Action Potential Recovery Research Articles

Questioning the Mechanism of Nerve Injury

We thank Lambert et al. for their interest in our article1and stimulating comments. We would like to take the opportunity to address the issues raised by their insights.Lambert et al. suggest that the mechanism of local anesthetic neurotoxicity is unrelated to membrane disruption caused by the detergent nature of highly concentrated local anesthetics, instead resulting from breakdown of the sodium conductance system (i.e. , absence of compound action potential generation) despite the presence of an intact membrane structure and normal ionic gradients and compound resting potential. The basis for their contention is that, although both compound action potential and compound resting potential in excised sciatic nerves of the bullfrog disappear permanently after bathing in sodium lauryl sulfate (typical detergent) solution,2compound resting potential recovers without compound action potential recovery after bathing in 5% lidocaine solution for 15 min, as noted in their earlier electrophysiologic study.3However, the study cited as grounds for their question about our conclusions seems to have an inherent limitation in that the period for bathing the specimen in 5% lidocaine is 15 min. Kanai et al. 4reported a similar study using a single crayfish axon and demonstrated that although resting potential recovers after bathing in 80 mm lidocaine solution for 15 min, resting potential permanently disappears after bathing for 30 min. They suggested that highly concentrated lidocaine causes membrane disruption and indicated in that time-dependent study that sufficient exposure (≥ 30 min) was needed for membrane disruption to be induced by lidocaine. Ready et al. 5also demonstrated successfully that lidocaine at a concentration of 4% or higher induces histopathologic changes in spinal nerves during a dose-dependent study of a spinal anesthesia model in rabbits. The fact that highly concentrated lidocaine causes membrane disruption is thus not in doubt.We advocated in our article that the mechanism of membrane disruption induced by highly concentrated local anesthetics would result from the detergent nature of these agents.1From the perspective of results from various investigators, including Lambert et al. , we speculate that an intermediate step may exist in the membrane disruption dynamics induced by 5% lidocaine, with resting membrane potential maintained despite permanent inhibition of action potential. The electrophysiologic phenomena described by Lambert et al. may represent an early phase in the sequence of neurotoxic dynamics induced by 5% lidocaine.* Saga Medical School, Nabeshima, Saga, Japan. kitagawa@mail.anes.saga-med.ac.jp

Endocochlear potentials and compound action potential recovery: functions in the C57BL/6J mouse

The C57BL/6J mouse suffers from cochlear degeneration beginning at an early age and has been used as a model of age-related hearing loss (presbyacusis). Here, the endocochlear potential (EP) and compound action potential (CAP) responses were determined in one-, four-, 12- and 24-month-old C57BL/6J mice. CAP measures included thresholds to tone pips, input/output (I/O) functions, and recovery functions to conditioning tones. EP values among the four age groups did not differ significantly ( P>0.05) in either the basal or apical turns. CAP thresholds were increased significantly by 10 to 30 dB in the four-month group compared to the one-month controls at 11.3, 16, 20, and 22.6 kHz. CAP I/O functions were shallower in the four-month group compared to controls at all frequencies. In the 12- and 24-month-old mice, CAP responses were absent, despite normal EP values in these animals. Recovery functions after conditioning tones were obtained at 8, 16, 20 and 22.6 kHz; the functions had fast and slow components at all frequencies tested in both the one- and four-month-old groups. The corresponding recovery curves were identical for both age groups, even with significant threshold shifts in the older group. The two component recovery curves provide the first physiological evidence that different spontaneous rate (SR) classes of auditory neurons exist in the C57BL/6J mouse. Moreover, the unchanged recovery functions in the older group suggest that there was no loss of activity of the low-SR fiber population with age under conditions where the EP remains stable, in contrast to the gerbil model of presbyacusis where there is a loss of low-SR fiber activity and EP does decline with age.

Polyethylene glycol rapidly restores physiological functions in damaged sciatic nerves of guinea pigs.

