Carsten Bantel

Neuronal Preconditioning by Inhalational Anesthetics: Evidence for the Role of Plasmalemmal Adenosine Triphosphate–sensitive Potassium Channels

Background:Ischemic preconditioning is an important intrinsic mechanism for neuroprotection. Preconditioning can also be achieved by exposure of neurons to K+ channel–opening drugs that act on adenosine triphosphate–sensitive K+ (KATP) channels. However, these agents do not readily cross the blood–brain barrier. Inhalational anesthetics which easily partition into brain have been shown to precondition various tissues. Here, the authors explore the neuronal preconditioning effect of modern inhalational anesthetics and investigate their effects on KATP channels. Methods:Neuronal–glial cocultures were exposed to inhalational anesthetics in a preconditioning paradigm, followed by oxygen–glucose deprivation. Increased cell survival due to preconditioning was quantified with the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction test. Recombinant plasmalemmal KATP channels of the main neuronal type (Kir6.2/SUR1) were expressed in HEK293 cells, and the effects of anesthetics were evaluated in whole cell patch clamp recordings. Results:Both sevoflurane and the noble gas xenon preconditioned neurons at clinically used concentrations. The effect of sevoflurane was independent of KATP channel activation, whereas the effect of xenon required the opening of plasmalemmal KATP channels. Recombinant KATP channels were activated by xenon but inhibited by halogenated volatiles. Modulation of mitochondrial K-ATP channels did not affect the activity of KATP channels, thus ruling out an indirect effect of volatiles via mitochondrial channels. Conclusions:The preconditioning properties of halogenated volatiles cannot be explained by their effect on KATP channels, whereas xenon preconditioning clearly involves the activation of these channels. Therefore, xenon might mimic the intrinsic mechanism of ischemic preconditioning most closely. This, together with its good safety profile, might suggest xenon as a viable neuroprotective agent in the clinical setting.

Noble Gas Xenon Is a Novel Adenosine Triphosphate-sensitive Potassium Channel Opener

Background:Adenosine triphosphate-sensitive potassium (KATP) channels in brain are involved in neuroprotective mechanisms. Pharmacologic activation of these channels is seen as beneficial, but clinical exploitation by using classic K+ channel openers is hampered by their inability to cross the blood–brain barrier. This is different with the inhalational anesthetic xenon, which recently has been suggested to activate KATP channels; it partitions freely into the brain. Methods:To evaluate the type and mechanism of interaction of xenon with neuronal-type KATP channels, these channels, consisting of Kir6.2 pore-forming subunits and sulfonylurea receptor-1 regulatory subunits, were expressed in HEK293 cells and whole cell, and excised patch-clamp recordings were performed. Results:Xenon, in contrast to classic KATP channel openers, acted directly on the Kir6.2 subunit of the channel. It had no effect on the closely related, adenosine triphosphate (ATP)-regulated Kir1.1 channel and failed to activate an ATP-insensitive mutant version of Kir6.2. Furthermore, concentration–inhibition curves for ATP obtained from inside-out patches in the absence or presence of 80% xenon revealed that xenon reduced the sensitivity of the KATP channel to ATP. This was reflected in an approximately fourfold shift of the concentration causing half-maximal inhibition (IC50) from 26 ± 4 to 96 ± 6 &mgr;m. Conclusions:Xenon represents a novel KATP channel opener that increases KATP currents independently of the sulfonylurea receptor-1 subunit by reducing ATP inhibition of the channel. Through this action and by its ability to readily partition across the blood–brain barrier, xenon has considerable potential in clinical settings of neuronal injury, including stroke.

Assessing pain objectively: the use of physiological markers.

Pain diagnosis and management would benefit from the development of objective markers of nociception and pain. Current research addressing this issue has focused on five main strategies, each with its own advantages and disadvantages. These encompass: (i) monitoring changes in the autonomic nervous system; (ii) biopotentials; (iii) neuroimaging; (iv) biological (bio‐) markers; and (v) composite algorithms. Although each strategy has shown areas of promise, there are currently no validated objective markers of nociception or pain that can be recommended for clinical use. This article introduces the most important developments in the field and highlights shortcomings, with the aim of allowing the reader to make informed decisions about what trends to watch in the future.

Role of adenosine receptors in spinal G-protein activation after peripheral nerve injury.

