Prospective validation of a new airway management algorithm and predictive features of intubation difficulty.

BACKGROUND: Some patients have features that indicate possible difficulty with direct laryngoscopy for tracheal intubation. Prediction of the likely outcome and selection of patients for an enhanced management algorithm would reduce the possible harm from failed intubation attempts. METHODS: Adult elective patients were assessed for seven features associated with difficult direct laryngoscopy, ranked in difficulty from 0 to 3. For a patient with at least one Class 3 feature, or two or more features of class 1 or higher, the enhanced management used a channelled videolaryngoscope Airtraq™ instead of a Macintosh laryngoscope. A long flexible angulated stylet and a flexible fibrescope would be used as the second and third steps. For patients with lesser difficulty scores, a Macintosh laryngoscope was used. Outcomes of enhanced management were analysed. Logistic regression and Random Forest algorithm, using the ranks of the predictive features, were used to predict difficulty during enhanced management. RESULTS: We prospectively studied 16 695 patients. We selected 1501 (9%) for enhanced management, and tracheal intubation was successful in all of them. Of these, 73% were intubated in less than 30 s, and only 4.5% required more than 4 min for intubation. Progression to the second and third steps of enhanced management was predicted by restriction of mouth opening and reduced cervical spine mobility. CONCLUSIONS: An enhanced management algorithm allowed successful tracheal intubation of all patients with anticipated difficult laryngoscopy. The need to combine the use of a stylet and a fibrescope with the Airtraq™ could be predicted with a high degree of certainty.

Effects of oleic acid on the high threshold barium current in seabass Dicentrarchus labrax ventricular myocytes.

The present study employed a patch clamp technique in isolated seabass ventricular myocytes to investigate the hypothesis that oleic acid (OA), a mono-unsaturated fatty acid, can exert direct effects upon whole-cell barium currents. Acute application of free OA caused a dose-dependent depression of the whole-cell barium current that was evoked by a voltage step to 0 mV from a holding potential of -80 mV. The derived 50% inhibitory concentration (IC50) was 12.49+/-0.27 micromol l(-1). At a concentration of 30 micromol l(-1), OA significantly reduced the current density to about 45% of control values, but did not modify either the shape of the current-density voltage relationship or the apparent reversal potential. In addition, OA did not modify the voltage dependence of either steady state inactivation or activation curves. Taken together, these results indicate that physiological concentrations of free OA decrease the conductance of the L-type inward current, without altering its properties of selectivity and its voltage dependence. The inhibitory effect of OA upon the L-type calcium channel may translate, in vivo, into a protective effect against arrhythmias induced by Ca2+ overload.

A flow cytometric approach to study intracellular-free Ca2+ in Crassostrea gigas haemocytes.

Bivalve haemocytes are essential in defence mechanisms including phagocytosis. They also produce molecules including hydrolytic enzymes and antimicrobial peptides that contribute to pathogen destruction. Although haemocyte activities have been extensively studied, relatively little is known about the intracellular signalling pathways that are evoked during haemocyte activation and especially the role of calcium. Flow cytometry has been used for the first time to define the effect of cell incubation in haemolymph and artificial sea water (ASW) on Pacific oyster, Crassostrea gigas, haemocytes. Cell viability, enzymatic activities (esterases and aminopeptidases), phagocytosis and granulocyte percentage were analysed. Viability and some activities were different in haemolymph and ASW. Cytoplasmic-free calcium in circulating haemocytes was then investigated by flow cytometry in both media using a calcium probe (Fluo-3/AM). To explore calcium homeostasis, different calcium modulators were tested. The calcium chelator Bapta/AM (10 microM) reduced significantly the percentage of Fluo-3-positive cells in ASW. In addition, ryanodine (5 microM) induced a significant enhancement of the percentage of Fluo-3 positive cells in haemolymph and in ASW. Flow cytometry may be used to study calcium movements in C. gigas haemocytes, but several haemocyte incubation media need to be tested in order to confirm results. The objective of the study should be considered before selecting a particular experimental medium.

Three-dimensional rotations of human three-joint fingers: an optoelectronic measurement. Preliminary results.

