Molecular mechanisms of action determine inhibition of paroxysmal depolarizing shifts by NMDA receptor antagonists in rat cortical neurons.

N-methyl-d-aspartate glutamate receptors (NMDARs) are involved in numerous central nervous system (CNS) processes, including epileptiform activity. We used a picrotoxin-induced epileptiform activity model to compare the action of different types of NMDAR antagonists in rat brain slices. Paroxysmal depolarizing shifts (PDS) were evoked by external stimulation in the medial prefrontal cortex (mPFC) slices and recorded in pyramidal cells (PC) and in fast-spiking interneurons (FSI). The NMDAR antagonists APV and memantine reduced the duration of PDS. However, the competitive antagonist APV caused similar effects on the PC and FSI, while the open-channel blocker memantine had a much stronger effect on the PDS in the FSI than in the PC. This difference cannot be explained by a corresponding difference in NMDAR sensitivity to memantine because the drug inhibited the excitatory postsynaptic current (EPSC) similarly in both cell types. Importantly, the PDS were significantly longer in the FSI than in the PC. The degree of PDS inhibition by memantine correlated with individual PDS durations in each cell type. Computer modeling of a synaptic network in the mPFC suggests that the different effects of memantine on the PDS in the PC and FSI can be explained by use dependence of its action. An open-channel blocking mechanism and competition with Mg2+ ions for the binding site result in pronounced inhibition of the long PDS, whereas the short PDS are weakly sensitive. Our results show that peculiarities of kinetics and the mechanism of action largely determine the effects of NMDAR antagonists on physiological and/or pathological processes.

Merits and Limitations of Studying Neuronal Depolarization-Dependent Processes Using Elevated External Potassium.

Elevated extracellular potassium chloride is widely used to achieve membrane depolarization of cultured neurons. This technique has illuminated mechanisms of calcium influx through L-type voltage sensitive calcium channels, activity-regulated signaling, downstream transcriptional events, and many other intracellular responses to depolarization. However, there is enormous variability in these treatments, including durations from seconds to days and concentrations from 3mM to 150 mM KCl. Differential effects of these variable protocols on neuronal activity and transcriptional programs are underexplored. Furthermore, potassium chloride treatments in vitro are criticized for being poor representatives of in vivo phenomena and are questioned for their effects on cell viability. In this review, we discuss the intracellular consequences of elevated extracellular potassium chloride treatment in vitro, the variability of such treatments in the literature, the strengths and limitations of this tool, and relevance of these studies to brain functions and dysfunctions.

Blockade of Hemichannels Normalizes the Differentiation Fate of Myoblasts and Features of Skeletal Muscles from Dysferlin-Deficient Mice.

Dysferlinopathies are muscle dystrophies caused by mutations in the gene encoding dysferlin, a relevant protein for membrane repair and trafficking. These diseases are untreatable, possibly due to the poor knowledge of relevant molecular targets. Previously, we have shown that human myofibers from patient biopsies as well as myotubes derived from immortalized human myoblasts carrying a mutated form of dysferlin express connexin proteins, but their relevance in myoblasts fate and function remained unknown. In the present work, we found that numerous myoblasts bearing a mutated dysferlin when induced to acquire myogenic commitment express PPARγ, revealing adipogenic instead of myogenic commitment. These cell cultures presented many mononucleated cells with fat accumulation and within 48 h of differentiation formed fewer multinucleated cells. In contrast, dysferlin deficient myoblasts treated with boldine, a connexin hemichannels blocker, neither expressed PPARγ, nor accumulated fat and formed similar amount of multinucleated cells as wild type precursor cells. We recently demonstrated that myofibers of skeletal muscles from blAJ mice (an animal model of dysferlinopathies) express three connexins (Cx39, Cx43, and Cx45) that form functional hemichannels (HCs) in the sarcolemma. In symptomatic blAJ mice, we now show that eight-week treatment with a daily dose of boldine showed a progressive recovery of motor activity reaching normality. At the end of this treatment, skeletal muscles were comparable to those of wild type mice and presented normal CK activity in serum. Myofibers of boldine-treated blAJ mice also showed strong dysferlin-like immunoreactivity. These findings reveal that muscle dysfunction results from a pathophysiologic mechanism triggered by mutated dysferlin and downstream connexin hemichannels expressed de novo lead to a drastic reduction of myogenesis and favor muscle damage. Thus, boldine could represent a therapeutic opportunity to treat dysfernilopathies.