We have studied the ability of the hydrophilic polymer polyethylene glycol (PEG) to anatomically and physiologically reconnect damaged axons of the adult guinea pig spinal cord. Here we have extended this approach to test whether completely severed guinea pig sciatic nerves in isolation could be fused and whether PEG was able to repair severe standardized crush injuries to sciatic nerves in vivo. The fusion test was performed with isolated sciatic nerves maintained in a double-sucrose gap recording chamber. For in vivo experiments, the sciatic nerve was surgically exposed in the hind leg of deeply anesthetized adult guinea pigs and was crushed proximal to its insertion in the gastrocnemius muscle. PEG was injected just beneath the epineurium with a 29-gauge needle, allowed to remain in the damaged axon region for 2 minutes, and removed. Sham-treated guinea pigs received an injection of water or Krebs' solution. Three indices of recovery were simultaneously monitored in response to electrical stimulation of the proximal nerve, i.e., 1) recovery of compound muscle action potentials (in millivolts), 2) contraction force of the muscle (in dynes), and 3) displacement of the muscle (in millimeters). When isolated sciatic nerves were severed within the double-sucrose gap chamber, compound action potential propagation through the transection plane was eliminated. After abutment of the two segments and 2-minute PEG application to this site, variable compound action potential recovery was measured in all four cases. The crush injuries to the sciatic nerve in vivo eliminated the three functional responses to sciatic nerve stimulation in all animals. Within the first 30 minutes after treatment, only 1 of 12 control animals exhibited spontaneous recovery in any of these measures, compared with six of eight PEG-treated animals. By 45 minutes, two more sham-treated animals and one more PEG-treated animal had recovered at least one functional response. This difference in proportions between PEG-treated and sham-treated animals was statistically significant (P < or =0.02). We conclude that these preliminary data suggest that PEG application may be a way to interfere with the steady dissolution of peripheral nerve fibers after mechanical damage and to even functionally fuse or reconnect severed proximal and distal segments.

Hydrogen peroxide decelerates recovery of action potential after high‐frequency fatigue in skeletal muscle

Hydrogen peroxide decelerates recovery of action potential after high‐frequency fatigue in skeletal muscle

Hydrogen peroxide decelerates recovery of action potential after high-frequency fatigue in skeletal muscle.

Effects of reactive oxygen species (ROS), especially hydrogen peroxide (H(2)O(2)), on recovery of action potential by resting for 30 min after high-frequency fatigue were studied using frog skeletal muscle fibers. After stimulation at a frequency of 50 HZ for 2 min, the action potential amplitude was decreased by 14.5 mV from controls, and resting membrane was depolarized by 15.4 mV. Action potential duration was also prolonged by high-frequency stimulation (1.5 ms in controls to 2.6 ms). The high-frequency stimulation used here caused no muscle damage. The action potential was partially improved after a 30-min rest. Addition of catalase at 500 units/ml or H(2)O(2) at 0.5 mM to sartorius muscle did not alter any of the parameters of the action potential after high-frequency stimulation. Treatment with catalase accelerated post-fatigue recovery of the action potential. Application of H(2)O(2) delayed post-fatigue recovery of resting and action potentials. When added to detubulated toe muscle fibers, catalase no longer improved the attenuation of action potential induced by high-frequency stimulation, even after a 30-min rest. These findings suggest that removal of H(2)O(2) from transverse tubules is effective for post-fatigue recovery of action potential in skeletal muscle.

Graded, irreversible changes in crayfish giant axon as manifestations of lidocaine neurotoxicity in vitro.

