, there is no reason why anesthesiologists should change the current practice.

Depression of spinal network activity by thiopental: shift from phasic to tonic GABA(A) receptor-mediated inhibition.

Interneuronal networks in the spinal ventral horn are plausible substrates for mediating anesthetic-induced immobility. Here, we investigated how their activity is affected by clinically relevant concentrations of thiopental, a barbiturate in clinical use. In cultured spinal cord slices from mice, thiopental reduced action potential activity with an EC(50) of 16.6+/-2.4microM. Recordings of GABA(A) and glycine receptor-mediated inhibitory currents indicated that the effect was largely mediated by GABA(A) receptors and that glycine receptors were not relevant targets. Specifically, 20microM thiopental prolonged the decay time of spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) more than twofold. Although this prolongation of decay time increased the inhibitory charge per sIPSC the concomitant strong reduction of sIPSC frequency resulted in less inhibitory current entering the neurons via this route. However, 20microM thiopental also induced a tonic current of 30+/-10pA, mediated by GABA(A) receptors; 50microM thiopental nearly abolished sIPSC activity but augmented tonic currents to 69+/-14pA. Furthermore, at this concentration, activity-depressing mechanisms independent of GABA(A) receptors came into play. The results suggest that in the spinal ventral horn thiopental acts mostly, but not exclusively, via GABA(A) receptors. With increasing concentrations of the drug, inhibition via sIPSCs is limited by negative feedback on interneuronal firing whereas action potential-independent GABAergic inhibition due to tonic currents gains progressively in impact.

[The GABA(A) receptor family: possibilities for the development of better anesthetics]. / Die GABA(A)-Rezeptor-Familie: Möglichkeiten für die Entwicklung besserer Anästhetika.

Clinically used anesthetics show amnestic, sedative, hypnotic and immobilizing properties. On a molecular level these drugs affect several receptors in the cell membrane of neurons. By using genetically engineered mice a linkage can now be made between actions on certain receptors and clinically desired and undesired effects. Experiments show that a certain GABA(A) receptor subtype mediates hypnosis and immobility, whereas another subtype is involved in side-effects like sedation and hypothermia. These findings form the basis for the development of new drugs, acting highly specific and with fewer side-effects.

Mitochondrial permeability transition can be directly monitored in living neurons.

Mitochondria have been suggested as key players in apoptotic cell death of neurons and many other tissues, since the release of proapoptotic molecules from mitochondria is implicated in caspase activation. As a potential release mechanism, the occurrence of a large pore opening in the inner membrane (mitochondrial permeability transition pore, PTP) has been proposed, but has not yet been observed directly in neurons. We investigated whether the calcein/Co2+-quenching technique introduced by Petronilli et al. [Biofactors 8 (1998) 263], which allows direct observation of PTP opening, can be applied to neurons. Exposure of calcein-loaded neurons to Co2+ ions resulted in the fading of diffuse cytoplasmic calcein fluorescence, with organelle-restricted fluorescent spots remaining. These spots were colocalized with mitochondrially-entrapped tetramethylrhodamineethylester (TMRE) fluorescence and corresponded to colocalization of calcein and TMRE fluorescence in digitonin-permeabilized neurons. Importantly, extensive neuronal calcium loading, which is assumed to induce PTP opening, resulted in significant fading of mitochondrial fluorescence, suggesting the occurrence of permeability transition. This fluorescence decrease could be completely prevented by the PTP blocker cyclosporin A.

4-bromopropofol decreases action potential generation in spinal neurons by inducing a glycine receptor-mediated tonic conductance.

