Degeneration of myelinated efferent fibers induces spontaneous activity in uninjured C-fiber afferents

Abstract

We demonstrated recently that uninjured C-fiber nociceptors in the L4 spinal nerve develop spontaneous activity after transection of the L5 spinal nerve. We postulated that Wallerian degeneration leads to an alteration in the properties of the neighboring, uninjured afferents from adjacent spinal nerves. To explore the role of degeneration of myelinated versus unmyelinated fibers, we investigated the effects of an L5 ventral rhizotomy in rat. This lesion leads to degeneration predominantly in myelinated fibers. Mechanical paw-withdrawal thresholds were assessed with von Frey hairs, and teased-fiber techniques were used to record from single C-fiber afferents in the L4 spinal nerve. Behavioral and electrophysiological data were collected in a blinded manner. Seven days after surgery, a marked decrease in withdrawal thresholds was observed after the ventral rhizotomy but not after the sham operation. Single fiber recordings revealed low-frequency spontaneous activity in 25% of the C-fiber afferents 8-10 d after the lesion compared with only 11% after sham operation. Paw-withdrawal thresholds were inversely correlated with the incidence of spontaneous activity in high-threshold C-fiber afferents. In normal animals, low-frequency electrocutaneous stimulation at C-fiber, but not A-fiber, strength produced behavioral signs of secondary mechanical hyperalgesia on the paw. These results suggest that degeneration in myelinated efferent fibers is sufficient to induce spontaneous activity in C-fiber afferents and behavioral signs of mechanical hyperalgesia. Ectopic spontaneous activity from injured afferents was not required for the development of the neuropathic pain behavior. These results provide additional evidence for a role of Wallerian degeneration in neuropathic pain.

Fig. 1.

Spontaneous activity in two typical C-fiber afferents recorded 8 d after an L5 ventral rhizotomy.A, Teased-fiber techniques were used to record activity from single nerve fibers of the L4 spinal nerve (R) in sham-operated animals and in animals after transection of the L5 ventral root (T). Electrical stimulation of the sciatic nerve (S) was used to identify and count the number of C-fibers at the recording electrode. B, The presence of spontaneous action potential activity was assessed over a 5 min recording interval. In this example, two C-fibers with low-grade spontaneous activity were recorded simultaneously. On the left, eachpanel represents the spontaneous activity of one fiber, and each vertical line corresponds to the time of occurrence of an action potential. The action potential waveforms are illustrated on the right. C, Suprathreshold electrical stimulation at the sciatic nerve produced three discrete action potential waveforms at C-fiber latencies. The action potential waveform starting at 21 msec (peak at 22 msec) had the same shape as the spontaneously active fiber 1, providing evidence that the spontaneous activity came from this C-fiber. The two other C-fibers had superimposed waveforms (from 26 to 32 msec). Fiber 2 corresponds to one of these C-fibers.

Fig. 2.

Histological confirmation of the ventral rhizotomy lesion. A, Spinal roots at the level of the dorsal root ganglion 7 d after ventral rhizotomy, demonstrating a selective lesion of the ventral root axons. The ventral root on theright (asterisk) shows ongoing Wallerian degeneration. The dorsal root ganglion on the left shows intermixed cells and axons of the DRG, with no evidence of injury or fiber degeneration. Magnification, 100×. B, Left lateral plantar nerve 7 d after ventral rhizotomy shows a mixture of degenerating and intact myelinated axons. A myelinated axon undergoing Wallerian degeneration (asterisk) is seen in close proximity to the unmyelinated axons of C-fibers (arrows), a small myelinated axon, and an intact large myelinated axon (on the right). Magnification, 4500×.

Fig. 3.

Ventral rhizotomy produces mechanical hyperalgesia on the side ipsilateral to the lesion and spontaneous activity in C-fibers. A, Mechanical withdrawal thresholds decreased after the lesion but not after the sham surgery. Measurements were made before (open bars) and 7 d after (filled bars) the surgery (*p< 0.05; Wilcoxon matched pairs test). B, Incidence of spontaneous activity in high-threshold C-fibers increases after the L5 ventral rhizotomy (***p < 0.001; χ²test). The proportion of low-threshold fibers with spontaneous activity that responded to gentle brushing of the skin did not change with the lesion. However, the proportion of high-threshold fibers with spontaneous activity that did not respond to gentle brushing of the skin significantly increased after the lesion. C, Hyperalgesia is associated with a high incidence of spontaneous activity. The paw-withdrawal threshold for each animal is plotted as a function of the incidence of spontaneous activity in high-threshold C-fiber afferents for that animal. Animals with low paw-withdrawal thresholds had a higher incidence of spontaneous activity in high-threshold C-fiber afferents. Open symbolscorrespond to sham-operated animals, and filled symbolscorrespond to lesioned animals. D, Distribution of discharge frequencies for the high-threshold, C-fiber afferents. The distribution for the lesioned animals (filled bars) was similar to the distribution for the sham-operated animals (open bars). Data are normalized by the total number of spontaneously active, high-threshold afferents (n = 36, lesioned; n = 13, sham). The experimenters were blinded to the type of surgery at the time of the behavioral and electrophysiological measurements. Seven sham-operated animals and seven lesioned animals were studied.

Fig. 4.

Low-frequency electrocutaneous stimulation at C-fiber strength produces behavioral signs of hyperalgesia. The incidence of paw withdrawal is plotted as a function of time. Electrical stimulation (ES) of the heel (1 pulse every 5 sec for 10 min) was applied at C-fiber strength (filled symbols) or at A-fiber strength (open symbols).A, The response to the 150 mN von Frey probe increased significantly immediately after the termination of the C-fiber stimulation and remained high for 15 min. B, The response to the 75 mN probe also increased significantly after the C-fiber stimulation. There was no significant change in response after the A-fiber stimulation. (* p ≤ 0.05 and **p ≤ 0.01 with respect to average baseline response; ANOVA, followed by paired t test)