Analgesic dorsal root ganglionic field stimulation blocks conduction of afferent impulse trains selectively in nociceptive sensory afferents

Abstract

Increased excitability of primary sensory neurons after peripheral nerve injury may cause hyperalgesia and allodynia. Dorsal root ganglion field stimulation (GFS) is effective in relieving clinical pain associated with nerve injury and neuropathic pain in animal models. However, its mechanism has not been determined. We examined effects of GFS on transmission of action potentials (APs) from the peripheral to central processes by in vivo single-unit recording from lumbar dorsal roots in sham injured rats and rats with tibial nerve injury (TNI) in fiber types defined by conduction velocity. Transmission of APs directly generated by GFS (20 Hz) in C-type units progressively abated over 20 seconds, whereas GFS-induced Aβ activity persisted unabated, while Aδ showed an intermediate pattern. Activity generated peripherally by electrical stimulation of the sciatic nerve and punctate mechanical stimulation of the receptive field (glabrous skin) was likewise fully blocked by GFS within 20 seconds in C-type units, whereas Aβ units were minimally affected and a subpopulation of Aδ units was blocked. After TNI, the threshold to induce AP firing by punctate mechanical stimulation (von Frey) was reduced, which was reversed to normal during GFS. These results also suggest that C-type fibers, not Aβ, mainly contribute to mechanical and thermal hypersensitivity (von Frey, brush, acetone) after injury. Ganglion field stimulation produces use-dependent blocking of afferent AP trains, consistent with enhanced filtering of APs at the sensory neuron T-junction, particularly in nociceptive units.

Conflict of interest statement

There is no conflict of interest to declare.

Figures

Figure 1.

Effects of GFS with different frequencies on nociceptive sensation on rats with TNI. Time course for effects on the testing day are shown in the left panels (A, B, C, D), and AUC analysis (E, F, G, H) for group comparisons are shown in the right panels, for sensitivity to noxious mechanical stimuli (pin, A, E), threshold mechanical stimuli (von Frey, B, F), brush (C, G), and acetone (F, H). The sham treatment (0Hz stimulation) group consisted of animals with GFS electrodes that were inserted but not activated. Red bars represent Means. Results are means ± SEM. The variance at the baseline timepoint is small such that the SEM is smaller than the radius of the data symbol. “1” P<0.05, “2” P<0.01, “3” P<0.001 compared to data immediately before GFS by the Dunnett’s test following two-way repeated-measures ANOVA. * P<0.05, *** P<0.001 by the Tukey test following one-way ANOVA. n represents animal numbers.

Figure 2.

Recordings from dorsal root teased fibers. (A) The GFS lead was implanted besides L4 DRG. Dorsal root was teased and recorded. Representative AP firing from teased fibers by stimulating DRG (B) and sciatic nerve (C). Solid black arrows represent the start point of stimulation. Neuronal subtypes were distinguished by conduction velocity. (D) Minimum intensities with GFS to activate Aβ, Aδ, C, and motor fibers. Recordings were from 9 rats. (E) Conduction velocities of those recordings with GFS. (F) Comparison between averaged intensity to activate C-type neurons and 80% intensity to activate motor fibers on the same rat. (G) Minimum intensities with sciatic nerve stimulation to activate Aβ, Aδ, C, and motor fibers. Recordings were from 5 rats. (H) Conduction velocities of those recordings with sciatic nerve stimulation. (I) Minimum intensities with saphenous nerve stimulation to activate Aβ, Aδ, and C fibers. Recordings were from 5 rats. (J) Conduction velocities of those recordings with saphenous nerve stimulation. (K) Comparison of difference of intensities to activate Aβ and C with saphenous nerve, sciatic nerve and DRG stimulation. Red bar represents Mean. * P<0.05, *** P<0.001 by the Tukey test following one-way ANOVA.

Figure 3.

Activation of dissociated neurons and in vivo intact DRG neurons by GFS with Ca2+ indicators. (A) Field stimulation (10Hz-0.5s) produces Ca2+ transients in DRG neurons loaded with the ratiometric Ca2+-sensitive fluorophore Fura-2 in a saturating pattern of Ca2+ transients that reach a maximum despite increasing voltage. The threshold (24V in this case) was defined as the minimal voltage necessary to induce full-size Ca2+ transient. Arrows indicate stimulation. (B) There is negative correlation (r2=0.20, P<0.001) between neuronal sizes and threshold intensities needed to activate the voltage-gated calcium channels. Red line is the linear regression line. n=172 neurons from 5 rats. (C) Ratios of intensities to activate small- and large-sized neurons on the same cover slip with in vitro field stimulation. Each data point represents the mean value for small neurons divided by the mean value of large neurons on a single slip. Red bar represent Mean for the group; per slip, n=8–16 small neurons and n=4–6 for large neurons. (D) GFS on in vivo intact DRG GCaMP-expressing neurons. Bright-field image with IR-LED (left), fluorescent image during stimulation (middle), and representative response trace (right) from cell #1. Numbers in the bright-field image represent cell recorded. Solid arrow represents GFS. (E) Summary of effects of GFS (20Hz-1s with intensity of 80% MT) on activation of DRG neurons. Data are shown for all fluorescent neurons in the examined fields regardless of the presence or absence of a response to stimulation. Red line is the linear regression line. n=66 total neurons from 5 rats.