Longitudinal axial rotations of phalanges during flexion motions of digits have scarcely been analyzed with current anatomical or radiological methods. Recent optoelectronic systems were developed for three-dimensional (3D) kinematic analysis of human motion. These systems have the advantages of being non-invasive and non-irradiating. The current study was based on the VICON optoelectronic system. A validation of the protocol was made among a sample of volunteers for further direct clinical applications. An experimental protocol was set up with adaptations to the requirements of finger analyses (multiple infrared markers inside small-sized capture volumes). The set-up and the protocol details are described. Kinematic studies consisted in recording the movements of the right hand of six volunteers (free from any visible pathology). Results were displayed for the joints of each three-joint finger with calculation of 3D rotations. Metacarpophalangeal (MCP), proximal interphalangeal (PIP) and distal interphalangeal (DIP) flexion angles ranged from 78 degrees to 118 degrees, 72 degrees to 119 degrees and 9 degrees to 66 degrees respectively. Lateral angles ranged from 5 degrees to 39 degrees (MCP), 4 degrees to 39 degrees (PIP) and 4 degrees to 30 degrees (DIP). Mean longitudinal axial rotations of MCP, PIP and DIP joints ranged from 11 degrees pronation to 26 degrees supination. The index finger was in a global pronation position (five of the six specimens). The fourth and fifth fingers were in a global supination position in every case. The third finger was in a more variable global rotation (pronation in four of the six specimens). An experimental protocol using an optoelectronic system (VICON) has been developed for a kinematic analysis of three-joint finger. A global measure study should be initiated among a wider sample of adults. A database should be created with direct clinical applications. Patients' kinematic deficits could be graded either for standard movements (flexion/extension and abduction/adduction) or for longitudinal axial rotations.

Calcium currents and transients in co-cultured contracting normal and Duchenne muscular dystrophy human myotubes.

1. The goal of the present study was to investigate differences in calcium movements between normal and Duchenne muscular dystrophy (DMD) human contracting myotubes co-cultured with explants of rat spinal cord with attached dorsal root ganglia. Membrane potential, variations of intracellular calcium concentration and T- and L-type calcium currents were recorded. Further, a descriptive and quantitative study by electron microscopy of the ultrastructure of the co-cultures was carried out. 2. The resting membrane potential was slightly less negative in DMD (-61.4 +/- 1.1 mV) than in normal myotubes (-65.5 +/- 0.9 mV). Both types of myotube displayed spontaneous action potentials (mean firing frequency, 0.42 and 0.16 Hz, respectively), which triggered spontaneous calcium transients measured with Indo-1. 3. The time integral under the spontaneous Ca(2+) transients was significantly greater in DMD myotubes (97 +/- 8 nM s) than in normal myotubes (67 +/- 13 nM s). 4. The L- and T-type current densities estimated from patch-clamp recordings were smaller in DMD cells (2.0 +/- 0.5 and 0.90 +/- 0.19 pA pF(-1), respectively) than in normal cells (3.9 +/- 0.7 and 1.39 +/- 0.30 pA pF(-1), respectively). 5. The voltage-dependent inactivation relationships revealed a shift in the conditioning potential at which inactivation is half-maximal (V(h,0.5)) of the T- and L-type currents towards less negative potentials, from -72.1 +/- 0.7 and -53.7 +/- 1.5 mV in normal cells to -61.9 +/- 1.4 and -29.2 +/- 1.4 mV in DMD cells, respectively. 6. Both descriptive and quantitative studies by electron microscopy suggested a more advanced development of DMD myotubes as compared to normal ones. This conclusion was supported by the significantly larger capacitance of the DMD myotubes (408 +/- 45 pF) than of the normal myotubes (299 +/- 34 pF) of the same apparent size. 7. Taken together, these results show that differences in T- and L-type calcium currents between normal and DMD myotubes cannot simply explain all observed alterations in calcium homeostasis in DMD myotubes, thus suggesting that other transmembrane calcium transport mechanisms must also be altered in DMD myotubes compared with normal myotubes.

The effect of methylprednisolone on intracellular calcium of normal and dystrophic human skeletal muscle cells.