Background Spinally injected adenosine induces antinociception in animal models of neuropathic but not acute pain. The reasons for this discrepancy remain unclear. Adenosine receptors are coupled to G proteins, and increased efficiency of adenosine-induced G-protein activity in neuropathic pain could contribute to the antinociceptive effect of adenosine. In this study the authors used [35S]guanosine-5′-O-(3-thiotriphosphate) ([35S]GTP&ggr;S) autoradiography in rat spinal cord sections to test this possibility. Methods The spinal cords of normal animals and those that underwent left L5 and L6 spinal nerve ligation (SNL) were removed and immediately frozen. Horizontal spinal cord sections were cut and mounted on chrom-alum gelatin-subbed slides. Sections were incubated with guanosine diphosphate, [35S]GTP&ggr;S, the adenosine A1 agonist R-N6-phenylisopropyladenosine, and various other drugs, apposed to films, and analyzed. Results Baseline and R-N6-phenylisopropyladenosine–stimulated [35S]GTP&ggr;S binding was predominantly localized to the superficial dorsal horns of both normal and SNL animals. This binding was significantly increased in SNL compared with normal animals. In contrast, no difference in R-N6-phenylisopropyladenosine–stimulated [35S]GTP&ggr;S binding was observed between SNL and normal animals. Blockade of adenosine A1 receptors by 1,3-dipropyl-8-cyclopentylxanthine, or adenosine destruction by added adenosine deaminase, reduced the increased basal activity in SNL to baseline levels of normal dorsal horns, whereas atropine and naloxone had no effect. Conclusion This study shows an increased basal G-protein activity in lumbar spinal cords during conditions of SNL. The data suggest that increased adenosine release during conditions of SNL results in an increased basal activity of G proteins in the spinal cord during neuropathic pain.

The role of the autonomic nervous system in acute surgical pain processing – what do we know?

For all anaesthetists, the link between peri-operative pain and sympathetic nervous system activity seems obvious, and has actually become second nature to their profession. Changes in heart rate and blood pressure in response to painful stimuli, for example, are routinely used as surrogate markers to assess analgesia in anaesthetised patients. If asked to describe the nature of the relationship between pain and sympathetic nervous system activity, most anaesthetists would therefore probably regard an increased sympathetic outflow as a mere epiphenomenon or symptom of pain. This school of thought is based not only on textbook knowledge but also predominantly on our day-to-day clinical experience. It implies a unidimensional, one-way, reflex pathway, with the obligatory noxious stimulus at the start and the observed sympathetic nervous system activity as the downstream endpoint. That this notion might be an oversimplification of pathway interactions, even in acute surgical pain, is one of the exciting findings of the case series of McDonnell et al. in this issue [1]. The authors showed a remarkable reduction in postoperative analgesic requirements after blockade of the sympathetic stellate ganglion with the local anaesthetic and sodium channel blocker, lidocaine. The stellate ganglion contains the somata of postganglionic sympathetic fibres and afferent fibres. Both of these might be inhibited by lidocaine. Therefore, either efferent or afferent pathways could be involved, thus leaving the question open as to whether modulation of pain sensation is achieved in the periphery or centrally. However, in any case, their work suggests an autonomic nervous system involvement in nociceptive processing after upper limb surgery. Based on these results, one can think of two main principles of how the autonomic nervous system might contribute to modulation of pain pathways: (i) through excitatory sympathetic or (ii) through inhibitory parasympathetic mechanisms. Employment of excitatory sympathetic nervous system mechanisms, for example, could mean that afferent sympathetic fibres are directly capable of either conducting noxious signals or enhancing Ador C-fibres activity. In contrast, parasympathetic inhibition would reduce these signals or nerve fibre activity. The participation of the sympathetic nervous system in pain generation is well recognised and best described in chronic, usually neuropathic, pain states such as complex regional pain syndrome, but also for instance in cancer, vascular or visceral pain [2, 3]. Subsequently, for those phenomena the term ‘sympathetically maintained pain’ has been coined [4]. However, whether or not the sympathetic nervous system is similarly involved in acute pain is still uncertain. Data from animal and human volunteer studies strongly indicate that under physiological conditions, sympathetic outflow has no effect on nociceptor function and activity [5, 6]. However, this might change under circumstances in which primary afferents have been sensitised by trauma or inflammation. In animal models, sympathomimetics have been found to increase nociceptioninduced behaviour after nerve or heat injury, as well as in response to chemically induced inflammatory processes, whilst sympatholytics reduce nociception [5–7]. These findings are in agreement with experiments where the influence of sympathetic stimulation on the electrical activity of Adand C-fibres was tested before and after sensitisation [8, 9]. The results thus obtained showed that inflammation and hence inflammatory mediators changed the excitation characteristics of these fibres. They subsequently became receptive to sympathetic modulation and therefore sensitised to sympathetic stimulation [8, 9]. In contrast to the animal data, surprisingly little is known about involvementof thesympatheticnervoussystem in acute somatic pain pathways in humans. To date, only one study has investigated the effect of sympathetic blockade on heat injury-induced inflammatorypaininhealthyvolunteers [10]. In this study, Pedersen and colleagues could find no effect of lumbar sympathetic blockade on acute pain, pain thresholds or hyperalgesia [10]. Therefore, although evidence from humans is lacking, animal research indicates thepossibility that the sympathetic nervous system is capable of modulating afferent pain pathways, probably in an excitatory pain-enhancing way. However, prior sensitisation of the nervous system through disease processes seems to be required. If this is the evidence for the sympathetic nervous system, what then can be said about the role of the parasympathetic nervous system, and especially its predominant component, the vagus nerve? Anatomically, the vagus nerve consists of afferent as well as efferent branches, supplying mostly visceral organs. Not surprisingly, its function Anaesthesia, 2011, 66, pages 539–549 Editorial .....................................................................................................................................................................................................................