A Chronology for the Identification and Disclosure of Adverse Effects of Succinylcholine.

BACKGROUND: New therapies are created to address specific problems and enjoy popularity as they enter widespread clinical use. Broader use can reveal unknown adverse effects and impact the life cycle significantly. Succinylcholine, a depolarizing neuromuscular blocker, was the product of decades of research surrounding the ancient compound, curare. It was introduced into practice in the 1950s by Burroughs Wellcome and Company (BW Co) and was welcomed due to its rapidly acting muscle relaxation effects. Global clinical use revealed adverse effects, both minor and major, in particular, hyperkalemia and malignant hyperthermia. We investigated when practitioners and the manufacturer became aware of these adverse effects, how information about these side effects was disseminated, and whether the manufacturer met the regulatory requirements of the time, specifically regarding the timely reporting of adverse effects. SOURCES: Primary literature search using online and archived documents was conducted at the Wood Library-Museum of Anesthesiology, Schaumburg, IL. We consulted documents submitted by BW Co to federal authorities, through the Freedom of Information Act (FOIA), Food and Drug Administration (FDA) reports, promotional advertisements, package inserts, published articles, and textbooks. RESULTS: Initial clinical testing in humans in 1952 found no adverse effects on cardiovascular or respiratory systems. Fasciculations and myalgia were early side effects described in case reports in 1952. Large-scale clinical trials in 1953 found abnormally long recovery times among some patients; the discovery of abnormal pseudocholinesterase enzyme activity was not fully demonstrated until the early 1960s. Bradycardia was first reported in 1957 in children, and in 1959 in adults. In 1960, animal studies reported a transient increase in plasma potassium; further experiments in 1969 clearly demonstrated succinylcholine-induced hyperkalemia in burn patients. Malignant hyperthermia was first described in 1966. Similar cases of elevated temperatures and muscle rigidity were described globally but the underlying mechanism was not elucidated until the 1990s. Standard anesthesia textbooks did not report major side effects of succinylcholine until 1960 and included newly documented side effects with each edition. BW Co's packaging contained warnings as early as the 1950s but were later updated in 1962 and beyond to reflect the newly discovered hyperkalemia and malignant hyperthermia. CONCLUSION: Particularly given the regulatory environment of the time, BW Co appropriately reported the adverse effects of succinylcholine after market entry; it updated promotional and packaging material in a timely manner to reflect newly discovered adverse effects. The toxicity, though alarming and put clinicians on alert, did not seem to heavily impact succinylcholine's use, given its various desirable properties. It is still a choice muscle relaxant used today, although there are efforts to develop superior agents to replace succinylcholine.

Magnesium sulfate - An effective agent could delay the progression of fulminant malignant hyperthermia.

Malignant hyperthermia (MH) is a life-threatening disease that occurs during general anaesthesia following exposure to succinylcholine (SCh), a depolarizing muscle relaxant, and volatile anaesthetics. Susceptibility to MH most commonly arises from mutations in the RyR1 gene, the Ca2+ release channel of skeletal muscle. Fulminant MH (f-MH) is the most dangerous form of MH, which presents a hypermetabolic cascade state, including very high temperature and carbon dioxide production, increased heart rate and oxygen consumption, mixed acidosis, rigid muscles, and rhabdomyolysis. Dantrolene is the only specific drug therapy for MH on the market. Without dantrolene, the reported mortality of f-MH is as high as 42.3%. Based on the participation of catecholamine in the hyperhaemodynamic response of f-MH and the demonstrated effective control of catecholamine release of magnesium sulfate, combined with the fact that magnesium and calcium have opposite effects on muscle contraction, I hypothesized that magnesium sulfate could be a choice for delaying the progression of f-MH while waiting for dantrolene treatment.

Lasting s-ketamine block of spreading depolarizations in subarachnoid hemorrhage: a retrospective cohort study.