BACKGROUND AND PURPOSE: Impaired function of spinal strychnine-sensitive glycine receptors gives rise to chronic pain states and movement disorders. Therefore, increased activity of glycine receptors should help to treat such disorders. Although compounds targeting glycine receptors with a high selectivity are lacking, halogenated analogues of propofol have recently been considered as potential candidates. Therefore we asked whether 4-bromopropofol attenuated the excitability of spinal neurons by promoting glycine receptor-dependent inhibition. EXPERIMENTAL APPROACH: The actions of sub-anaesthetic concentrations of propofol and 4-bromopropofol were investigated in spinal tissue cultures prepared from mice. Drug-induced alterations in action potential firing were monitored by extracellular multi-unit recordings. The effects on GABAA and glycine receptor-mediated inhibition were quantified by whole-cell voltage-clamp recordings. KEY RESULTS: Low concentrations of 4-bromopropofol (50 nM) reduced action potential activity of ventral horn neurons by about 30%, compared with sham-treated slices. This effect was completely abolished by strychnine (1 µM). In voltage-clamped neurons, 4-bromopropofol activated glycine receptors, generating a tonic current of 65 ± 10 pA, while GABAA - and glycine receptor-mediated synaptic transmission remained unaffected. CONCLUSIONS AND IMPLICATIONS: The highest glycine levels in the CNS are found in the ventral horn of the spinal cord, a region mediating pain-induced motor reflexes and participating in the control of muscle tone. 4-Bromopropofol may serve as a starting point for the development of non-sedative, non-addictive, muscle relaxants and analgesics to be used to treat low back pain.

Organotypic cultures as tools for testing neuroactive drugs - link between in-vitro and in-vivo experiments.

The development of neuroactive drugs is a time consuming procedure. Candidate drugs must be run through a battery of tests, including receptor studies and behavioural tests on animals. As a rule, numerous substances with promising properties as assessed in receptor studies must be eliminated from the development pipeline in advanced test phases because of unforeseen problems like intolerable side-effects or unsatisfactory performance in the whole organism. Clearly, test systems of intermediate complexity would alleviate this inefficiency. In this review, we propose cultured organotypic brain slices as model systems that could bridge the 'interpolation gap' between receptors and the brain, with a focus on the development of new general anaesthetics with lesser side effects. General anaesthesia is based on the modulation of neurotransmitter receptors and other conductances located on neurons in diverse brain regions, including cerebral cortex and spinal cord. It is well known that different components of general anaesthesia, e.g. hypnosis and immobility, are produced by the depression of neuronal activity in distinct brain regions. The ventral horn of the spinal cord is an important structure for the induction of immobility. Thus, the potentially immobilizing effects of a newly designed drug can be estimated from its depressant effect on neuronal network activity in cultured spinal slices. A drug's sedative and hypnotic potential can be examined in cortical cultures. Combined with genetically engineered mice, this approach can point to receptor subtypes most relevant to the drug's intended net effect and in return can help in the design of more selective drugs. In conclusion, the use of organotypic cultures permits predictions of neuroactive properties of newly designed drugs on an intermediate level, and should therefore open up avenues for a more creative and economic drug development process.

Effects of isoflurane and enflurane on GABAA and glycine receptors contribute equally to depressant actions on spinal ventral horn neurones in rats.

BACKGROUND: Volatile anaesthetics are widely used agents in clinical anaesthesia, although their mechanism of action is poorly understood. In particular, the dominant molecular mechanisms by which volatile anaesthetics depress spinal neurones and thereby mediate spinal effects such as immobility have recently become a matter of dispute. As GABAA and glycine receptors are potential candidates we investigated the impact of both receptor systems in mediating the depressant effects of isoflurane and enflurane on spinal neurones in rats. METHODS: The effects of isoflurane and enflurane on spontaneous action potential firing were investigated by extracellular voltage recordings from ventral horn interneurones in cultured spinal cord tissue slices obtained from embryonic rats (E 14-15). RESULTS: Isoflurane and enflurane reduced spontaneous action potential firing. Concentrations causing half-maximal effects (isoflurane: 0.17 mM; enflurane: 0.50 mM) were less than EC50-immobility (isoflurane: 0.32 mM; enflurane: 0.62 mM). Effects of isoflurane were mediated by 39% by glycine receptors and 36% by GABAA receptors. The effects of enflurane were mediated 26% by GABAA receptors and 29% by glycine receptors. CONCLUSION: These results demonstrate that the effects of isoflurane and enflurane on GABAA and glycine receptors contribute almost equally to their depressant actions on spinal ventral horn neurones in rats. The fraction of inhibition mediated by both receptor systems differs between specific volatile anaesthetics. Our data argue against the theory that a dominant molecular mechanism accounts for spinal effects of volatile anaesthetics.