Clinical trials have shown that a glucocorticoid, the methyiprednisolone (PDN), has a beneficial effect on muscle strength and function in Duchenne muscular dystrophy (DMD) patients. The aim of this study was to test if the effect of PDN could be mediated via a possible action on intracellular calcium. The intracellular calcium activity, at rest and during calcium mobilizing drug superfusion protocols was recorded in normal and dystrophic human cocultured muscle cells. PDN (10 microM) pretreatment induced an elevation of the resting calcium concentration of 51, 34 and 38% in proliferating normal myoblasts, DMD myoblasts and DMD myotubes, respectively, while normal myotubes resting [Ca2+]i was not altered.

Hypoosmotic shocks induce elevation of resting calcium level in Duchenne muscular dystrophy myotubes contracting in vitro.

In Duchenne muscular dystrophy (DMD) muscle cells which lack dystrophin, contraction seems to be a dominant factor contributing to the abnormal elevated intracellular calcium level. Human normal and DMD contracting myotubes cocultured with nervous cells were exposed to a hypotonic medium to mimic contraction-induced mechanical stress on the membrane, and the cytoplasmic calcium activity was simultaneously monitored (Indo-1). Hypotonic shocks induced a reversible [Ca2+]i increase in 81% of the DMD cells vs. 54% of control. In addition, responses were qualitatively different: most of DMD myotubes displayed a fast increase of Ca2+ flowing from the edge of the myotube while the response in normal cells was slow and diffuse. The fact that these responses were not affected by ryanodine, was in favour of an external source of Ca2+ involved in the hypoosmotic shocks. The localized increase of Ca2+ in DMD myotubes, inhibited by Gd3+, could result from sites of high mechanosensitive channel activity or density which could constitute a pathway for Ca2+ entry provided these cells contract.

Abnormal calcium homeostasis in Duchenne muscular dystrophy myotubes contracting in vitro.

Resting intracellular calcium activity was recorded in three kinds of human muscle cells in culture: normal (control) and dystrophic (DMD and FSH), by means of a ratiometric fluorescence method using the calcium probe Indo-1 under laser illumination. DMD cells are characterized by a lack of dystrophin whereas FSH cells express normal dystrophin. The aim of this study was to determine whether, in dystrophin-deficient muscle cells (DMD), contraction destabilized internal calcium homeostasis. Muscle cells were cocultured with rat spinal cord explants to improve the maturation of human myotubes up to the stage where contraction appears. The resting intracellular calcium level was significantly higher in contracting DMD cells (107 +/- 8 nM; n = 44) compared to control cells (66 +/- 6 nM; n = 43) or in FSH cells (56 +/- 6 nM; n = 35). DMD myotubes cocultured in the presence of TTX which inhibited contractile activity, did not develop an increase in free cytosolic Ca2+ concentration. The amplitudes of calcium transients elicited by exposure to acetylcholine (ACh) or high K+ medium (100K) were significantly higher in contracting DMD myotubes than in control ones. The extra-responses were not observed in DMD myotubes cocultured with TTX. This study strongly suggest that: (i) contraction is a dominant factor contributing to Ca2+ abnormalities in DMD cells; and (ii) contracting dystrophin-deficient cells have defective calcium handling mechanisms during electrical events which involve sarcolemma.

Cultured rat skeletal muscle cells treated with cytochalasin exhibit normal dystrophin expression and intracellular free calcium control.

Many studies performed to elucidate the molecular and cellular processes involved in muscular dystrophies have led to the working hypothesis of a key role for the cytoskeleton elements linking the extracellular matrix to myofibrils. It was recently suggested that cytochalasin B treatment of mouse soleus muscle promoted cell damage mediated by a cytosolic increase in free calcium concentration. Since intracellular calcium overload may be a primary event resulting from the alteration of cytoskeletal structure, this study was intended to evaluate whether or not the integrity of the F-actin microfilament network is necessary for calcium homeostasis. The developmental establishment of the normal cytoarchitecture was altered by treatment of myoblasts with the actin-disrupting agents cytochalasin B and D, and the effects were compared with those in myoblasts treated with colchicine. These drugs modified the morphogenesis in that they prevented the formation of elongated myotubes by myoblast fusion, but did not prevent the maturation of contractile myogenic cells. The subcellular organisation of actin filaments visualised by confocal fluorescence microscopy was modified by colchicine and cytochalasins, but appearance of contractile apparatus and mechanical activity were not precluded. Sarcolemmal addressing of dystrophin, the subsarcolemmal protein lacking in Duchenne muscular dystrophy, was not prevented by cytochalasin. The evaluation of the basal activity of cytosolic calcium measured with indo-1 suggested that the disruption of actin or microtubules did not prevent developing muscle cells to maintain a low basal calcium activity. We propose that the global integrity of the cytoskeleton network is not crucial for the maintenance of calcium homeostasis in muscle cells developing in vitro. These results are discussed with regard to current theories attempting to understand the functional consequences of an abnormal expression of the dystrophin-glycoprotein complex interacting with the extracellular matrix and the cytoskeleton.

