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, 21 (6), 1868-75

Upregulation of Dorsal Root Ganglion (alpha)2(delta) Calcium Channel Subunit and Its Correlation With Allodynia in Spinal Nerve-Injured Rats

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Upregulation of Dorsal Root Ganglion (alpha)2(delta) Calcium Channel Subunit and Its Correlation With Allodynia in Spinal Nerve-Injured Rats

Z D Luo et al. J Neurosci.

Abstract

Peripheral nerve injury can lead to a persistent neuropathic pain state in which innocuous tactile stimulation elicits pain behavior (tactile allodynia). Spinal administration of the anticonvulsant gabapentin suppresses allodynia by an unknown mechanism. In vitro studies indicate that gabapentin binds to the alpha(2)delta-1 (hereafter referred to as alpha(2)delta) subunit of voltage-gated calcium channels. We hypothesized that nerve injury may result in altered alpha(2)delta subunit expression in spinal cord and dorsal root ganglia (DRGs) and that this change may play a role in neuropathic pain processing. Using a rat neuropathic pain model in which gabapentin-sensitive tactile allodynia develops after tight ligation of the left fifth and sixth lumbar spinal nerves, we found a >17-fold, time-dependent increase in alpha(2)delta subunit expression in DRGs ipsilateral to the nerve injury. Marked alpha(2)delta subunit upregulation was also evident in rats with unilateral sciatic nerve crush, but not dorsal rhizotomy, indicating a peripheral origin of the expression regulation. The increased alpha(2)delta subunit expression preceded the allodynia onset and diminished in rats recovering from tactile allodynia. RNase protection experiments indicated that the DRG alpha(2)delta regulation was at the mRNA level. In contrast, calcium channel alpha(1B) and beta(3) subunit expression was not co-upregulated with the alpha(2)delta subunit after nerve injury. These data suggest that DRG alpha(2)delta regulation may play an unique role in neuroplasticity after peripheral nerve injury that may contribute to allodynia development.

Figures

Fig. 1.
Fig. 1.
Development of tactile allodynia after L5/L6 spinal nerve ligation and the effects of intrathecal gabapentin infusion on established allodynia. Left L5/L6 spinal nerve tight ligation was performed in Harlan Sprague Dawley rats, and the PWT to mechanical stimulation was tested daily as indicated. Allodynic rats (1 week after nerve ligation) were treated with saline or gabapentin for 1 week through an intrathecal (i.t.) pump. Data are presented as the means ± SEM from at least 5 treated to 20 nontreated rats in each group.
Fig. 2.
Fig. 2.
Calcium channel protein levels in L5/L6 DRG and spinal cord of nerve-ligated rats. Total proteins were extracted from ipsilateral or contralateral DRGs (two each) or lumbar dorsal spinal cord quadrants of control or allodynic rats 1 week after the spinal nerve ligation. The protein levels were identified using indicated monoclonal antibodies (for α2 and eNOS) or polyclonal antibodies (for β3 and α1B).A, Representative Western blots each from at least four independent experiments showing protein levels in DRG and lumbar dorsal spinal cord of control and allodynic rats. Membrane extracts of stable HEK293 cells expressing transfected human recombinant α2bδ, α1B, and β3cDNAs were used as respective positive (+) controls. Extracts of endothelial cells were used as positive control for eNOS.Lanes are labeled C, for the contralateral side, and Ip, for the ipsilateral (nerve-ligated) side. B, Nerve ligation-induced increase of the α2 subunit in ipsilateral DRG and spinal cord compared with respective contralateral (contral.) samples. Data are presented as the means ± SEM from at least 11 (sham) to 16 (ligated) independent experiments (*p< 0.05 by Student's t test and Mann–Whitney test; #p < 0.05 by Student's t test but not by Mann–Whitney test). S.C., Spinal cord.
Fig. 3.
Fig. 3.
DRG α2 expression levels in rats with 1 week dorsal rhizotomy or sciatic nerve crush. Pooled DRGs were collected from both sides of rats with unilateral rhizotomy (Rhizot.; L5/L6) or sciatic nerve crush (L4–L6). Total proteins were extracted and subjected to Western blot analyses as described. The insetsabove thebar graph are representative Western blots.Lanes are labeled C, for the contralateral side, and Ip, for the ipsilateral (nerve-injured) side. The bar graph shows the percentage changes of the α2 subunit levels in ipsilateral DRGs (Ipsilat.) compared with those in contralateral DRGs (Contral.) that were assigned the value of 100%. Data presented are the means ± SEM averaged from four (rhizotomy) to six (crush) independent determinations (*p < 0.05 by Student's t test and Mann–Whitney test).
Fig. 4.
Fig. 4.
Time-dependent increases in the α2subunit from L5/L6 DRGs ipsilateral to spinal nerve ligation. Ipsilateral or contralateral pooled L5/L6 DRGs were collected from nerve-ligated rats at the designated times, and total proteins were extracted and subjected to Western blot analysis as described.A, A representative Western blot. Thelower nonspecific bands indicate an equivalent protein loading within each pair of samples. Lanes are labeledC, for the contralateral side, and Ip, for the ipsilateral (nerve-ligated) side. B, Percentage increase of the α2 subunit in ipsilateral DRGs compared with that in contralateral (contral.) DRGs that was assigned the value of 100%. Data presented are the means ± SEM averaged from 4 to 16 independent determinations.
Fig. 5.
Fig. 5.
Time-dependent increases of DRG α2δ mRNA in nerve-ligated rats. Total RNA was extracted from two pooled L5/L6 DRGs or one L4 DRG at the designated time after nerve ligation, and α2δ mRNA levels were examined by RNase protection assays. A, Representative autoradiography showing the α2δ and cyclophilin (Cyclo.) probes protected by their corresponding mRNAs. α2δ bands had longer exposure times than did cyclophilin bands because of the low abundance of the α2δ mRNA. Each pair of samples was taken from the same rat on the contralateral side (lane labeledC) or surgery side (lane labeledIp). B, Summarized time-dependent increase of α2δ mRNA after nerve ligation. The percentage increase of α2δ mRNA was defined by comparing α2δ band densities in the injury side with those in the contralateral (contral.) side after using the ratio of the α2δ band to the cyclophilin band to correct for differences in RNA loading. Data presented are the means ± SEM averaged from four independent experiments.wk, Week.
Fig. 6.
Fig. 6.
Upregulated DRG α2 subunit diminishes in nerve-ligated rats recovering from tactile allodynia. Nerve ligation injury was induced as described in Harlan Sprague Dawley rats, and the PWT to mechanical stimulation was tested up to 6 months after the surgery. Total protein was extracted from two pooled L5/L6 DRGs from each side of the rat at the designated time points, and DRG α2 subunit levels were examined by Western blot analyses.A, Recovery of nerve-ligated rats from tactile allodynia. Data presented are the means ± SEM from 4 to 16 rats in each group. B, Representative Western blots showing expression levels of α2 and nNOS in L5/L6 DRG of nerve-ligated rats at time points after the nerve injury. In some experiments, these blots were stripped and then reblotted with eNOS antibodies as shown in Figure 2, and eNOS expression was not increased in DRGs ipsilateral to the nerve ligation (data not shown).Lanes are labeled C, for the contralateral side, and Ip, for the ligation side.C, Summarized Western blot data shown inB. Data are presented as the percentage of maximal increase of α2 and nNOS expression in DRGs ipsilateral to the nerve ligation in each experiment and are expressed as the means ± SEM of six independent determinations. wk, Week.

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