Effects of propofol on N-methyl-D-aspartate receptor-mediated calcium increase in cultured rat cerebrocortical neurons.

BACKGROUND AND OBJECTIVE: The intravenous anaesthetic propofol has been reported to exert neuroprotective actions by several mechanisms. This study has been designed to investigate the effects of propofol on intracellular calcium increase in cultured cerebrocortical neurons after exposure to pathological concentrations of N-methyl-D-aspartate (NMDA) mediated by potential direct interactions of propofol with NMDA receptors. METHODS: The effects of propofol (0.1-100 micromol) on intracellular calcium increase induced by 300 micromol NMDA (180 s) were measured in cultured cerebrocortical neurons using the calcium-sensitive fluorochrome calcium green-5N-acetoxymethylester with confocal laser scanning microscopy. RESULTS: The intraneuronal calcium increase after exposure to 300 micromol NMDA depended on extracellular calcium concentration. Propofol reduced the increase of NMDA receptor-induced intraneuronal calcium concentration dependently with a threshold concentration for a significant effect of 10 micromol. The overall effect was small, since even high concentrations of propofol (100 micromol) diminished intraneuronal calcium rise by only 50%. CONCLUSIONS: The threshold concentration for significant effects of propofol on the NMDA-induced increase of intraneuronal calcium turned out to be in the upper limit of propofol concentrations that are considered to be clinically relevant. However, in the presence of high propofol concentrations, inhibition of NMDA receptor-mediated calcium increase might contribute to neuroprotective effects observed with propofol.

Modulation of ventral pallidal dopamine and glutamate release by the intravenous anesthetic propofol studied by in vivo microdialysis.

The intravenous anesthetic propofol is reported to have various psychological side effects as hallucinations, sexual disinhibition, or euphoria. Hedonic and rewarding states like these are modulated by the dopaminergic system in the nucleus accumbens, prefrontal cortex and also in the ventral pallidum and by the glutamatergic system in the neocortex and limbic system. In the present study, propofol was administered either alone or in combination with the GABAA receptor antagonist bicuculline via reverse microdialysis into the ventral pallidum of freely moving rats. Dialysis fractions were taken every 20 min and analyzed for dopamine and glutamate using high performance liquid chromatography. Application of propofol decreased dopamine levels in the ventral pallidum. This effect seems to be mainly mediated through GABAA receptors, since it was compensated by the GABAA receptor antagonist bicuculline. Propofol and propofol plus bicuculline exerted no effect on glutamate release in this brain region. The reduced dopamine release in ventral pallidum was most probably mediated through a GABAergic feedback loop from the ventral pallidum via the nucleus accumbens to the dopaminergic neurons of the ventral tegmental area or by long loop feedback. As an increase but not a decrease of dopamine release in the ventral pallidum is involved in hedonic and rewarding properties, similar symptoms induced by propofol seem to be unrelated to an action of propofol in the ventral pallidum.

Internal standard high-performance liquid chromatography method for the determination of obidoxime in urine of organophosphate-poisoned patients.

Obidoxime is an antidote approved for reactivation of inhibited acetylcholinesterase in organophosphate poisoning. HPLC methods were described for its determination in blood or aqueous solutions but not for the determination in urine. Since data for renal obidoxime excretion ranged from 2.2 to 84% of administered dose in healthy volunteers depending on the route of administration and little is known about pharmacokinetics of obidoxime in severely intoxicated patients we developed an internal standard (HI 6) reversed-phase HPLC method for determining obidoxime in urine. The mobile phase consisted of methanol, the counter ion 1-heptane sulfonic acid and tetrabutylammonium phosphate, the stationary phase involved a 5 microm reversed-phase column (125x4 mm). Obidoxime was detected spectrophotometrically at 288 nm. The limit of quantification (LOQ) was 1 microM, the limit of detection (LOD) 0.5 microM. Linear calibration curves were obtained in a concentration range from 1 to 1000 microM. Intra- and inter-day precision C.V.s were below 4%. Accuracy was 95.9% in the LOQ range. Using this method, we were able to quantify obidoxime in urine of an organophosphate poisoned patient. Based on this data we calculated that 58% of the administered dose was excreted in the urine.