High concentrations of lidocaine induce irreversible conduction block with little effect on resting membrane potential (Em). We assumed the mechanism of persistent neurologic deficit caused by local anesthetics may result from neural death, as represented by the loss of Em. We investigated the effects of lidocaine on Em and action potential (AP) in single crayfish giant axons in vitro. Axons were perfused with two doses of lidocaine for either 15 or 30 min, and they were continuously washed. No axons exposed to 80 mM lidocaine for 30 min showed recovery of AP and Em. Those exposed to 40 mM for 30 min and 80 mM for 15 min showed a return to baseline for Em, but no recovery of AP. Those exposed to 40 mM lidocaine for 15 min showed full recovery of Em and AP immediately after washing. The membrane depolarization was significantly greater during exposure to 80 mM lidocaine for 30 min than in other groups. We conclude that lidocaine has a direct neurotoxic effect on crayfish giant axons and that the generation of AP is more vulnerable than the maintenance of Em. The irreversibility of AP and Em is dose- and time-dependent. Highly concentrated lidocaine induced an irreversible conduction block and a complete loss of resting membrane potential in crayfish giant axons in vitro. Our results may represent a possible explanation for various grades of local anesthetic-induced neurotoxicity in clinical cases if the same toxicity occurs in mammalian nerves in vivo.

Graded, Irreversible Changes in Crayfish Giant Axon as Manifestations of Lidocaine Neurotoxicity In Vitro

High concentrations of lidocaine induce irreversible conduction block with little effect on resting membrane potential (Em).We assumed the mechanism of persistent neurologic deficit caused by local anesthetics may result from neural death, as represented by the loss of Em. We investigated the effects of lidocaine on Em and action potential (AP) in single crayfish giant axons in vitro. Axons were perfused with two doses of lidocaine for either 15 or 30 min, and they were continuously washed. No axons exposed to 80 mM lidocaine for 30 min showed recovery of AP and Em. Those exposed to 40 mM for 30 min and 80 mM for 15 min showed a return to baseline for Em, but no recovery of AP. Those exposed to 40 mM lidocaine for 15 min showed full recovery of Em and AP immediately after washing. The membrane depolarization was significantly greater during exposure to 80 mM lidocaine for 30 min than in other groups. We conclude that lidocaine has a direct neurotoxic effect on crayfish giant axons and that the generation of AP is more vulnerable than the maintenance of Em. The irreversibility of AP and Em is dose- and time-dependent. Implications: Highly concentrated lidocaine induced an irreversible conduction block and a complete loss of resting membrane potential in crayfish giant axons in vitro. Our results may represent a possible explanation for various grades of local anesthetic-induced neurotoxicity in clinical cases if the same toxicity occurs in mammalian nerves in vivo. (Anesth Analg 1998;86:569-73)

Protection by hypoxic preconditioning against hypoxia-reoxygenation injury in guinea-pig papillary muscles.

Developed tension in guinea-pig papillary muscles is depressed by prolonged hypoxia; subsequent reoxygenation leads to a partial recovery that stabilizes after an early period of arrhythmia. We have investigated whether hypoxic preconditioning in these muscles (1) improves the recovery of developed tension, (2) protects against arrhythmia, and (3) causes other significant electromechanical changes. Papillary muscles stimulated at 1 Hz were superfused with oxygenated Krebs solution for 60 min and either preconditioned (5 min of 3 Hz pacing substrate-free hypoxic conditions, 10 min of normoxic recovery) or equilibrated for an extra 15 min. Muscles were subsequently challenged with substrate-free hypoxia (1 Hz), and reoxygenated (1 Hz) for 60 min. Contractile performance, action potential parameters, and indicators of arrhythmic activity were measured in 10 preconditioned and 10 non-preconditioned muscles. Developed tension in preconditioned muscles declined to the same level (10-15% control) as in non-preconditioned muscles after 60 min hypoxia. A notable difference was that developed tension in the preconditioned muscles failed to rebound during mid-hypoxia, a hallmark feature in non-preconditioned muscles. The action potential duration and overshoot collapsed at a significantly faster rate in hypoxic preconditioned muscles. Action potential recovery during reoxygenation was similar in the two groups of muscles, but recovery of developed tension was significantly stronger in preconditioned (76.7 +/- 5.4%) than in non-preconditioned (42.9 +/- 1.7%) muscles (P < 0.001). Reoxygenation provoked arrhythmic activity in all muscles, but the summed average duration was shorter (5.5 +/- 1.0 vs. 9.4 +/- 1.5 min) (P < 0.05) in the preconditioned muscles. Hypoxic preconditioning can significantly enhance post-hypoxia recovery of developed tension, and significantly attenuate arrhythmic activity, in guinea-pig papillary muscles.