OBJECTIVE: Spreading depolarizations (SD) are characterized by breakdown of transmembrane ion gradients and excitotoxicity. Experimentally, N-methyl-D-aspartate receptor (NMDAR) antagonists block a majority of SDs. In many hospitals, the NMDAR antagonist s-ketamine and the GABAA agonist midazolam represent the current second-line combination treatment to sedate patients with devastating cerebral injuries. A pressing clinical question is whether this option should become first-line in sedation-requiring individuals in whom SDs are detected, yet the s-ketamine dose necessary to adequately inhibit SDs is unknown. Moreover, use-dependent tolerance could be a problem for SD inhibition in the clinic. METHODS: We performed a retrospective cohort study of 66 patients with aneurysmal subarachnoid hemorrhage (aSAH) from a prospectively collected database. Thirty-three of 66 patients received s-ketamine during electrocorticographic neuromonitoring of SDs in neurointensive care. The decision to give s-ketamine was dependent on the need for stronger sedation, so it was expected that patients receiving s-ketamine would have a worse clinical outcome. RESULTS: S-ketamine application started 4.2 ± 3.5 days after aSAH. The mean dose was 2.8 ± 1.4 mg/kg body weight (BW)/h and thus higher than the dose recommended for sedation. First, patients were divided according to whether they received s-ketamine at any time or not. No significant difference in SD counts was found between groups (negative binomial model using the SD count per patient as outcome variable, p = 0.288). This most likely resulted from the fact that 368 SDs had already occurred in the s-ketamine group before s-ketamine was given. However, in patients receiving s-ketamine, we found a significant decrease in SD incidence when s-ketamine was started (Poisson model with a random intercept for patient, coefficient - 1.83 (95% confidence intervals - 2.17; - 1.50), p < 0.001; logistic regression model, odds ratio (OR) 0.13 (0.08; 0.19), p < 0.001). Thereafter, data was further divided into low-dose (0.1-2.0 mg/kg BW/h) and high-dose (2.1-7.0 mg/kg/h) segments. High-dose s-ketamine resulted in further significant decrease in SD incidence (Poisson model, - 1.10 (- 1.71; - 0.49), p < 0.001; logistic regression model, OR 0.33 (0.17; 0.63), p < 0.001). There was little evidence of SD tolerance to long-term s-ketamine sedation through 5 days. CONCLUSIONS: These results provide a foundation for a multicenter, neuromonitoring-guided, proof-of-concept trial of ketamine and midazolam as a first-line sedative regime.

Mammalian skeletal muscle does not express functional voltage-gated H+ channels.

High metabolic activity and existence of a large transmembrane inward electrochemical gradient for H+ at rest promote intracellular acidification of skeletal muscle. Exchangers and cotransports efficiently contend against accumulation of intracellular H+ and associated deleterious effects on muscle functions. Voltage-gated H+ channels have also been found to represent another H+ extrusion pathway in cultured muscle cells. Up to now, the skeletal muscle cell was therefore the unique vertebrate excitable cell in which voltage-gated H+ currents have been described. In this study, we show that, unlike cultured cells, single mouse muscle fibers do not generate H+ currents in response to depolarization. In contrast, expression of human voltage-gated H+ channels in mouse muscle gives rise to robust outward voltage-gated H+ currents. This result excludes that inappropriate experimental conditions may have failed to reveal voltage-gated H+ currents in control muscle. This work therefore demonstrates that fully differentiated mammalian muscle fibers do not express functional voltage-gated H+ channels and consequently can no longer be considered as the only vertebrate excitable cells exhibiting voltage-gated H+ currents.

Succinylcholine and Intracranial Pressure.

Intracranial and Hemodynamic Changes after Succinylcholine Administration in Cats. By Cottrell JE, Hartung J, Giffin JP, and Shwiry B. Anesthesia & Analgesia 1983; 62:1006-9. Reprinted with permission.Bolus injections of succinylcholine (1.5 mg/kg) significantly increased intracranial pressure (ICP) in cats under normal conditions from control levels of 8 +/- 1 mmHg to 16 +/- 3 mmHg (+/- SEM, P less than 0.01), and in the presence of artificially increased ICP from control levels of 27 +/- 1 mmHg to 47 +/- 4 mmHg (P less than 0.01). These approximately 100% increases in ICP were accompanied by a transitory decrease in mean arterial pressure (approximately 10 s), followed by a 15 to 20% increase (P less than 0.05). Pulmonary arterial pressure increased 20 to 30% (P less than 0.05). These results, when considered in conjunction with results previously obtained in humans, suggest that succinylcholine may be contraindicated in neurosurgical patients.

