Differential effects of peripheral versus central coadministration of QX-314 and capsaicin on neuropathic pain in rats

Abstract

Background: Neuropathic pain is common and difficult to treat. Recently a technique was developed to selectively inhibit nociceptive inputs by simultaneously applying two drugs: capsaicin, a transient receptor potential vanilloid receptor-1 channel activator, and QX-314, a lidocaine derivative that intracellularly blocks sodium channels. We used this technique to investigate whether transient receptor potential vanilloid receptor 1-expressing nociceptors contribute to neuropathic pain.

Methods: The rat chronic constriction injury model was used to induce neuropathic pain in order to test the analgesic effects of both peripheral (perisciatic) and central (intrathecal) administration of the QX-314/capsaicin combination. The Hargreaves and von Frey tests were used to monitor evoked pain-like behaviors and visual observations were used to rank spontaneous pain-like behaviors.

Results: Perisciatic injections of the QX-314/capsaicin combination transiently increased the withdrawal thresholds by approximately 3-fold, for mechanical and thermal stimuli in rats (n = 6/group) with nerve injuries suggesting that peripheral transient receptor potential vanilloid receptor 1-expressing nociceptors contribute to neuropathic pain. In contrast, intrathecal administration of the QX-314/capsaicin combination did not alleviate pain-like behaviors (n = 5/group). Surprisingly, intrathecal QX-314 alone (n = 9) or in combination with capsaicin (n = 8) evoked spontaneous pain-like behaviors.

Conclusions: Data from the perisciatic injections suggested that a component of neuropathic pain was mediated by peripheral nociceptive inputs. The role of central nociceptive terminals could not be determined because of the severe side effects of the intrathecal drug combination. We concluded that only peripheral blockade of transient receptor potential vanilloid receptor 1-expressing nociceptive afferents by the QX-314/capsaicin combination was effective at reducing neuropathic allodynia and hyperalgesia.

Figure 1

The QX-314/capsaicin combination reduced the sensitivity of normal rats to mechanical stimuli. The normalized peak threshold (A) and the time course of the threshold to mechanical stimuli (B) show that perisciatic injection of QX-314 (0.2 %) with capsaicin (0.5 mg/ml) increased the withdrawal threshold for ~2 h. The mechanical threshold was also increased by lidocaine (2 %), however lidocaine also caused motor deficits. QX-314 alone or capsaicin alone did not change the normalized peak mechanical threshold. The normalized peak latency (C) and the time course of the latency to thermal stimuli (D) show that perisciatic injection of the QX-314/capsaicin did not change the withdrawal threshold to thermal stimulation. The thermal threshold was increased by lidocaine, whereas it was decreased by QX-314 and capsaicin alone. The peak response to each drug during the time course was normalized to facilitate comparisons between the different groups and graphed as the normalized threshold or latency (A and C). Data graphed in this figure and all subsequent figures are averages ± SEM. For the normalized threshold and latency (A and C), single asterisks indicate that p < 0.05 and double asterisks indicate that p < 0.01 compared to the baseline before the injection. For the time courses (B and D), an asterisk indicates that p < 0.05 for the QX-314/capsaicin combination, q indicates that p < 0.05 for the QX-314 alone, and c indicates that p < 0.05 for the capsaicin alone. For each group, n = 6. Cap = capsaicin; Lido = lidocaine; QX = QX-314; Veh = vehicle.

Figure 2

The QX-314/capsaicin combination reduced the sensitivity of rats with injured sciatic nerves to mechanical and thermal stimuli. The normalized peak threshold (A) and the time course of the threshold to mechanical stimuli (B) show that perisciatic injection of QX-314 (0.2 %) with capsaicin (0.5 mg/ml) increased the withdrawal threshold to mechanical stimulation for ~1 h. The mechanical threshold was also increased by lidocaine (2 %), however lidocaine also caused motor deficits (data not shown). QX-314 alone or capsaicin alone dramatically lowered the mechanical threshold. The normalized peak latency (C) and the time course of the latency to thermal stimuli (D) show that perisciatic injection of the QX-314/capsaicin increased the withdrawal threshold to thermal stimulation. The thermal threshold was decreased by QX-314 and capsaicin alone. The peak response to each drug during the time course was normalized to facilitate comparisons between the different groups and graphed as the normalized threshold or latency (A and C). For the normalized threshold and latency (A and C), single asterisks indicate that p < 0.05 and double asterisks indicate that p < 0.01 compared to the baseline before the injection. For the time courses (B and D), an asterisk indicates that p < 0.05 for the QX-314/capsaicin combination. For each group, n = 6. Cap = capsaicin; Lido = lidocaine; QX = QX-314;Veh = vehicle.

Figure 3

Ethanol/Tween containing vehicle enhances the antinociceptive effects of the QX-314/capsaicin combination in rats with a nerve ligation. Time course shows that withdrawal threshold to mechanical stimuli (A) was increased more by the QX-314/capsaicin combination dissolved in ethanol/Tween containing vehicle than dimethyl sulfoxide (DMSO) containing vehicle. The composition of the vehicle had little effect on the withdrawal threshold to thermal stimuli (B). Asterisks indicate that p < 0.05 for ethanol/Tween containing vehicle compared to the DMSO containing vehicle at the same time point. For each group, n = 6.