Cycle length dependence of the electrophysiological effects of increased load on the myocardium.

Mechanoelectric feedback, the process by which changes in mechanical activity change the electrophysiology of the myocardium, has been linked to the genesis of arrhythmias. We investigated possible arrhythmogenic mechanisms by measuring changes in steady-state action potential duration and, more particularly, electrical restitution on a transiently applied load change, because action potential recovery may provide clues to arrhythmogenesis. Pigs were anesthetized and their hearts exposed. A snare was placed around the aorta, and the right atrium was paced. Ventricular pressure, monophasic action potential, and segment motion were recorded from the left ventricle. The action potential duration was measured before and during transient aortic occlusion. Electrical restitution curves were constructed from the records obtained during normal loading or during transient aortic occlusion. The degree of shortening of action potential duration on aortic occlusion decreased with decreases in the steady-state beat-to-beat interval (P = .0008). Control restitution curves had the typical configuration, with a rapid initial, usually monotonic, rise toward a plateau. Some curves showed a marginal "supernormal" section. Increased load reduced the action potential duration at the plateau of the restitution curve (9.4 ms, P < .0001) but increased the action potential duration at the start of the restitution curve (8.7 ms, P = .03). Increased loading increased the maximum slope of the electrical restitution curve by 32 ms/100 ms (P = .04). Increased load also increased the supernormal period of the electrical restitution curves. Mechanoelectric feedback produces changes in rate-dependent electrophysiology, which could favor a matrix conducive to arrhythmogenesis.

Role of extracellular calcium in anoxic injury of mammalian central white matter.

White matter (WM) of the mammalian brain is susceptible to anoxic injury, but little is known about the pathophysiology of this process. We studied the mechanisms of anoxic injury in WM using the isolated rat optic nerve, a typical central nervous system WM tract. Optic nerve function, measured as the area under the compound action potential, rapidly failed when exposed to anoxia and recovered to 28.5% of control after a standard 60-min period of anoxia. Irreversible anoxic injury was critically dependent on the molar concentration of extracellular calcium [( Ca2+]o); maintaining the tissue in Ca2(+)-free solution throughout the anoxic period resulted in 100% compound action potential recovery. Increasing perfusion [Ca2+] during anoxia from zero to 4 mM resulted in progressively less recovery. As the introduction of the Ca2(+)-free solution was progressively delayed with respect to the onset of anoxia, a graded decrease in recovery of function was observed, suggesting that in WM the deleterious effects of Ca2+ accumulate slowly during anoxia. At the time of reoxygenation, an additional stepwise increase in injury was observed that was also Ca2(+)-dependent. Mammalian WM, which is relatively resistant to anoxic injury compared with gray matter, is damaged by anoxia in a manner that appears to depend on the gradual accumulation of Ca2+ in a cytoplasmic compartment.

Effect of Na+- or Ca2+-filled liposomes on electrical activity of cultured heart cells.

Phosphatidylcholine (PC) liposomes were used to deliver their entrapped ions into spontaneously contracting cultured heart cells (reaggregates) prepared from 15-day embryonic chick ventricles (superfused at 8 ml/min). With slowly rising action potentials (AP) (+Vmax less than 30 V/s), when Na+ liposomes were added (13.2% vol/vol), there was a progressive decrease in slope of the pacemaker potential and in firing frequency. Recovery on washout was rapid. When vesicles containing Ca2+ (6.6% vol/vol) were added, there was an immediate decrease in AP duration. Within 10 min, +Vmax, overshoot, maximum diastolic potential, and frequency decreased, and all spontaneous activity stopped within 30 min. Electrical stimulation could not elicit AP responses. Partial recovery of APs occurred after 60 min of washout, but the recovering APs initially were abnormal. Lower liposome concentration (3.3%) had a similar but slower effect. The rapidity of the effects depended on the Ca2+ concentration inside the vesicles (0.15 and 1 M). K+-containing liposomes had no effect. This difference between liposomes containing Na+, Ca2+, and K+ suggests that the effects observed were due to the ions and not to the phosphatidylcholine. Injection of Ca2+ could cause depolarization by the Ca2+-activation of a nonspecific Na+-K+ channel (i.e., gNa, K, (Ca]; inhibition of automaticity by injection of Na+ or Ca2+ could be due to diminution of the electrochemical gradient for inward background depolarizing current. Thus PC liposomes provide a good means for delivering substances inside the cells without altering the electrical properties of the membrane by the lipid vehicle.

