Marko Ljubkovic

Distinct membrane effects of spinal nerve ligation on injured and adjacent dorsal root ganglion neurons in rats.

Background:Painful peripheral nerve injury results in disordered sensory neuron function that contributes to the pathogenesis of neuropathic pain. However, the relative roles of neurons with transected axons versus intact adjacent neurons have not been resolved. An essential first step is identification of electrophysiologic changes in these two neuronal populations after partial nerve damage. Methods:Twenty days after spinal nerve ligation (SNL), intracellular recordings were obtained from axotomized fifth lumbar (L5) dorsal root ganglion neurons and adjacent, intact L4 neurons, as well as from control neurons and others subjected to sham-SNL surgery. Results:Pronounced electrophysiologic changes were seen only in L5 neurons after SNL. Both Aα/β and A&dgr; neuron types showed increased action potential duration, decreased afterhyperpolarization amplitude and duration, and decreased current threshold for action potential initiation. Aα/β neurons showed resting membrane potential depolarization, and increased repetitive firing during sustained depolarization developed in A&dgr; neurons. The afterhyperpolarization duration in neurons with C fibers shortened after axotomy. In contrast to the axotomized L5 neurons, neighboring L4 neurons showed no changes in action potential duration, afterhyperpolarization dimensions, or excitability after SNL. Depolarization rate (dV/dt) increased after SNL in L4 Aα/β and A&dgr; neurons but decreased in L5 neurons. Time-dependent rectification during hyperpolarizing current injection (sag) was greater after SNL in Aα/β L4 neurons compared with L5. Sham-SNL surgery produced only a decreased input resistance in Aα/β neurons and a decreased conduction velocity in medium-sized cells. In the L5 ganglion after axotomy, a novel set of neurons, consisting of 24% of the myelinated population, exhibited long action potential durations despite myelinated neuron conduction velocities, particularly depolarized resting membrane potential, low depolarization rate, and absence of sag. Conclusions:These findings indicate that nerve injury–induced electrical instability is restricted to axotomized neurons and is absent in adjacent intact neurons.

Microparticle production, neutrophil activation, and intravascular bubbles following open-water SCUBA diving

The goal of this study was to evaluate annexin V-positive microparticles (MPs) and neutrophil activation in humans following decompression from open-water SCUBA diving with the hypothesis that changes are related to intravascular bubble formation. Sixteen male volunteer divers followed a uniform profile of four daily SCUBA dives to 18 m of sea water for 47 min. Blood was obtained prior to and at 80 min following the first and fourth dives to evaluate the impact of repetitive diving, and intravascular bubbles were quantified by trans-thoracic echocardiography carried out at 20-min intervals for 2 h after each dive. MPs increased by 3.4-fold after each dive, neutrophil activation occurred as assessed by surface expression of myeloperoxidase and the CD18 component of β(2)-integrins, and there was an increased presence of the platelet-derived CD41 protein on the neutrophil surface indicating interactions with platelet membranes. Intravascular bubbles were detected in all divers. Surprisingly, significant inverse correlations were found among postdiving bubble scores and MPs, most consistently at 80 min or more after the dive on the fourth day. There were significant positive correlations between MPs and platelet-neutrophil interactions after the first dive and between platelet-neutrophil interactions and neutrophil activation documented as an elevation in β(2)-integrin expression after the fourth dive. We conclude that MPs- and neutrophil-related events in humans are consistent with findings in an animal decompression model. Whether there are causal relationships among bubbles, MPs, platelet-neutrophil interactions, and neutrophil activation remains obscure and requires additional study.