Mode of action of piperovatine, an insecticidal piperamide isolated from Piper piscatorum (Piperaceae), against voltage-gated sodium channels.

Piperamides, which are secondary metabolites in the genus Piper, have potent insecticidal activity, and have thus inspired the development of novel insecticides. In this study, piperovatine, a piperamide from Piper piscatorum (Piperaceae), was investigated using a two-electrode voltage clamp to clarify its detailed mode of action against voltage-gated sodium channels, a classic target. In Xenopus oocytes expressing voltage-gated sodium channels from German cockroach (Blattella germanica), piperovatine induced inward currents depending on repetitive openings. For instance, maximal currents were generated with 10 µM piperovatine following 100 trains of depolarizing pulses with frequency 25 Hz. Piperovatine also shifted the half-activation voltage after conditioning pulses from -35 mV to -45 mV. In addition, piperovatine-modified currents were correlated with not only the number of prior conditioning pulses but also the proportion of activated channels. Finally, piperovatine was found to stabilize voltage-gated sodium channels in the fast-inactivated state after opening, and inhibit transition to the slow-inactivated state. These results suggest that piperovatine preferably binds to activated channels and stabilizes voltage sensors at the conformation acquired during depolarization.

Depolarization and electrical stimulation enhance in vitro and in vivo sensory axon growth after spinal cord injury.

Activity dependent plasticity is a key mechanism for the central nervous system (CNS) to adapt to its environment. Whether neuronal activity also influences axonal regeneration in the injured CNS, and whether electrical stimulation (ES) can activate regenerative programs in the injured CNS remains incompletely understood. Using KCl-induced depolarization, in vivo ES followed by ex-vivo neurite growth assays and ES after spinal cord lesions and cell grafting, we aimed to identify parameters important for ES-enhanced neurite growth and axonal regeneration. Using cultures of sensory neurons, neurite growth was analyzed after KCl-induced depolarization for 1-72h. Increased neurite growth was detected after short-term stimulation and after longer stimulation if a sufficient delay between stimulation and growth measurements was provided. After in vivo ES (20Hz, 2× motor threshold, 0.2ms, 1h) of the intact sciatic nerve in adult Fischer344 rats, sensory neurons showed a 2-fold increase in in vitro neurite length one week later compared to sham animals, an effect not observed one day after ES. Longer ES (7h) and repeated ES (7days, 1h each) also increased growth by 56-67% one week later, but provided no additional benefit. In vivo growth of dorsal column sensory axons into a graft of bone marrow stromal cells 4weeks after a cervical spinal cord lesion was also enhanced with a single post-injury 1h ES of the intact sciatic nerve and was also observed after repeated ES without inducing pain-like behavior. While ES did not result in sensory functional recovery, our data indicate that ES has time-dependent influences on the regenerative capacity of sensory neurons and might further enhance axonal regeneration in combinatorial approaches after SCI.

Perioperative Gabapentin Improves Patient-Centered Outcomes After Inguinal Hernia Repair.

INTRODUCTION: Over 5,300 inguinal hernia repairs (IHR) were performed in the Military Health System in 2015. Chronic pain can be a debilitating complication, occurring in up to 34% of patients after IHR and impacts mission readiness. Gabapentin has been shown to be effective for postoperative analgesia in a variety of operations. We evaluated the effect of a short course of perioperative gabapentin on chronic pain after IHR. METHODS: This was a double-blinded, randomized study involving male patients ≥18 years old with an initial inguinal hernia and no history of chronic pain or psychiatric disorder. Patients chose laparoscopic or open surgery and were then randomized to receive gabapentin 300 mg before surgery, then three times daily for 6 doses or placebo. There were 50 patients randomized to both the gabapentin and placebo groups for a total of 100 patients. Main outcomes were pain and health status, assessed with a visual analogue scale (VAS) and the Short Form-12v2 (SF-12v2). Assessments were performed preoperatively and 1, 6, 12, and 24 months postoperatively. Analysis of variance was used to compare groups. RESULTS: From the initial 100 patients, 19 withdrew or were excluded. Thus, 81 patients remained, 40 receiving gabapentin and 41 placebo. Throughout the 24-month follow-up, there was no difference in VAS pain scores between the gabapentin and placebo groups (p = 0.867). Beyond 1 month of follow-up, SF-12v2 physical component score (PCS) scores were improved in the gabapentin group (p = 0.039). When comparing open to laparoscopic groups, SF-12v2 PCS scores were improved in the laparoscopic group (p = 0.046) and SF-12v2 mental component summary scores were improved in the open group (p = 0.041). CONCLUSIONS: Perioperative gabapentin was not effective in decreasing chronic pain after IHR; however, patient perception of physical health, as measured by SF-12v2, did improve.