Intrathecal adenosine following spinal nerve ligation in rat: Short residence time in cerebrospinal fluid and no change in A 1 receptor binding

Background Intrathecal adenosine produces a remarkably prolonged effect to relieve mechanical hypersensitivity after peripheral nerve injury in animals. The purpose of the current study was to investigate whether this reflected an alteration in kinetics of adenosine in cerebrospinal fluid or in the number of spinal A1 adenosine receptors after nerve injury. Methods Male rats were anesthetized, and the left L5 and L6 spinal nerves were ligated. Two weeks later, a lumbar intrathecal catheter and intrathecal space microdialysis catheter were inserted. Adenosine, 20 &mgr;g, was injected intrathecally in these and in normal rats, and microdialysates of the intrathecal space were obtained. Radioligand binding studies of adenosine A1 receptors were determined in spinal cord tissue from other normal and spinal nerve–ligated rats. Results Adenosine disappeared from rat cerebrospinal fluid within 30 min after intrathecal injection, with no difference between normal and spinal nerve–ligated animals. A1 adenosine receptor binding sites in the spinal cord were increased after spinal nerve ligation. This increase disappeared when adenosine deaminase was added to the membrane homogenates, suggestive of decreased endogenous adenosine in the membranes of nerve-ligated animals. Conclusion These data show that prolonged alleviation of hypersensitivity observed with intrathecal adenosine in this animal model of neuropathic pain is not due to prolonged residence in cerebrospinal fluid, although pharmacokinetics in tissues are unknown. Similarly, there is no evidence for up-regulation in spinal A1 adenosine receptors after spinal nerve ligation, and the adenosine deaminase experiment is consistent with a depletion of adenosine in spinal cord tissue after spinal nerve ligation.

An obligatory role for spinal cholinergic neurons in the antiallodynic effects of clonidine after peripheral nerve injury.

Background Indirect evidence supports a role of spinal cholinergic neurons in tonically reducing response to noxious mechanical stimulation and in effecting analgesia from &agr;2-adrenergic agonists. This study directly assessed the role of cholinergic neurons in regulating the level of mechanical allodynia and in participating in the antiallodynic effect of the clinically used &agr;2-adrenergic agonist, clonidine, in an animal model of neuropathic pain. Methods Allodynia was produced in rats by ligation of the left L5 and L6 spinal nerves. Rats received a single intrathecal injection of saline or one of three different doses of the cholinergic neurotoxin, ethylcholine mustard aziridinium ion (AF64-A; 2, 5, and 15 nmol). Seven days later, allodynia was assessed before and after intrathecal injection of 15 &mgr;g clonidine. The spinal cord was removed, and spinal cord acetylcholine content, cholinergic neuron number and distribution, and &agr;2-adrenergic receptor expression were determined. Results AF64-A administration reduced both the number of cholinergic cells and the acetylcholine content of the lumbar dorsal spinal cord by 20–50% but did not affect level of mechanical allodynia. AF64-A did, however, completely block the antiallodynic effect of clonidine. AF64-A did not reduce &agr;2-adrenergic ligand binding in dorsal lumbar cord. Conclusions These data suggest that spinal cholinergic tone does not affect the level of mechanical allodynia after peripheral nerve injury. There is a quantitative reliance on spinal cholinergic neurons in the allodynia relieving properties of intrathecal clonidine, and this reliance does not depend on &agr;2-adrenergic receptors colocalized on spinal cholinergic interneurons.