Differences in Production of Reactive Oxygen Species and Mitochondrial Uncoupling as Events in the Preconditioning Signaling Cascade Between Desflurane and Sevoflurane

BACKGROUND: Signal transduction cascade of anesthetic-induced preconditioning has been extensively studied, yet many aspects of it remain unsolved. Here, we investigated the roles of reactive oxygen species (ROS) and mitochondrial uncoupling in cardiomyocyte preconditioning by two modern volatile anesthetics: desflurane and sevoflurane. METHODS: Adult rat ventricular cardiomyocytes were isolated enzymatically. The preconditioning potency of desflurane and sevoflurane was assessed in cell survival experiments by evaluating myocyte protection from the oxidative stress-induced cell death. ROS production and flavoprotein fluorescence, an indicator of flavoprotein oxidation and mitochondrial uncoupling, were monitored in real time by confocal microscopy. The functional aspect of enhanced ROS generation by the anesthetics was assessed in cell survival and confocal experiments using the ROS scavenger Trolox. RESULTS: Preconditioning of cardiomyocytes with desflurane or sevoflurane significantly decreased oxidative stress-induced cell death. That effect coincided with increased ROS production and increased flavoprotein oxidation detected during acute myocyte exposure to the anesthetics. Desflurane induced significantly greater ROS production and flavoprotein oxidation than sevoflurane. ROS scavenging with Trolox abrogated preconditioning potency of anesthetics and attenuated flavoprotein oxidation. CONCLUSION: Preconditioning with desflurane or sevoflurane protects isolated rat cardiomyocytes from oxidative stress-induced cell death. Scavenging of ROS abolishes the preconditioning effect of both anesthetics and attenuates anesthetic-induced mitochondrial uncoupling, suggesting a crucial role for ROS in anesthetic-induced preconditioning and implying that ROS act upstream of mitochondrial uncoupling. Desflurane exhibits greater effect on stimulation of ROS production and mitochondrial uncoupling than sevoflurane.

Modulators of Calcium Influx Regulate Membrane Excitability in Rat Dorsal Root Ganglion Neurons

BACKGROUND: Chronic neuropathic pain resulting from neuronal damage remains difficult to treat, in part, because of incomplete understanding of underlying cellular mechanisms. We have previously shown that inward Ca2+ flux (ICa) across the sensory neuron plasmalemma is decreased in a rodent model of chronic neuropathic pain, but the direct consequence of this loss of ICa on function of the sensory neuron has not been defined. We therefore examined the extent to which altered membrane properties after nerve injury, especially increased excitability that may contribute to chronic pain, are attributable to diminished Ca2+ entry. METHODS: Intracellular microelectrode measurements were obtained from A-type neurons of dorsal root ganglia excised from uninjured rats. Recording conditions were varied to suppress or promote ICa while biophysical variables and excitability were determined. RESULTS: Both lowered external bath Ca2+ concentration and blockade of ICa with bath cadmium diminished the duration and area of the after-hyperpolarization (AHP), accompanied by decreased current threshold for action potential (AP) initiation and increased repetitive firing during sustained depolarization. Reciprocally, elevated bath Ca2+ increased the AHP and suppressed repetitive firing. Voltage sag during neuronal hyperpolarization, indicative of the cation-nonselective H-current, diminished with decreased bath Ca2+, cadmium application, or chelation of intracellular Ca2+. Additional recordings with selective blockers of ICa subtypes showed that N-, P/Q, L-, and R-type currents each contribute to generation of the AHP and that blockade of any of these, and the T-type current, slows the AP upstroke, prolongs the AP duration, and (except for L-type current) decreases the current threshold for AP initiation. CONCLUSIONS: Taken together, our findings show that suppression of ICa decreases the AHP, reduces the hyperpolarization-induced voltage sag, and increases excitability in sensory neurons, replicating changes that follow peripheral nerve trauma. This suggests that the loss of ICa previously demonstrated in injured sensory neurons contributes to their dysfunction and hyperexcitability, and may lead to neuropathic pain.

Bubbles, microparticles, and neutrophil activation: changes with exercise level and breathing gas during open-water SCUBA diving