A study of intubating conditions: Sevoflurane versus propofol-suxamethonium in children.

BACKGROUND: Endotracheal intubation is an integral part of general anaesthesia in children, and the choice of induction agents and technique may affect the ease of intubation and thus the outcome of paediatric patients. We compared the ease of endotracheal intubation following sevoflurane and propofol-suxamethonium induction using Helbo-Hansen score. PATIENTS AND METHODS: A prospective, randomized double-blinded comparative study conducted on sixty-six children (two groups of 33 each) between the ages of 3-10 years undergoing different elective surgeries. Group I received intravenous propofol and intravenous suxamethonium while Group II had inhalational induction with sevoflurane in 60% nitrous oxide and oxygen. Data including intubating conditions, time to tracheal intubation and haemodynamic changes were analysed using SPSS version 18, with statistical significance set at P < 0.05. RESULTS: Using the Helbo-Hansen intubation score, the study reveals that 28 patients (85%) scored 4, 5 (15.2%) scored 5 and no patient scored 6 in Group I whereas 15 (45.5%) scored 4, 16 (48.5%) scored 5 and 2 (6.1%) scored 6 in Group II with P = 0.002. The mean time taken from induction to laryngoscopy was 91.27 ± 29.96 s in Group I and 219.09 ± 63.88 s in Group II (with P < 0.0001); mean time taken from laryngoscopy to completion of intubation was 29.03 ± 10.61 s and 28.09 ± 9.48 s which was not statistically significant with P = 0.71. CONCLUSION: Sevoflurane provides clinically acceptable intubating conditions and can be a suitable alternative to propofol-suxamethonium for endotracheal intubation in children. We recommend the use of sevoflurane to facilitate intubation in elective procedures in children.

Inhibition of glial hemichannels by boldine treatment reduces neuronal suffering in a murine model of Alzheimer's disease.

The contribution of reactive gliosis to the pathological phenotype of Alzheimer's disease (AD) opened the way for therapeutic strategies targeting glial cells instead of neurons. In such context, connexin hemichannels were proposed recently as potential targets since neuronal suffering is alleviated when connexin expression is genetically suppressed in astrocytes of a murine model of AD. Here, we show that boldine, an alkaloid from the boldo tree, inhibited hemichannel activity in astrocytes and microglia without affecting gap junctional communication in culture and acute hippocampal slices. Long-term oral administration of boldine in AD mice prevented the increase in glial hemichannel activity, astrocytic Ca2+ signal, ATP and glutamate release and alleviated hippocampal neuronal suffering. These findings highlight the important pathological role of hemichannels in AD mice. The neuroprotective effect of boldine treatment might provide the basis for future pharmacological strategies that target glial hemichannels to reduce neuronal damage in neurodegenerative diseases.

Neonatal domoic acid alters in vivo binding of [11C]yohimbine to α2-adrenoceptors in adult rat brain.