Repeated Dosing with Oral Allosteric Modulator of Adenosine A1 Receptor Produces Tolerance in Rats with Neuropathic Pain

BackgroundThe positive allosteric adenosine receptor modulator, T62 (2-amino-3-(4-chlorobenzoyl)-5,6,7,8-tetrahydrobenzothiophene), has been shown to reduce mechanical allodynia in a rat model of neuropathic pain. However, whether chronic oral T62 retains efficacy in this pain model has not been examined. Therefore, the authors studied antiallodynic effects of chronic oral T62 in spinal nerved–ligated rats, as well as motor and sedative behavioral effects. MethodsOral T62, 100 mg/kg, or oral oil was applied daily to spinal nerve–ligated rats for 4 weeks, with rat weights examined daily. Sedation, placing and ambulation scores, and withdrawal threshold were observed for 3 h daily for the first 2 weeks and then once a week. At the end of observation, the animals were killed, and the spinal tissues were collected for radioligand binding. In addition, withdrawal thresholds were also observed in rats with 5 days of treatment with 50 mg/kg oral T62. Furthermore, the effects of intrathecal adenosine on rats with oral T62 or oil treatment were compared. ResultsChronic oral T62, at 100 mg/kg, initially returned the withdrawal threshold to mechanical testing to preinjury levels, with minor or no sedative or motor effects. Tolerance was observed, with a 60% loss of most possible effects in antiallodynia within 5 days of daily administration. Similarly, tolerance also occurred with chronic oral T62 at 50 mg/kg but did not alter the effect of intrathecal adenosine. Furthermore, 4 weeks of exposure to 100 mg/kg T62 resulted in a small reduction in spinal cord A1 receptor number. ConclusionThe results imply that chronically administered A1 adenosine modulators lose efficacy over time, partly as a result of receptor down-regulation.

Intraspinal adenosine induces spinal cord norepinephrine release in spinal nerve-ligated rats but not in normal or sham controls.

Background Intrathecal adenosine is antinociceptive under conditions of central sensitization, but not in response to acute stimuli in normals. The reasons for this selective circumstance of action remain unclear, but some evidence links adenosines antinociceptive effects to release of norepinephrine by terminals in the spinal cord. The purpose of this study was to test whether spinal adenosine induces norepinephrine release selectively in settings of hypersensitivity. Methods Rats randomly assigned to spinal nerve ligation, sham operation, or no operation were anesthetized. A microdialysis fiber was implanted in the spinal cord dorsal horn at the L5–L6 level and perfused with artificial cerebrospinal fluid. After washout and a baseline sample period, adenosine at various concentrations was infused through the fiber for 150 min, and samples were collected every 15 min. Results In ligated, but not in sham or normal animals, adenosine perfusion increased norepinephrine in spinal cord microdialysates in a concentration-dependent manner. The effects of adenosine plateaued after 75 min and remained stable until the end of the experiment. Intravenous injection of selective adenosine A1 and A2 receptor antagonists revealed that adenosines effect on spinal norepinephrine release was A1 receptor mediated. Conclusions This is the first study to provide direct evidence that adenosine is able to release norepinephrine in spinal cord dorsal horns in living animals. However, this effect was only seen in animals after spinal nerve ligation. These data are consistent with behavioral studies demonstrating that adenosines antinociceptive effects in rats after spinal nerve ligation is totally dependent on intact spinal noradrenergic terminals and can be blocked by spinal &agr;2-adrenergic receptor antagonists.

Is number sense impaired in chronic pain patients

Background Recent advances in imaging have improved our understanding of the role of the brain in painful conditions. Discoveries of morphological changes have been made in patients with chronic pain, with little known about the functional consequences when they occur in areas associated with ‘number-sense’; thus, it can be hypothesized that chronic pain impairs this sense. Methods First, an audit of the use of numbers in gold-standard pain assessment tools in patients with acute and chronic pain was undertaken. Secondly, experiments were conducted with patients with acute and chronic pain and healthy controls. Participants marked positions of numbers on lines (number marking), before naming numbers on pre-marked lines (number naming). Finally, subjects bisected lines flanked with ‘2’ and ‘9’. Deviations from expected responses were determined for each experiment. Results Four hundred and ninety-four patients were audited; numeric scores in the ‘moderate’ and ‘severe’ pain categories were significantly higher in chronic compared with acute pain patients. In experiments (n=150), more than one-third of chronic pain patients compared with 1/10th of controls showed greater deviations from the expected in number marking and naming indicating impaired number sense. Line bisection experiments suggest prefrontal and parietal cortical dysfunction as cause of this impairment. Conclusions Audit data suggest patients with chronic pain interpret numbers differently from acute pain sufferers. Support is gained by experiments indicating impaired number sense in one-third of chronic pain patients. These results cast doubts on the appropriateness of the use of visual analogue and numeric rating scales in chronic pain in clinics and research.