Changes in cytosolic resting ionized calcium level and in calcium transients during in vitro development of normal and Duchenne muscular dystrophy cultured skeletal muscle measured by laser cytofluorimetry using indo-1.

Intracellular calcium activity was recorded during in vitro myogenesis of human normal and DMD muscle, using the calcium probe Indo-1 under laser illumination, at rest and during different kinds of stimulation (acetylcholine, high K+, caffeine). In myoblasts, the resting intracellular calcium level was significantly larger in DMD cells (89 +/- 9 nM; n = 40 vs 37 +/- 5 nM; n = 22) but there was no significant difference in myotubes, after fusion (44 +/- 4 nM; n = 34 vs 36 +/- 4 nM; n = 52). A similar evolution was observed in cells cultured from FSH biopsies. The amplitude of ACh- and high K(+)-induced calcium transients was significantly halved in DMD myotubes as compared to control ones and non-significantly decreased for caffeine responses. Some alterations in the kinetics of responses were observed in DMD muscle: the rising phases of ACh- and high K(+)-elicited transients and the decaying phase of the ACh-responses were significantly slowed down. It is concluded that: (i) in aneurally cultured human muscle, an increase in the basal level of internal calcium can occur at early stages of myogenesis before the expression of the dystrophin gene; and (ii) the changes in calcium transients induced by depolarization or direct stimulation of sarcoplasmic reticulum are not susceptible of inducing a calcium overload in DMD cells.

Appearance and evolution of calcium currents and contraction during the early post-fusional stages of rat skeletal muscle cells developing in primary culture.

Primary cultures from enzymatically dissociated satellite cells of newborn rat skeletal muscles enabled developmental in vitro studies of mechanical and electrical properties during the first steps of myogenesis. The present work focused on the appearance, evolution and roles of two types of calcium currents (ICa,T and ICa,L) and of depolarization-induced contractile activity during the early stages of muscle cell development in primary culture. Prefusional mononucleated cells (myoblasts), young myotubes of 1 day (with less than 10 nuclei) or 2-3 days (more than 9 nuclei) and myoballs from 4-6, 7-9, 10-12 and 13-16 days cultures were patch-clamped (whole-cell configuration), and calcium currents and contraction simultaneously recorded. Sodium but not calcium currents could be recorded at the myoblast stage. In young myotubes (1 day), ICa,L was present with high incidence as compared to ICa,T, which was poorly expressed. Contractile responses appeared at the next stage (2-3 days) while the occurrence of ICa,T progressively increased. This developmental evolution of the calcium currents and contraction expression was accompanied by some changes in their characteristics: the ICa,T/ICa,L amplitudes ratio progressively increased and the time-to-peak of contraction progressively decreased with the age of myoballs. Physiological functions for calcium currents in developing muscle are suggested and discussed: ICa,T, which is transiently expressed, could be involved in the pacemaker-like activity while ICa,L could serve as an early contraction triggering mechanism and/or initially to fill and then to maintain the intracellular calcium stores.

A third type of calcium current in cultured human skeletal muscle cells.

A third type of calcium current could be recorded on a non-negligible number of human skeletal muscle cells (normal and Duchenne dystrophic (DMD)) in primary culture. This transient current exhibited a maximum at 0 mV, a time-to-peak around 30 ms, an inactivation time constant around 70 ms and was insensitive to nifedipine. On these basis, it differentiates from T- and L-type previously described and looks like the neuronal N-type. However, this third type of current was not sensitive to omega-Cgtx, a specific N-type blocker. The occurrence and the possible role of this current are briefly discussed.