RATIONALE: Epilepsy is a debilitating seizure disorder that affects approximately 50 million people. Noradrenaline reduces neuronal excitability, has anticonvulsant effects and is protective against seizure onset. OBJECTIVE: We investigated the role of α2-adrenoceptors in vivo in a neonatal domoic acid (DOM) rat model of epilepsy. METHODS: We injected male Sprague-Dawley rats daily from postnatal day 8-14 with saline or one of two sub-convulsive doses, 20 µg/kg (DOM20) or 60 µg/kg (DOM60) DOM, an AMPA/kainate receptor agonist. The rats were observed in open field, social interaction and forced swim tests at day 50, 75 and 98, respectively. At ~120 days of age, four rats per group were injected and scanned with [11C]yohimbine, an α2-adrenoceptor antagonist, and scanned in a Mediso micro positron emission tomography (PET) scanner to measure α2-adrenoceptor binding. RESULTS: DOM60-treated rats spent more time in the periphery during the open field test and had a significant 26-33 % reduction in [11C]yohimbine binding in the hypothalamus, hippocampus and orbital prefrontal cortex compared to saline-treated rats. On the other hand, DOM20 rats had a significant 34-40 % increase in [11C]yohimbine binding in the hypothalamus, amygdala and entorhinal cortex compared to saline-treated rats, with no obvious behavioural differences. CONCLUSIONS: The current data clearly indicate that low concentrations of DOM given to rats in their second week of life induces long-term changes in α2-adrenoceptor binding in rat brain that may have relevance to the progression of an epilepsy phenotype.

Patients with prolonged effect of succinylcholine or mivacurium had novel mutations in the butyrylcholinesterase gene.

INTRODUCTION: Mutations in the butyrylcholinesterase enzyme (BChE) can result in prolonged duration of action of the neuromuscular blocking agents, succinylcholine and mivacurium, as BChE hydrolyses these drugs. Hereditary low BChE activity can cause extensively prolonged apnoea during general anaesthesia when these drugs are used. The aim of this study was to describe novel mutations in the butyrylcholinesterase gene (BCHE) in patients who have experienced prolonged duration of action of mivacurium or succinylcholine. METHODS: The Danish Cholinesterase Research Unit registers patients with prolonged duration of action to succinylcholine and mivacurium. Patients were studied if they had equivocal phenotypes on the basis of BChE activity, biochemical inhibitor reactions and with pedigree if possible. Complete nucleotide sequencing was performed to describe the genotype and pedigree was used to separate the alleles. Multiple sequence alignment of BChE was performed for comparison with other species. RESULTS: Genotyping indicated seven novel mutations in the BCHE (I373T, G467S, W518R, L184S, V421A, M462I and R577H). CONCLUSION: We have found seven new variants of the BCHE, which seem to reduce the activity of BChE in patients undergoing anaesthesia involving succinylcholine or mivacurium.

[Danish Cholinesterase Research Unit diagnoses patients with prolonged paralysis after succinylcholine and mivacurium]. / Dansk Kolinesterase Kartotek udreder patienter med forlænget paralyse efter suxamethon og mivacurium.

The Danish Cholinesterase Research Unit (DCRU) is a nationwide unit for patients carrying mutations in the butyrylcholinesterase enzyme (BChE). BChE hydrolyzes the neuromuscular blocking drugs succinylcholine and mivacurium. Patients with mutations in the butyrylcholinesterase gene are at risk of experiencing a prolonged effect of the drugs, such as weakness or paralysis for hours. In order to diagnose the referred patients correctly, DCRU combines results such as BChE activity, genotyping, pedigree and clinical reactions to succinylcholine or mivacurium.

Effects of skeletal muscle denervation on the potency of succinylcholine.

The aim of the present study was to investigate the time­dependent effects of denervation on the sensitivity of skeletal muscles to the relaxant succinylcholine (SuCh) and to assess the possible association of the de novo expression of γ­acetylcholine receptor (AChR). Innervated as well as denervated mouse muscle cells and human embryonic kidney (HEK293) cells expressing γ­AChR and ε­AChR were used in the present study. The effects of SuCh on the current of nicotinic (n)AChRs were examined using a whole­cell patch clamp technique. Compared with innervated skeletal muscle cells, the SuCh concentration producing 50% of the maximal response (EC50) were decreased by 20, 56, 73, 66, 60 and 62% (P<0.05), and current responses induced by 30 µM SuCh were increased by 1.9­, 4.6­, 9.4­, 7.1­, 5.2­ and 5.1­fold (P<0.05) at days 1, 4, 7, 14, 21 and 28 after denervation, respectively. However, SuCh was equipotent regarding γ­AChR and ε­AChR (P>0.05). These results indicated that short­term denervation led to a change in the sensitivity of muscle cells to SuCh, which, however, was unlikely to be associated with the de novo expression of γ­AChR.