The study goal was to evaluate responses in humans following decompression from open-water SCUBA diving with the hypothesis that exertion underwater and use of a breathing mixture containing more oxygen and less nitrogen (enriched air nitrox) would alter annexin V-positive microparticle (MP) production and size changes and neutrophil activation, as well as their relationships to intravascular bubble formation. Twenty-four divers followed a uniform dive profile to 18 m of sea water breathing air or 22.5 m breathing 32% oxygen/68% nitrogen for 47 min, either swimming with moderately heavy exertion underwater or remaining stationary at depth. Blood was obtained pre- and at 15 and 120 min postdive. Intravascular bubbles were quantified by transthoracic echocardiography postdive at 20-min intervals for 2 h. There were no significant differences in maximum bubble scores among the dives. MP number increased 2.7-fold, on average, within 15 min after each dive; only the air-exertion dive resulted in a significant further increase to 5-fold over baseline at 2 h postdive. Neutrophil activation occurred after all dives. For the enriched air nitrox stationary at depth dive, but not for other conditions, the numbers of postdive annexin V-positive particles above 1 μm in diameter were correlated with intravascular bubble scores (correlation coefficients ∼0.9, P < 0.05). We conclude that postdecompression relationships among bubbles, MPs, platelet-neutrophil interactions, and neutrophil activation appear to exist, but more study is required to improve confidence in the associations.

Venous and Arterial Bubbles at Rest after No-Decompression Air Dives

PURPOSE During SCUBA diving, breathing at increased pressure leads to a greater tissue gas uptake. During ascent, tissues may become supersaturated, and the gas is released in the form of bubbles that typically occur on the venous side of circulation. These venous gas emboli (VGE) are usually eliminated as they pass through the lungs, although their occasional presence in systemic circulation (arterialization) has been reported and it was assumed to be the main cause of the decompression sickness. The aims of the present study were to assess the appearance of VGE after air dives where no stops in coming to the surface are required and to assess their potential occurrence and frequency in the systemic circulation. METHODS Twelve male divers performed six dives with 3 d of rest between them following standard no-decompression dive procedures: 18/60, 18/70, 24/30, 24/40, 33/15, and 33/20 (the first value indicates depth in meters of sea water and the second value indicates bottom time in minutes). VGE monitoring was performed ultrasonographically every 20 min for 120 min after surfacing. RESULTS Diving profiles used in this study produced unexpectedly high amounts of gas bubbles, with most dives resulting in grade 4 (55/69 dives) on the bubble scale of 0-5 (no to maximal bubbles). Arterializations of gas bubbles were found in 5 (41.7%) of 12 divers and after 11 (16%) of 69 dives. These VGE crossovers were only observed when a large amount of bubbles was concomitantly present in the right valve of the heart. CONCLUSIONS Our findings indicate high amounts of gas bubbles produced after no-decompression air dives based on standardized diving protocols. High bubble loads were frequently associated with the crossover of VGE to the systemic circulation. Despite these findings, no acute decompression-related pathology was detected.

Successive deep dives impair endothelial function and enhance oxidative stress in man

The aim of this study was to assess the effects of successive deep dives on endothelial function of large conduit arteries and plasma pro‐oxidant and antioxidant activity. Seven experienced divers performed six dives in six consecutive days using a compressed mixture of oxygen, helium and nitrogen (trimix) with diving depths ranging from 55 to 80 m. Before and after first, third and sixth dive, venous gas emboli formation and brachial artery function (flow‐mediated dilation, FMD) was assessed by ultrasound. In addition, plasma antioxidant capacity (AOC) was measured by ferric reducing antioxidant power, and the level of oxidative stress was assessed by thiobarbituric acid‐reactive substances (TBARS) method. Although the FMD was reduced to a similar extent after each dive, the comparison of predive FMD showed a reduction from 8·6% recorded before the first dive to 6·3% before the third (P = 0·03) and 5·7% before the sixth dive (P = 0·003). A gradual shift in baseline was also detected with TBARS assay, with malondialdehyde values increasing from 0·10 ± 0·02 μmol l−1 before the first dive to 0·16 ± 0·03 before the sixth (P = 0·005). Predive plasma AOC values also showed a decreasing trend from 0·67 ± 0·20 mmol l−1 trolox equivalents (first day) to 0·56 ± 0·12 (sixth day), although statistical significance was not reached (P = 0·08). This is the first documentation of acute endothelial dysfunction in the large conduit arteries occurring after successive deep trimix dives. Both endothelial function and plasma pro‐oxidant and antioxidant activity did not return to baseline during the course of repetitive dives, indicating possible cumulative and longer lasting detrimental effects.

