Alteration of primary afferent activity following inferior alveolar nerve transection in rats

Abstract

Background: In order to evaluate the neural mechanisms underlying the abnormal facial pain that may develop following regeneration of the injured inferior alveolar nerve (IAN), the properties of the IAN innervated in the mental region were analyzed.

Results: Fluorogold (FG) injection into the mental region 14 days after IAN transection showed massive labeling of trigeminal ganglion (TG). The escape threshold to mechanical stimulation of the mental skin was significantly lower (i.e. mechanical allodynia) at 11-14 days after IAN transection than before surgery. The background activity, mechanically evoked responses and afterdischarges of IAN Adelta-fibers were significantly higher in IAN-transected rats than naive. The small/medium diameter TG neurons showed an increase in both tetrodotoxin (TTX)-resistant (TTX-R) and -sensitive (TTX-S) sodium currents (INa) and decrease in total potassium current, transient current (IA) and sustained current (IK) in IAN-transected rats. The amplitude, overshoot amplitude and number of action potentials evoked by the depolarizing pulses after 1 muM TTX administration in TG neurons were significantly higher, whereas the threshold current to elicit spikes was smaller in IAN-transected rats than naive. Resting membrane potential was significantly smaller in IAN-transected rats than that of naive.

Conclusions: These data suggest that the increase in both TTX-S INa and TTX-R INa and the decrease in IA and Ik in small/medium TG neurons in IAN-transected rats are involved in the activation of spike generation, resulting in hyperexcitability of Adelta-IAN fibers innervating the mental region after IAN transection.

Figure 1

Dark filed photomicrographs of FG-labeled TG neurons (A, B and C), the number of FG-labeled TG neurons in naive and IAN-transected rats (D) and change in the threshold intensity for eliciting escape behavior following mechanical stimulation to the mental skin reinnervated by the transected IAN (n = 5, E, F). The threshold intensity was plotted related to the stimulus intensity applied to rats face. A: naive rats, B: 7 days after IAN transection, C: 14 days after IAN transection. D: Total number of FG-labeled TG neurons in naive, 7 days and 14 days IAN-transected rats. * p < 0.05 (7 days vs. 14 days), ** p < 0.01 (vs. naive), E: ipsilateral side to IAN transection, F: contralateral side to IAN transection, before: before IAN transection. The escape threshold is shown as the medial value of the stimulus intensity. Burs in A, B and C indicate 100 μm. * p < 0.05, ** p < 0.01 (vs. before)

Figure 2

Conduction velocity (CV) of A- and C-fiber units recorded from the IAN. A: The antidromic spikes following 330 Hz electrical stimulation of the trigeminal spinal nucleus. Arrows indicate stimulus onset and closed circles represent antidromic spikes. B: The collision test for antidromic spikes. The open circle indicates the expected time point where antidromic spike should have appeared. C and D: Frequency histogram of CV in naive rats and IAN-transected rats, respectively. Inset figures in C and D indicate the receptive field in each unit.

Figure 3

Background activities (A) and afterdischarges (B) and mechanical responses (C and D) of Aδ-units to pressure, brushing or pinching of the receptive fields. Aa and Ba: IAN fibers in naive rats, Ab and Bb: background activity (Ab) and afterdischarge (Bb) of IAN fibers with receptive fields at 14 days after IAN transection, Ac: background activity of the IAN fiber without receptive field at 14 days after IAN transection, Ad and Bc: mean background activities and afterdischarges in naive, IAN-transected rats without behavioral changes and IAN-transected rats, respectively. Note that background activities and afterdischarges of A-units in IAN-transected rats showed significantly higher firing frequency than those of naive rats. C: typical mechanical responses in naive and IAN-transected rats. D: mean mechanical responses of A-units in naive and IAN-transected rats. Note that A-units showed significantly higher responses to non-noxious and noxious mechanical stimulation and also IAN-transected rats without behavioral changes showed any changes in spike frequency. -behav.change: IAN-transected rats without behavioral changes after IAN transection, +behav.change: IAN-transected rats which showed mechano-allodynia like behavior after IAN transection. RF+: area of receptive field can be defined by mechanical simulation to face. RF-: area of receptive field cannot be defined by mechanical simulation to face. * p < 0.05 (vs. naive)

Figure 4

Classification of TTX-S I_Na and TTX-R I_Na in naive and IAN-transected rats. A: TTX-S I_Na was isolated by digitally subtracting TTX-R I_Na (in 1 μM TTX) from the total I_Na (without TTX). B: The current-voltage (I-V) relationship of total I_Na (without TTX) and TTX-R I_Na (in 1 μM TTX) in both naive and IAN-transected rats. Mean values (mean ± SEM) of total and TTX-R I_Na in TG neurons were illustrated in B. C: Peak current densities of total I_Na, TTX-R I_Na and TTX-S I_Na in naive and IAN-transected rats. Open column: naive rats. Solid column: rats with IAN transection. * p < 0.05 (vs. naive)

Figure 5

Changes in spike form and the action potential firing in the TG neurons during application of depolarizing pulses in naive and IAN-transected rats under current clamp conditions. The stimulus currents were applied at 50 pA steps under current clamp conditions. A and B: Sample recordings of action potentials of TG neurons from naive and IAN-transected rats. Spike amplitudes of action potentials were calculated from the distance between two dotted lines in B. The action potential was induced at the threshold (1T), two-times (2T) and three-times (3T) the threshold level. C: Mean spike amplitude of TG neurons from naive rats and IAN-transected rats. D: Mean overshoot of TG neurons from naive rats and IAN-transected rats. E: Mean threshold current in naive and IAN-transected rats. F: Mean number of spikes evoked in naive and IAN-transected rats during depolarization step pulses at 1T, 2T and 3T. * p < 0.05 (vs. naive)

Figure 6

The changes in voltage-gated K⁺ currents of TG neurons with IAN-transected and naive rats. Separation of total outward currents (a) into I_A(a-b) and I_K (b). (a) Initiated via a prepulse of -120 mV. (b) Initiated via a prepulse of -40 mV to +60 mV. Subtract a-b to reveal I_A. Subtraction of Ab from Aa reveals a transient K⁺ current (I_A). B: Current-voltage relationships on I_K and I_A in TG neurons from naive (open circles) and IAN-transected rats (solid circles). Each value represents the mean ± SEM. C: Peak current densities for total K⁺, I_K and I_A in TG neurons from naive (open column) and the rats with IAN transection (solid column). Each value represents the mean ± SEM.* p < 0.05.

Figure 7

The experimental diagram and time-course of the present study. A: The schematic illustration of the FG injection site, electrode placement, B: Top view of the experimental set-up, C: The time-course of the present experiment. Ca: FG tracing experiment, Cb: single fiber recording experiment, Cc: patch-clamp recording experiment.