Inactivation of sodium channels underlies reversible neuropathy during critical illness in rats

Abstract

Neuropathy and myopathy can cause weakness during critical illness. To determine whether reduced excitability of peripheral nerves, rather than degeneration, is the mechanism underlying acute neuropathy in critically ill patients, we prospectively followed patients during the acute phase of critical illness and early recovery and assessed nerve conduction. During the period of early recovery from critical illness, patients recovered from neuropathy within days. This rapidly reversible neuropathy has not to our knowledge been previously described in critically ill patients and may be a novel type of neuropathy. In vivo intracellular recordings from dorsal root axons in septic rats revealed reduced action potential amplitude, demonstrating that reduced excitability of nerve was the mechanism underlying neuropathy. When action potentials were triggered by hyperpolarizing pulses, their amplitudes largely recovered, indicating that inactivation of sodium channels was an important contributor to reduced excitability. There was no depolarization of axon resting potential in septic rats, which ruled out a contribution of resting potential to the increased inactivation of sodium channels. Our data suggest that a hyperpolarized shift in the voltage dependence of sodium channel inactivation causes increased sodium inactivation and reduced excitability. Acquired sodium channelopathy may be the mechanism underlying acute neuropathy in critically ill patients.

Figure 1. Rats develop reversible neuropathy following induction of sepsis.

(A) Example of 3 tail nerve responses before and 3 days after cecal ligation and puncture to induce sepsis. There is a range of responses in the 3 rats. In rat 1, there was a 70% drop in amplitude and a substantial prolongation of distal latency. In rat 2, there was a 20% drop in amplitude and moderate prolongation of distal latency and duration. In rat 3, there was little effect on amplitude, but distal latency and duration were prolonged. (B) Paired scatter plot of tail nerve response amplitude before and after 3 days of sepsis for each of 29 rats (P < 0.01, paired Student’s t test). (C) An example of the tail nerve response in a rat before, during, and after recovery from sepsis. The amplitude is reduced by 20% days 2 and 3 following cecal ligation and puncture but recovers by day 7. The dotted line is placed to aid in comparison of amplitudes. (D) A plot of the normalized average tail nerve response prior to cecal ligation and puncture (CLP), 2, 3, and 7 days later demonstrates the recovery of nerve amplitude as the rats (n = 8) recovered from sepsis. At day 2, the reduction in tail nerve amplitude is statistically significant relative to both the baseline and post-recovery amplitude (P < 0.01 for both). Data are presented as mean ± SEM.

Figure 2. Nerve morphology is normal in septic rats with neuropathy.

Toluidine blue–stained sural (A and B) and distal tibial (C–E) nerve sections from a rat 7 days after induction of sepsis. The nerves have normal axon morphology and myelination despite a 35% reduction in tail nerve amplitude on nerve conduction studies (on day 3 following sepsis) in the rat from which the nerves were harvested. A–D show 1-μm cross sections, and E shows a 1-μm longitudinal section. The arrow in E indicates a normal node of Ranvier. Scale bars: 50 μm (A and C); 20 μm (B, D, and E).

Figure 3. Amplitude of action potentials is reduced in dorsal root axons 3 days after induction of sepsis.

(A) Representative dorsal root axon action potentials from untreated and septic rats, showing the range of amplitudes in each group. While the largest action potentials from axons in septic rats were normal in amplitude, half the axons had action potentials that were smaller than those normally found in untreated rats. The horizontal line represents 0 mV. In untreated rats, 53% of axons had action potentials that exceeded 0 mV. In septic rats only 27% of axons had action potentials that exceeded 0 mV. Scale bars: 20 mV (vertical); 0.5 ms (horizontal). (B) Left: Action potential amplitude for axons from all untreated and septic rats (P < 0.01 by the Kolmogorov-Smirnov test, n = 60 axons from untreated rats and 33 axons from septic rats). Right: Action potential amplitude when resting potentials were matched between –50 and –55 mV. The difference in action potential amplitudes is unchanged when resting potential is matched (P < 0.01, n = 13 axons from untreated rats and n = 13 axons from septic rats).

Figure 4. Increase in action potential amplitude following anode break excitation suggests that inactivation of sodium channels is an important contributor to reduced excitability.

(A) Examples of depolarization-induced (rheobase) and anode break–induced action potentials from individual axons in septic rats 3 days following induction of sepsis. In axon 1, the small action potential following rheobase excitation gets much larger after anode break. In axon 2, the large action potential following rheobase excitation is only modestly larger following anode break. The horizontal line represents 0 mV. (B) Plot of the mean (±SEM) increase in action potential amplitude following anode break excitation for untreated and septic axons. The increase in action potential amplitude following anode break was greater in septic axons with small action potentials (P < 0.01 vs. both axons from untreated rats and axons from septic rats with normal action potentials; n = 58 normal action potentials [AP] from untreated rats, n = 15 small action potentials from septic rats, and n = 18 normal action potentials from septic rats). (C) Plot of the percent increase in action potential amplitude following anode break versus rheobase action potential amplitude. The increase in action potential amplitude following anode break is inversely correlated with rheobase action potential amplitude in septic rats (r = –0.78, P < 0.01, solid line). In control rats, there was also a relationship, but it was not as strong (r = –0.63, P < 0.01, dashed line). (D) The increase in action potential amplitude following anode break is not related to resting potential (r = 0.04, P = 0.69 for axons from septic rats).

Figure 5. The effect of shifting the voltage dependence of sodium channel inactivation on action potentials triggered by both depolarizing and hyperpolarizing current injection.

The black curve represents the hypothetical voltage dependence of sodium channel inactivation in an axon from an untreated rat. The sodium channel inactivation curve (gray) for an axon with reduced excitability from a septic rat is shifted to the left. In the septic axon, at a resting potential of –55 mV, only 30% of sodium channels are available to participate in generating an action potential. In the normal axon, at the same resting potential, 80% of sodium channels are available. The difference in percentage of sodium channels available leads to a small action potential in the axon from the septic rat (Rheobase AP, gray curve) and a large action potential in the normal axon (Rheobase AP, black curve). An anode break pulse hyperpolarizes each axon by 5 mV. In the axon from the septic rat, the resting potential is on the steep part of the inactivation curve, so a 5-mV hyperpolarization causes substantial relief of sodium channel inactivation and a large increase in action potential amplitude (Anode break AP, gray curve). In the normal axon, the resting potential is on the shallow part of the curve, so a 5 mV hyperpolarization does not greatly relieve inactivation and causes only a small increase in action potential amplitude (Anode break AP, black curve).