Failure of action potential propagation in sensory neurons: mechanisms and loss of afferent filtering in C-type units after painful nerve injury

The peripheral terminals of sensory neurons encode physical and chemical signals into trains of action potentials (APs) and transmit these trains to the CNS. Although modulation of this process is thought to predominantly reside at synapses, there are also indications that AP trains are incompletely propagated past points at which axons branch. One such site is the T‐junction, where the single sensory neuron axon branches into peripheral and central processes. In recordings from sensory neurons of dorsal root ganglia excised from adult rats, we identified use‐dependent failure of AP propagation between the peripheral and central processes that results in filtering of rapid AP trains, especially in C‐type neurons. Propagation failure was regulated by membrane input resistance and Ca2+‐sensitive K+ and Cl− currents. Following peripheral nerve injury, T‐junction filtering is reduced in C‐type neurons, which may possibly contribute to pain generation.

Involuntary breathing movements improve cerebral oxygenation during apnea struggle phase in elite divers

We investigated whether the involuntary breathing movements (IBM) during the struggle phase of breath holding, together with peripheral vasoconstriction and progressive hypercapnia, have a positive effect in maintaining cerebral blood volume. The central hemodynamics, arterial oxygen saturation, brain regional oxyhemoglobin (bHbO(2)), deoxyhemoglobin, and total hemoglobin changes and IBM were monitored during maximal dry breath holds in eight elite divers. The frequency of IBM increased (by approximately 100%), and their duration decreased ( approximately 30%), toward the end of the struggle phase, whereas the amplitude was unchanged (compared with the beginning of the struggle phase). In all subjects, a consistent increase in brain regional deoxyhemoglobin and total hemoglobin was also found during struggle phase, whereas bHbO(2) changed biphasically: it initially increased until the middle of the struggle phase, with the subsequent relative decline at the end of the breath hold. Mean arterial pressure was elevated during the struggle phase, although there was no further rise in the peripheral resistance, suggesting unchanged peripheral vasoconstriction and implying the beneficial influence of the IBM on the cardiac output recovery (primarily by restoration of the stroke volume). The IBM-induced short-lasting, sudden increases in mean arterial pressure were followed by similar oscillations in bHbO(2). These results suggest that an increase in the cerebral blood volume observed during the struggle phase of dry apnea is most likely caused by the IBM at the time of the hypercapnia-induced cerebral vasodilatation and peripheral vasoconstriction.

Isoflurane activates human cardiac mitochondrial adenosine triphosphate-sensitive K+ channels reconstituted in lipid bilayers.

BACKGROUND:Activation of the mitochondrial adenosine triphosphate (ATP)-sensitive K+ channel (mitoKATP) has been proposed as a critical step in myocardial protection by isoflurane-induced preconditioning in humans and animals. Recent evidence suggests that reactive oxygen species (ROS) may mediate isoflurane-mediated myocardial protection. In this study, we examined the direct effect of isoflurane and ROS on human cardiac mitoKATP channels reconstituted into the lipid bilayers. METHODS:Inner mitochondrial membranes were isolated from explanted human left ventricles not suitable for heart transplantation and fused into lipid bilayers in symmetrical potassium glutamate solution (150 mM). ATP-sensitive K+ currents were recorded before and after exposure to isoflurane and H2O2 under voltage clamp. RESULTS:The human mitoKATP was identified by its sensitivity to inhibition by ATP and 5-hydroxydecanoate. Addition of isoflurane (0.8 mM) increased the open probability of the mitoKATP channels, either in the presence or absence of ATP inhibition (0.5 mM). The isoflurane-mediated increase in K+ currents was completely inhibited by 5-hydroxydecanoate. Similarly, H2O2 (200 &mgr;M) was able to activate the mitoKATP previously inhibited by ATP. CONCLUSIONS:These data confirm that isoflurane, as well as ROS, directly activates reconstituted human cardiac mitoKATP channel in vitro, without apparent involvement of cytosolic protein kinases, as commonly proposed. Activation of the mitoKATP channel may contribute to the myocardial protective effect of isoflurane in the human heart.