The Effect of Intravenous Injection of Lidocaine on the Auditory System

Lidocaine hydrochloride was intravenously injected into guinea pigs. Auditory evoked brain stem response (ABR) to clicks, whole nerve action potential (AP) and summating potential (SP) to clicks and 4 kHz tone bursts, and cochlear microphonics (CM) to 4 kHz tone bursts were recorded during a 60 min period following injection. Injection of a small dosage of lidocaine (4 mg/kg body weight) failed to produce any significant change in ABR wave III, AP and CM. However, a large dosage of lidocaine (20 mg/kg) influenced ABR and AP, but had no observable effect on CM. Latency of ABR wave III was prolonged, but its amplitude remained unchanged. Change in the case of 4 kHz tone burst-evoked AP was more pronounced than that in the case of the click-evoked AP. The 4 kHz tone burst-evoked AP showed an initial increase in amplitude, which was followed by a gradual decrease. Latency of both click and 4 kHz tone burst-evoked AP did not show any changes. The amplitude of SP showed more variation during the course of the test period following lidocaine injection than did either ABR or AP, however, SP decreased in its amplitude approximately 60 min after lidocaine injection. Complete disappearance of ABR and AP, followed by recovery of ABR and AP, was observed in animals given a larger dose (30 mg/kg) of lidocaine. CM did not completely die out, and in fact sustained only about a 75% decay in its amplitude. From the above results, it should be concluded that lidocaine, when injected intravenously, affects ABR and AP significantly more than CM.

Desensitization of the muscarinic receptor controlling action potential of bullfrog atrial muscles.

Action potentials of the bullfrog atrial muscle, being depressed by carbachol in concentrations of (1-5) X 10(-7) M, were found to show a slow recovery when application of the drug was sustained. The rate of onset of recovery largely varied depending on individual preparations. The recovery of action potentials was neither due to changes in the ionic distribution across the membrane nor to a secondary action of catecholamine which was released by the nicotinic action of carbachol on sympathetic nerve terminals. These results suggested that the muscarinic ACh receptor responsible for the depression of action potentials showed desensitization to the action of its agonist. The slow inward current recorded by the voltage-clamp experiment showed a decrease and subsequent slow recovery in the presence of carbachol. This suggested that the muscarinic ACh receptor associating with the ionic channel of the slow inward current showed desensitization. It may be suggested on the basis of these experimental results that 1) the muscarinic ACh receptor of bullfrog atrial muscle may compose a receptor-ionic channel complex (RICC) with voltage-dependent CA2+ channel and 2) the molecular reaction between this RICC and agonist may be comparable to that occurring in the nicotinic RICC of the frog end-plate.

