Upregulation of the voltage-gated sodium channel beta2 subunit in neuropathic pain models: characterization of expression in injured and non-injured primary sensory neurons

Abstract

The development of abnormal primary sensory neuron excitability and neuropathic pain symptoms after peripheral nerve injury is associated with altered expression of voltage-gated sodium channels (VGSCs) and a modification of sodium currents. To investigate whether the beta2 subunit of VGSCs participates in the generation of neuropathic pain, we used the spared nerve injury (SNI) model in rats to examine beta2 subunit expression in selectively injured (tibial and common peroneal nerves) and uninjured (sural nerve) afferents. Three days after SNI, immunohistochemistry and Western blot analysis reveal an increase in the beta2 subunit in both the cell body and peripheral axons of injured neurons. The increase persists for >4 weeks, although beta2 subunit mRNA measured by real-time reverse transcription-PCR and in situ hybridization remains unchanged. Although injured neurons show the most marked upregulation,beta2 subunit expression is also increased in neighboring non-injured neurons and a similar pattern of changes appears in the spinal nerve ligation model of neuropathic pain. That increased beta2 subunit expression in sensory neurons after nerve injury is functionally significant, as demonstrated by our finding that the development of mechanical allodynia-like behavior in the SNI model is attenuated in beta2 subunit null mutant mice. Through its role in regulating the density of mature VGSC complexes in the plasma membrane and modulating channel gating, the beta2 subunit may play a key role in the development of ectopic activity in injured and non-injured sensory afferents and, thereby, neuropathic pain.

Figure 1.

Spared nerve injury increases expression of the β2 subunit of VGSCs in the DRGs. A, Deglycosylated lysates of HEK293 cells transfected with β2, β1, or β3 subunit expression constructs or control expression vector only (ctrl). A single band is detected in β2-expressing cells only (GW93 antibody). B, Immunoblot with GW93 anti-β2 subunit antibody on deglycosylated brain tissue from β2 subunit null mutant (-/-) or wild-type (+/+) mice. The antibody was preincubated with a specific β2 peptide at increasing concentrations (0-100 nm) before Western blotting. C-E, Three antibodies specific for the β2 subunit all recognize a single band at 25 kDa in Western blot analyses of L4/L5 DRG tissue from SNI-operated and control rats. Enhanced β2 subunit immunoreactivity 1 week after SNI is observed using β2 subunit β2-ec (C), GW2690 (D), and GW93 (E) antibodies. To control for loading, the same blots were reprobed with an antibody recognizing both ERK1 (44 kDa) and ERK2 (42 kDa), which are constitutively expressed (bottom or middle panels in B-E) and confirmed with an GAPDH antibody (37 kDa) (E, bottom panel).

Figure 2.

Peripheral nerve injury induces a rapid and persistent increase in β2 subunit immunoreactivity in the DRG. A, Western blot analyses of the L4/L5 DRGs 1 week after an SNI, total axotomy of the sciatic nerve (Axo), or 48 h and 1 week (1W) after intraplantar CFA induced inflammation (longer exposure). B, D, Representative Western blots from at least four independent experiments at 1, 3, 5, and 7 d after SNI and 1, 2, 3, and 4 weeks after SNI. C, Quantitation of β2 subunit protein levels in the L4/L5 DRGs at the indicated days after SNI. Fold represents comparative increase over control level after normalizing to total ERK2 (n = 4 for each time point; ^*p < 0.01). ctrl, Control.

Figure 3.

Redistribution of the β2 subunit to the plasma membrane. Western blot analyses of cytosolic (left) and membrane (right) fractions show that SNI causes an increase in the amount of β2 subunit at the plasma membrane, whereas signal in this fraction is almost absent in controls (ctrl) (L4 and L5 DRGs; n = 4 in each lane conditions).

Figure 4.

