Injury discharges regulate calcium channel alpha-2-delta-1 subunit upregulation in the dorsal horn that contributes to initiation of neuropathic pain

Abstract

Previous studies have shown that peripheral nerve injury in rats induces increased expression of the voltage gated calcium channel (VGCC) alpha-2-delta-1 subunit (Ca v alpha2 delta1) in spinal dorsal horn and sensory neurons in dorsal root ganglia (DRG) that correlates to established neuropathic pain states. To determine if injury discharges trigger Ca v alpha2 delta1 induction that contributes to neuropathic pain initiation, we examined allodynia onset and Ca v alpha2 delta1 levels in DRG and spinal dorsal horn of spinal nerve ligated rats after blocking injury induced neural activity with a local brief application of lidocaine on spinal nerves before the ligation. The lidocaine pretreatment blocked ligation-induced discharges in a dose-dependent manner. Similar pretreatment with the effective concentration of lidocaine diminished injury-induced increases of the Ca v alpha2 delta1 in DRG and abolished that in spinal dorsal horn specifically, and resulted in a delayed onset of tactile allodynia post-injury. Both dorsal horn Ca v alpha2 delta1 upregulation and tactile allodynia in the lidocaine pretreated rats returned to levels similar to that in saline pretreated controls 2 weeks post the ligation injury. In addition, preemptive intrathecal Ca v alpha2 delta1 antisense treatments blocked concurrently injury-induced allodynia onset and Ca v alpha2 delta1 upregulation in dorsal spinal cord. These findings indicate that injury induced discharges regulate Ca v alpha2 delta1 expression in the spinal dorsal horn that is critical for neuropathic allodynia initiation. Thus, preemptive blockade of injury-induced neural activity or Ca v alpha2 delta1 upregulation may be a beneficial option in neuropathic pain management.

Fig. 1. Blockage of injury discharges by local lidocaine application

In three different experiments (A–D, E–H, and I–L), multi-unit activity was recorded from a fascicle of the L5 dorsal root before brief application of saline or one of the two concentrations (2% or 10%) of lidocaine to the left L5 spinal nerve for 15 min. During this period, the footpad (receptive field) was tapped with a glass rod at the times indicated by arrows - before lidocaine application (top row), and at 6 (2nd row) and 10 min (3rd row) after lidocaine (or saline) application. After 15 min, the spinal nerve was washed with saline, and then tightly ligated with a 6-0 silk suture (during the periods indicated by brackets in the bottom row). Calibration bars on the right column indicate 0.2 sec for each row. Note that nerve conduction (response to tapping) or injury discharges were completely eliminated with 10%, but not 2%, lidocaine. Signals shown in L represent an artifact associated with spinal nerve movement during the ligation.

Fig. 2. Brief local-application of lidocaine delayed injury-induced neuropathic allodynia

Paw withdrawal thresholds (PWT) to von Frey filament stimulations were tested in rats before and at designated times after L5 spinal nerve ligation following pretreatment with saline or 10% lidocaine at the ligation site for 15 min. The arrow indicates the time of local lidocaine pretreatment before the ligation. Data presented are the means ± SEM from seven rats in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with saline pretreated group.

Fig. 3. Influence of brief local-application of lidocaine on nerve injury-induced Ca_vα₂δ₁ upregulation in spinal dorsal horn and DRG

Western blot analysis was performed in L4/5 DRG (A) and spinal dorsal horn (B) samples taken at indicated time points post injury from injury (I) and contralateral (C) sides of spinal nerve ligated rats, pretreated with either saline or 10% lidocaine at the ligation site for 15 min. Representative Western blot images from each group were presented at the top of each panel of summarized Western blot data. Band density ratios of the Ca_vα₂δ₁ band over the GAPDH band within each sample were calculated for loading normalization before data comparison between injury and non-injury sides. Data presented are the means ± SEM from at least six independent determinations. DSC – dorsal spinal cord. Sal. – Saline pretreated. Lid. – 10% lidocaine pretreated. * p < 0.05, *** p < 0.001 compared with saline pretreatment within each group. # p < 0.05, ### p < 0.001 compared with the group of post-injury day five received the same pretreatment at the ligation site. + p < 0.05 compared with values from the injury (ipsilateral) side of saline pretreated rats within the same time point.

Fig. 4. Influence of brief local-application of lidocaine on nerve injury-induced GFAP upregulation in dorsal spinal cord

Western blot analysis was performed in L4/5 spinal dorsal horn samples taken at indicated time points post injury from injury, or ipsilateral, (I) side and contralateral (C) side of SNL rats, pretreated with either saline or 10% lidocaine at the ligation site for 15 min before the nerve ligation. Representative Western blot images from each group were presented at the top of each panel of summarized Western blot data. Band density ratios of the GFAP band over the GAPDH band within each sample were calculated for loading normalization before data comparison between injury and non-injury sides. Data presented are the means ± SEM from at least six independent determinations. DSC – dorsal spinal cord. Sal. – Saline pretreated. Lid. – 10% lidocaine pretreated. * p < 0.05, ** p < 0.01 compared with saline pretreatment within each group.

Fig. 5. Effects of preemptive intrathecal Ca_vα₂δ₁ antisense treatment on spinal nerve ligation induced tactile allodynia

Ca_vα₂δ₁ antisense or mismatch oligodeoxynucleotides (50 µg/rat, once daily) were injected directly into the L5/6 lumbar spinal regions for five consecutive days, starting one-day before SNL. Paw withdrawal thresholds (PWT) to von Frey filament stimulations were tested blindly in the rats before the daily intrathecal injection, then daily after the last intrathecal injection for a total of 14 days after the ligation injury. Data presented are the means ± SEM from 14 rats in the antisense group (not including one rat in the antisense-insensitive group shown in dashed lines) and 15 rats in the mismatch group up to post SNL day 4 (one-day after the last intrathecal injection), and from four (mismatch) to five (antisense) rats from post SNL day 5 to 14. C – Contralateral (non-injury) side. I – Ipsilateral (injury) side. *** p < 0.001 between data from the antisense (filled square) and mismatch (open circle) oligodeoxynucleotide treatments at the injury side.

Fig. 6. Influence of intrathecal preemptive Ca_vα₂δ₁ antisense treatment on nerve injury-induced Ca_vα₂δ₁ upregulation in spinal dorsal horn

Western blot analysis was performed in L5/6 spinal dorsal horn samples taken from injury, or ipsilateral, (I) side and contralateral (C) side of four-day or 14-day SNL rats that were pretreated with either Ca_vα₂δ₁ antisense or mismatch oligodeoxynucleotides for five days, starting one-day before the SNL as described for Fig. 5. Representative Western blot images from each group were presented at the top of each panel of summarized Western blot data. Band density ratios of the Ca_vα₂δ₁ band over the GAPDH band within each sample were calculated for loading normalization before data comparison between injury and non-injury sides. Data presented are the means ± SEM from five (antisense) to six (mismatch) rats, and the mean of two independent Western blots from one antisense-insensitive rat, in the four-day post SNL groups; and the means ± SEM from four (mismatch) to five (antisense) rats in the 14-day post SNL groups. DSC – dorsal spinal cord. Anti. – antisense oligodeoxynucleotide pretreated. Mis. – mismatch oligodeoxynucleotide pretreated. * p < 0.05, ** p < 0.01 compared with contralateral side within each group. ### p < 0.001 compared with the ligation site of the four-day post-SNL group that was sensitive to the antisense pretreatment. + p < 0.05 compared with values from the injury (ipsilateral) side of mismatch oligodeoxynucleotide pretreated rats within the same time point.