Figure 4.

Effects of GFS on AP propagation from soma to central. (A-C) Representative traces showing blocked APs from C-type not Aβ units during 20Hz GFS. (D) Effects of GFS with different frequencies on APs from Aβ units. (E) Comparison of the times to failure of AP propagation (i.e. time from beginning of GFS to the last induced AP) in Aβ units during GFS. (F) Effects of GFS with different frequencies on APs from Aδ units. (G) Comparison of onset of blockade of APs in Aδ units during GFS. (H) Effects of GFS with different frequencies on APs in C-type units. (I) Comparison of onset of blockade of APs from C-type units during GFS. (J) A representative pattern of progressively failed firing following 20Hz GFS. Those recordings were from 32 rats. Red bars represent Means. * P<0.05, ** P<0.01, *** P<0.001. In D, F, and H, Chi-Squared test was used to compare the incidence of blockade effects of GFS upon different groups and the post hoc correction for the P-value was multiplied by the total number of comparisons that were made. In G and I, one-way ANOVA followed by post hoc Tukey test.

Figure 5.

Effects of 20Hz GFS on AP propagation by sciatic nerve stimulation. (A) The GFS lead was implanted besides L4 DRG. Dorsal root is teased and recorded. Representative traces show firings from Aδ and C-type units with DRG stimulation (B), sciatic nerve stimulation (C), and with both stimulations (D). (E) Sequence of events. 20Hz-4s sciatic nerve stimulations were given 30s before GFS, and 30s, 60s, 90s, 120s, 150s, and 180s after initiation of GFS. (F) Effects of GFS on recordings from sham surgery and rats with TNI. The incidence of full blockade of AP propagation were compared on different subtypes, Aβ (G), Aδ (H), and C (I). Those recordings were from 10 rats with sham injury and 9 rats with TNI. Arrows represent stimulation. 3 P<0.001 compared to data immediately before GFS by the Dunnett’s test following two-way repeated-measures ANOVA.

Figure 6.

Effects of 20Hz GFS on von Frey threshold to initiate AP firing by touching the receptive field. (A) The GFS lead was implanted besides L4 DRG. Dorsal root was teased and recorded. von Frey fibers were used to initiate AP firing. Effects of 20Hz GFS on threshold of AP firing by von Frey threshold in C-type fibers (B) and in Aδ (C). (D) Receptive fields of those recordings in the hind paw plantar glabrous skin. Those recordings were from 10 rats with sham surgery and 10 rats with TNI surgery. * P<0.05, ** P<0.01, *** P<0.001 by the Dunnett’s test following two-way repeated-measures ANOVA.

Figure 7.

Effects of 20Hz GFS on AP firing by mechanical stimulation. (A-D) Representative traces showing AP firing before and after GFS in rats with sham surgery and rats with TNI. Effects of 20Hz GFS on AP firing by blunt (E) and sharp (F) puncture in C-type fibers. Effects of 20Hz GFS on AP firing by blunt (G) and sharp (H) in Aδ fibers. Those recordings were from 10 rats with sham surgery and 10 rats with TNI surgery. Red bar represents Mean. * P<0.05, ** P<0.01 by the Dunnett’s test following two-way repeated-measures ANOVA.

Figure 8.

Effects of 20Hz GFS on spontaneous firing after nerve injury. (A) Injury increases incidence of SA. ** P<0.01 by Chi-Squared test. (B) GFS reduces increased spontaneous firing rates in C-type units after injury. Those recordings were from 6 rats with sham surgery and 5 rats with TNI surgery. Red bar represents Mean. * P<0.05, ** P<0.01 by the Dunnett’s test following two-way repeated-measures ANOVA.

Figure 9.

Proposed mechanism of GFS-induced augmented T-junction filtering. Field stimulation (1) induces depolarization of the somatic membrane that initiates APs (2), which travel down the stem axon to the T-junction (3), inducing use-dependent reduction of membrane excitability at the T-junction (4), which increases the probability of failure for both AP propagation from the stem axon to the central process fails, and for centripetal propagation of APs originating in the periphery (5).