β2 subunit augmentation and distribution in primary sensory neuron populations after SNI. A, Representative photomicrograph of fluorescent immunohistochemistry for β2 subunit in L4 DRGs 1 week after SNI compared with control rats. B, Percentage of total neuronal profile that express β2 subunit in control DRGs and DRGs 1 week after SNI. ^*p < 0.001; n = 4 in each group. C, Double immunohistochemistry for β2 subunit (red) and NF-200 (green) shows a distribution of β2 immunoreactivity in both large cells (colocalization with NF-200 appears yellow on the merged image) and small cells (absence of colocalization, red). D, Profile area frequency histogram constructed from 575 cell profiles shows an increase of β2 subunit-IR neurons 1 week after SNI in small-sized (<600 μm²), medium-sized (600-1200 μm²), and large-sized (> 1200 μm²) neurons. Scale bars, 100 μm.

Figure 5.

Regulation of VGSC subunit mRNA expression after nerve injury. A, Relative quantification by real-time reverse transcription-PCR amplification of mRNA for β2 subunit and for α subunits Na_v1.3 and Na_v1.8 at indicated times. The SNI/control ratio refers to the ratio of mRNA levels in L4/L5 DRG tissue of SNI-injured and sham-operated animals (triplicate analysis of 4-6 rats for each time point; ^*p < 0.05). B, C, Representative dark-field photomicrographs of isotopic in situ hybridization in L4 DRGs from control animals or 1 week after SNI, as well as L4 (intact) DRGs and L5 (injured) DRGs 2 weeks after SNL. B, In situ hybridization for β2 subunit mRNA. C, In situ hybridization for Na_v1.3 α subunit mRNA. Scale bar, 350 μm.

Figure 6.

Characterization of β2 immunoreactivity in injured and non-injured DRG neurons in the SNI model of neuropathic pain. Colocalization of immunoreactivity for β2 with immunoreactivity for ATF3 and retrograde labeling with FG. The majority of β2 immunoreactivity (red) is seen in ATF3-IR neurons (green), corresponding to injured cell population (green). To a limited extent, β2 immunoreactivity is also seen in non-injured neurons of the sural nerve (white arrow) retrogradely traced with FG (bright gray). Scale bars, 100 μm.

Figure 7.

Characterization of β2 immunoreactivity in injured and non-injured neurons in the L5 SNL model of neuropathic pain. A, β2 immunoreactivity in L5 and L4 DRGs 1 week after SNL and in control. B, Immunohistochemistry for β2 subunit in sections of L4 and L5 DRGs 1 week after SNL and in a section of a control DRG. C, Percentage of neuronal profiles in injured (L5) and intact (L4) DRGs that express β2 subunit compared with control contralateral DRG (^*p < 0.01; ^**p < 0.001; n = 3). Fold represents comparative increase over control level after normalizing to ERK2 loading control.

Figure 8.

β2 subunit is increased in peripheral nerves after SNI and is transported along the injured nerve to the neuroma. Western blot (A) and immunohistochemistry (B, C) analyses for β2 subunit in injured and non-injured nerves 1 week (W) after SNI. A, Representative blots, each from at least three independent experiments from control and SNI groups (n = 4-6 in each group). The signal is augmented in the injured tibial and common peroneal nerves after SNI at all time points compared with nerves from control rats. B, C, Nerves were probed with anti-β2 subunit antibody, and confocal images of representative sections show β2 staining in injured common peroneal and tibial nerves 1 week after SNI (B) and in the spared sural nerve after SNI (C). High-magnification photomicrographs are of portions of nerves at a distance ∼2.5 mm away from the distal extremity of the neuroma. Fold represents comparative increase over control level after normalizing to ERK2 loading control.

Figure 9.

Attenuation of mechanical allodynia-like behavior after SNI in β2 subunit -/- mice. A, B, Paw withdrawal frequencies to mechanical stimuli using a series of calibrated monofilaments with logarithmic increment. Frequency of response (paw withdrawal) to each monofilament in wild-type (+/+) and β2 subunit knock-out mice (-/-) are shown before (A; two baselines, BL1 and BL2) and after SNI (B; 7, 14, 21, and 28 d). Baseline curves did not differ significantly between genotypes, whereas the response curves for the +/+ and -/- mice at day 7 and after are significantly different, with the -/- mice showing an attenuated response compared with the +/+ mice (p < 0.001; n = 12 and 10, respectively). C, The 50% withdrawal threshold 4 weeks after SNI. The mechanical threshold is higher in β2 subunit -/- mice than wild-type mice ipsilateral but not contralateral to the nerve injury (^*p = 0.02; n = 12 and 10, respectively).