4-Aminopyridine: A Single-Dose Diagnostic Agent to Differentiate Axonal Continuity in Nerve Injuries

Abstract

Introduction: Traumatic peripheral nerve injuries (TPNIs) are increasingly prevalent in battlefield trauma, and the functional recovery with TPNIs depends on axonal continuity. Although the physical examination is the main tool for clinical diagnosis with diagnostic work up, there is no diagnostic tool available to differentiate nerve injuries based on axonal continuity. Therefore, treatment often relies on "watchful waiting," and this leads to muscle weakness and further reduces the chances of functional recovery. 4-aminopyridine (4-AP) is clinically used in multiple sclerosis patients for walking performance improvement. Preliminary results in conscious mice suggested a diagnostic role of 4-AP in distinguishing axonal continuity. In this study, we thought to evaluate the diagnostic potential of 4-AP on the axonal continuity in unawake/sedated animals.

Materials and methods: Rat sciatic nerve crush and transection injuries were used in this study. Briefly, rats were anesthetized with isoflurane and mechanically ventilated with oxygen-balanced vaporized isoflurane. Sciatic nerve and triceps surae muscles were exposed by blunt dissection, and a stimulating electrode was placed under a sciatic nerve proximal to the crush injury. A force transducer measured muscle tension response to electrical stimulation of sciatic nerve. Muscle response was measured before crush, after crush, and 30 minutes after systemic 4-AP (150 µg/kg) or local (4-AP)-poly(lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PLGA-PEG) treatment.

Results: We found that both crush and transection injuries in sciatic nerve completely abolished muscle response to electrical stimulation. Single dose of systemic 4-AP and local (4-AP)-PLGA-PEG treatment with crush injury significantly restored muscle responses to electrical stimulation after 30 minutes of administration. However, systemic 4-AP treatment had no effect on muscle response after nerve transection. These results clearly demonstrate that 4-AP can restore nerve conduction and produce muscle response within minutes of administration only when there is a nerve continuity, even in the sedated animal.

Conclusions: We conclude that 4-AP could be a promising diagnostic agent in differentiating TPNI based on axonal continuity.

FIGURE 1.

Experimental set-up for in situ muscle force measurement. Stimulating electrode is placed under the sciatic nerve and triceps surae muscle tension is measured by stimulating the sciatic nerve. Setup is equipped with a force transducer (FT10, Grass Instruments) to measure muscle tension.

FIGURE 2.

Representative muscle tension tracings of triceps surae muscles before and after crush injury with or without systemic 4-aminopyridine (4-AP) (150 μg/kg, ip) (A) and local 4-AP thermogel (C) treatments. Figures (B) and (D) show the muscle tension as % increase over crush after systemic and local 4-AP treatments, respectively. Bar graphs showing area under the curve (E, G) and peak muscle tension (F, H) before and after systemic or local 4-AP treatment, respectively. Figures E and F are for systemic, and figures G and H are for local 4-AP treatments. Data are represented as mean ± SEM. n = 6/group for systemic 4-AP, n = 3/group for local 4-AP. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test.

FIGURE 3.

Representative muscle tension tracings of triceps surae muscles after nerve cut injury and 4-aminopyridine (4-AP) (150 μg/kg, ip) treatment (A) and area under the curve (B) derived from muscle tension experiments. Data are represented as mean ± SEM, n = 3/group. ***P < 0.001 by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test.

FIGURE 4.

Representative muscle tension tracings of triceps surae muscles 3 days after crush injury with or without systemic 4-aminopyridine (4-AP) (150 μg/kg, ip) (A) and local 4-AP thermogel (C) treatments. Figures (B) and (D) show the muscle tension as % increase over crush after systemic and local 4-AP treatments, respectively. Bar graphs showing area under the curve (E, G) and peak muscle tension (F, H) before and after systemic or local 4-AP treatment, respectively. Figures E and F are for systemic, and figures G and H are for local 4-AP treatments. Data are represented as mean ± SEM. n = 6/group for systemic 4-AP, n = 3/group for local 4-AP. *P < 0.05 by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test.