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Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels ([Na.sub.v]s) expressed in dorsal root ganglion (DRG) sensory neurons. [...]

Abstract Background Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. Results Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependant manner. More than 90% of transduced cells were small and medium sized neurons (< 700 μm2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (≈ 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types. Conclusion We have found that rAAV2/6 is an efficient vector to deliver transgenes to nociceptive neurons in mice. Furthermore, the characterization of the transduction profile may facilitate gene transfer studies to dissect mechanisms behind neuropathic pain.

Abstract Background Clinical and experimental studies of neuropathic pain support the hypothesis that a functional coupling between postganglionic sympathetic efferent and sensory afferent fibers contributes to the pain. We investigated whether neuropathic pain-related behavior in the spared nerve injury (SNI) rat model is dependent on the sympathetic nervous system. Results Permanent chemical sympathectomy was achieved by daily injection of guanethidine (50 mg/kg s.c.) from age P8 to P21. SNI was performed at adulthood followed by 11 weeks of mechanical and thermal hypersensitivity testing. A significant but limited effect of the sympathectomy on SNI-induced pain sensitivity was observed. The effect was delayed and restricted to cold allodynia-like behavior: SNI-related cold scores were lower in the sympathectomized group compared to the control group at 8 and 11 weeks after the nerve injury but not before. Mechanical hypersensitivity tests (pinprick and von Frey hair threshold tests) showed no difference between groups during the study period. Concomitantly, pericellular tyrosine-hydroxylase immunoreactive basket structures were observed around dorsal root ganglia (DRG) neurons 8 weeks after SNI, but were absent at earlier time points after SNI and in sham operated controls. Conclusion These results suggest that the early establishment of neuropathic pain-related behavior after distal nerve injury such as in the SNI model is mechanistically independent of the sympathetic system, whereas the system contributes to the maintenance, albeit after a delay of many weeks, of response to cold-related stimuli.

Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav α-subunit also directly affects Navs gating. β-subunits and glycosylation thus comodulate Nav α-subunit gating. We hypothesized that β-subunits could directly influence α-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both β1- a n d β3-subunits coexpression shifted V½ of steady-state activation and inactivation and increased Nav1.7-mediated INa density. Biotinylation of cell surface proteins, combined with the use of deglycosydases, confirmed that Nav1.7 α-subunits exist in multiple glycosylated states. The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at ∼250 kDa. At the plasma membrane, in addition to the core-glycosylated form, a fully glycosylated form of Nav1.7 (∼280 kDa) was observed. This higher band shifted to an intermediate band (∼260 kDa) when β1-subunits were coexpressed, suggesting that the β1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the β1-subunit increased the expression of this alternative glycosylated form and the β3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channel (Nav1) β2 subunits modulate channel gating, assembly, and cell-surface expression in CNS neurons in vitro and in vivo. β2 expression increases in sensory neurons after nerve injury, and development of mechanical allodynia in the spared nerve injury model is attenuated in β2-null mice. Thus, we hypothesized that β2 modulates electrical excitability in dorsal root ganglion (DRG) neurons in vivo. We compared sodium currents (INa ) in small DR Gneurons from β2+/+ and β2+/+ mice to determine the effects of β2 on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Nav1 in vivo. Small-fast DRG neurons acutely isolated from β2+/+ mice showed significant decreases in TTX-S INa compared with β2+/+ neurons. This decrease included a 51% reduction in maximal sodium conductance with no detectable changes in the voltage dependence of activation or inactivation. TTX-S, but not TTX-R, INa activation and inactivation kinetics in these cells were slower in β2-/- mice compared with controls. The selective regulation of TTX-S INa was supported by reductions in transcript and protein levels of TTX-S Nav1s, particularly Nav1.7. Low-threshold mechanical sensitivity was preserved in β2-/- mice, but they were more sensitive to noxious thermal stimuli than wild type whereas their response during the late phase of the formalin test was attenuated. Our results suggest that β2 modulates TTX-S Nav1 mRNA and protein expression resulting in increased TTX-S INa and increases the rates of TTX-S Nav1 activation and inactivation in small-fast DRG neurons in vivo. TTX-R INa were not significantly modulated by β2. [ABSTRACT FROM AUTHOR]

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 ß2 subunit of VGSCs participates in the generation of neuropathic pain, we used the spared nerve injury (SNI) model in rats to examine ß2 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 ß2 subunit in both the cell body and peripheral axons of injured neurons. The increase persists for >4 weeks, although ß2 subunit mRNA measured by real-time reverse transcription- PCR and in situ hybridization remains unchanged. Although injured neurons show the most marked upregulation, ß2 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 ß2 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 ß2 subunit null mutant mice. Through its role in regulating the density of mature VGSC complexes in the plasma membrane and modulating channel gating, the ß2 subunit may play a key role in the development of ectopic activity in injured and non-injured sensory afferents and, thereby, neuropathic pain. [ABSTRACT FROM AUTHOR]

The antidiabetic drug metformin has been shown to reduce pain hypersensitivity in preclinical models of chronic pain and in neuropathic pain in humans. Multiple intracellular pathways have been described as metformin targets. Among them, metformin is an activator of the adenosine 5'-monophosphate protein kinase that can in turn modulate the activity of the E3 ubiquitin ligase NEDD4-2 and thus post-translational expression of voltage-gated sodium channels (Na V s). In this study, we found that the bulk of the effect of metformin on Na1.7 is dependent on NEDD4-2. In HEK cells, the expression of Na V 1.7 at the membrane fraction, obtained by a biotinylation approach, is only reduced by metformin when cotransfected with NEDD4-2. Similarly, in voltage-clamp recordings, metformin significantly reduced Na V 1.7 current density when cotransfected with NEDD4-2. In mouse dorsal root ganglion (DRG) neurons, without changing the biophysical properties of Na V 1.7, metformin significantly decreased Na V 1.7 current densities, but not in Nedd4L knock-out mice ( SNS-Nedd4L -/- ). In addition, metformin induced a significant reduction in NEDD4-2 phosphorylation at the serine-328 residue in DRG neurons, an inhibitory phosphorylation site of NEDD4-2. In current-clamp recordings, metformin reduced the number of action potentials elicited by DRG neurons from Nedd4L fl/fl , with a partial decrease also present in SNS-Nedd4L -/- mice, suggesting that metformin can also change neuronal excitability in an NEDD4-2-independent manner. We suggest that NEDD4-2 is a critical player for the effect of metformin on the excitability of nociceptive neurons; this action may contribute to the relief of neuropathic pain., (Copyright © 2022 Deftu et al.)