Figure 4

Neuropathic pain is not altered by the intrathecal application of the QX-314/capsaicin combination. After ligating the right sciatic nerve, the withdrawal thresholds to mechanical (A, C, and E) and thermal (B, D, and F) stimuli decreased for the side ipsilateral to the nerve ligation (Ligated). The thresholds were not reduced for the paw contralateral to the ligation (Control, n = 5). Typically, this hyperalgesia developed within 4 to 10 days after the surgery. For the paw ipsilateral to the nerve ligation, the QX-314/capsaicin combination (0.2 % QX-314 + 0.5 mg/ml capsaicin, n = 5) did not change the withdrawal threshold to mechanical or thermal stimuli. Surprisingly, the QX-314/capsaicin combination decreased the mechanical threshold of the control (nonligated) leg. QX-314 (0.2 %, n = 6) alone also reduced the mechanical threshold of the control leg without relieving the mechanical allodynia. However, QX-314 did partially reverse the thermal hyperalgesia for the ipsilateral foot. The vehicle had no effect on the mechanical or thermal thresholds for the ligated or control paws. Insertion of osmotic pumps containing drugs is indicated by the dashed line. Asterisks indicate that p < 0.05 compared to the control (unligated) paw. Cap = capsaicin; QX = QX-314.

Figure 5

Differential changes in the withdrawal thresholds of normal rats were produced by intrathecal QX-314, capsaicin, and the QX-314/capsaicin combination. A. Capsaicin alone and the QX-314/capsaicin combination produced statistically significant elevation of the withdrawal threshold for mechanical stimuli compared to the vehicle controls. QX-314 did not produce statistically significant changes in the mechanical withdrawal threshold. B. QX-314, capsaicin alone, and the QX-314/capsaicin combination had little effect on the withdrawal latency to thermal stimuli. Single asterisks indicate that p < 0.05 compared to the vehicle at the same time point. For mechanical stimulation: n = 7 for vehicle and the QX-314/capsaicin combination, n = 8 for QX-314 alone and capsaicin alone. For thermal stimulation: n = 7 for the QX-314/capsaicin combination, n = 8 for vehicle, QX-314 alone, and capsaicin alone. The break in the line connecting the QX-314 data points indicated that data could not be collected for some of the animals at later test sessions.

Figure 6

Intrathecal QX-314 induced abnormal motor behaviors. A. Frequency of abnormal agitated behaviors after the intrathecal administration of QX-314, capsaicin, and the QX-314/capsaicin combination. Rats administered QX-314 alone or the QX-314/capsaicin combination were more likely to display agitated behaviors like vocalization, running, circling, and jumping. None of the rats treated with vehicle or capsaicin alone displayed abnormal behaviors. Single asterisks indicate that p < 0.05 (Kruskal-Wallis one-way ANOVA on ranks followed by post hoc comparisons using Dunn’s Method). B. Time course of the agitated behaviors after intrathecal administration of the drugs. The rats were observed and the behavioral responses were ranked for a 10-min period. The scores were recorded for every minute of observation and summed at the end of the experiment. Notice that the cumulative behavioral scores were higher for QX-314 (n = 9) and the QX-314/capsaicin combination (n = 7) groups, whereas they were unchanged for the capsaicin (n = 8) and vehicle (n = 8) groups. Single asterisks indicate that p < 0.05 compared to the baseline before the injection and compared to the vehicle control. The break in the line connecting the QX-314 data points indicated that data could not be collected for some of the animals at later test sessions. Cap = capsaicin; Lido = lidocaine; QX = QX-314; Veh = vehicle.

Figure 7

Intrathecal QX-314 did not produce overt lesions of the spinal cord. A small segment of the spinal cord near the opening of the catheter was removed, fixed and processed for histology. No obvious lesions were detected in the transverse sections of the spinal cord from animals treated with vehicle, QX-314, capsaicin, or the QX-314/capsaicin combination. Scale bar = 50 µm.

Figure 8

Intrathecal QX-314 did not produce increased activation of microglia and astrocytes. Antibodies directed against ionized calcium-binding adapter-1 (Iba1) and glial fibrillary acidic protein (GFAP) were used to label microglia and astrocytes in samples from the same segments of the spinal cord used for histology staining. No noticeable differences were detected in ionized calcium-binding adapter-1 or glial fibrillary acidic protein expression patterns in the spinal cord dorsal horn between vehicle, QX-314, capsaicin, or the QX-314/capsaicin combination treated animals. No evidence of increased activation of microglia or astrocytes was detected based on the morphology of the cell bodies and processes (A) and the numbers of microglia (B) and astrocytes (C) counted per microscopic field (at 40X) within the dorsal horn of the spinal cords (Kruskal-Wallis one-way ANOVA. p = 0.117 for microglia, n = 4; p = 0.913 for astrocytes, n = 4). Cap = capsaicin; QX = QX-314; Veh = vehicle.