Effect of cryoprotective agents on rat cutaneous nerves

Desheathed rat cutaneous nerves were exposed to various concentrations of ethylene glycol (EG), glycerol and dimethyl sulfoxide (DMSO) at temperatures of 1, 24, and 38 °C for periods of time ranging from 5 to 60 min. Measurements of the percent recovery of the original action potential (AP) were determined after removal of the cryoprotective agent (CPA) under various conditions, i.e., temperature, time and sequence of rinsing. A comparison of the results obtained after the nerves were exposed directly to a 15% concentration of the three CPAs at 1 °C for a 15-min period showed that the percentage of recovery of the AP was 90, 69, and 36% of the original values when treated with DMSO, EG, or glycerol, respectively. In all three groups, the nerves were rinsed at 1 °C for 15 min. If the exposure to glycerol at 1 °C was carried out in a gradual stepwise manner, the recovery of the AP in 10 and 15% solutions ranged from 58 to 64%. If the temperatures of the exposure and rinse were increased to 24 and 38 °C, glycerol produced some toxicity within 10 min and after 25 min no recovery of AP was obtained. The results of a 10-min direct exposure to EG at 1 °C showed a moderate decrease in recovery of the AP as the concentration was increased from 10 to 15–20%. Increasing the exposure time to 15 and 30 min at 1 °C also contributed to further reduction in recovery. DMSO, however, in concentrations of 10, 15, and 20% produced only a slight decline of AP after a 5–15 min exposure at 1 °C. Recovery ranged from 96% after 10 min in a 10% solution to 88% after 15 min in a 20% solution. Toxicity became more apparent with DMSO when nerves were exposed to 30% concentrations for 5–10 min; the latter time resulted in a 49% recovery of the AP. Exposure of nerves to a CPA solution containing isotonic concentrations of electrolytes resulted in a 10–30% improvement in recovery when compared with specimens treated with lower levels of salt. The effect of raising the temperature of the rinse to 38 °C and increasing the wash time to 20 min was studied in a few selected experiments. After a direct 15-min exposure to a 15% solution of a CPA at 1 °C the recovery in the case of glycerol was significantly increased with such treatment whereas with EG and DMSO it remained unchanged. There was no evidence of thermal or cold shock in this work.

The recovery of auditory nerve action potentials after masking.

It is well known that the action potentials of the auditory nerve, as recorded from the round window in the cat or guinea pig in response to a train of click stimuli, are immediately reduced in amplitude in the presence of a wide band of noise of suitable intensity. We have observed that when such a masking noise is suddenly turned off, the action potentials of the click response do not regain their original size at once, but only after a period of 0.1 to 1.0 sec., depending upon the relative intensities of clicks and noise. The microphonic component of the response is similarly affected only at noise levels sufficiently intense to cause reflex contraction of the intra‐aural muscles; when this occurs the recovery of the action potentials is even longer delayed. The failure of the nerve response to regain full amplitude immediately after masking appears to bear upon the nature of the masking process itself. If masking were dependent exclusively upon the all‐or‐none properties of the nerve fiber and the maintenance of the refractory state by the noise, recovery might be expected to be complete within a few msec. after the noise ceases. We are inclined to attribute the fact that recovery is not instantaneous rather to a temporary depletion of the mechanism responsible for the initiation of excitation in the auditory nerve. The phenomenon appears to be more closely related to auditory fatigue than to intra‐cochlear inhibition, although the latter explanation has not been excluded.

The Recovery of Auditory Nerve Action Potentials After Masking

It is well known that the action potentials of the auditory nerve, as recorded from the round window in the cat or guinea pig in response to a train of click stimuli, are immediately reduced in amplitude in the presence of a wide band of noise of suitable intensity. We have observed that when such a masking noise is suddenly turned off, the action potentials of the click response do not regain their original size at once, but only after a period of 0.1 to 1.0 sec., depending upon the relative intensities of clicks and noise. The microphonic component of the response is similarly affected only at noise levels sufficiently intense to cause reflex contraction of the intra-aural muscles; when this occurs the recovery of the action potentials is even longer delayed. The failure of the nerve response to regain full amplitude immediately after masking appears to bear upon the nature of the masking process itself. If masking were dependent exclusively upon the all-or-none properties of the nerve fiber and the maintenance of the refractory state by the noise, recovery might be expected to be complete within a few msec. after the noise ceases. We are inclined to attribute the fact that recovery is not instantaneous rather to a temporary depletion of the mechanism responsible for the initiation of excitation in the auditory nerve. The phenomenon appears to be more closely related to auditory fatigue than to intra-cochlear inhibition, although the latter explanation has not been excluded.