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Virus-mediated shRNA knockdown of Na(v)1.3 in rat dorsal root ganglion attenuates nerve injury-induced neuropathic pain.

ABSTRACT: Neuropathic pain is a chronic condition that is often refractory to treatment with available therapies and thus an unmet medical need. We have previously shown that the voltage-gated sodium channel Na(v)1.3 is upregulated in peripheral and central nervous system (CNS) of rats following nerve injury, and that it contributes to nociceptive neuron hyperexcitability in neuropathic conditions. To evaluate the therapeutic potential of peripheral Na(v)1.3 knockdown at a specific segmental level, we constructed adeno-associated viral (AAV) vector expressing small hairpin RNA against rat Na(v)1.3 and injected it into lumbar dorsal root ganglion (DRG) of rats with spared nerve injury (SNI). Our data show that direct DRG injection provides a model that can be used for proof-of-principle studies in chronic pain with respect to peripheral delivery route of gene transfer constructs, high transduction efficiency, flexibility in terms of segmental localization, and limited behavioral effects of the surgical procedure. We show that knockdown of Na(v)1.3 in lumbar 4 (L4) DRG results in an attenuation of nerve injury-induced mechanical allodynia in the SNI model. Taken together, our studies support the contribution of peripheral Na(v)1.3 to pain in adult rats with neuropathic pain, validate Na(v)1.3 as a target, and provide validation for this approach of AAV-mediated peripheral gene therapy.

SUBMITTER: Samad OA

PROVIDER: S-EPMC3538302 | biostudies-other | 2013 Jan

REPOSITORIES: biostudies-other

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Project description:17β-estradiol plays a role in pain sensitivity, analgesic drug efficacy, and neuropathic pain prevalence, but the underlying mechanisms remain unclear. Here, we investigated whether voltage-gated chloride channel-3 (ClC-3) impacts the effects of 17β-estradiol (E2) on spared nerve injury (SNI)-induced neuropathic pain in ovariectomized (OVX) female Sprague Dawley rats that were divided into OVX, OVX + SNI, OVX + SNI + E2, OVX + SNI + E2 + DMSO (vehicle, dimethyl sulfoxide), or OVX + SNI + E2+Cltx (ClC-3-blocker chlorotoxin) groups. Changes in ClC-3 protein expression were monitored by western blot analysis. Behavioral testing used the paw withdrawal threshold to acetone irritation and paw withdrawal thermal latency (PWTL) to thermal stimulation. Immunofluorescence indicated the localization and protein expression levels of ClC-3. OVX + SNI + E2 rats were subcutaneously injected with 17β-estradiol once daily for 7 days; a sheathed tube was implanted, and chlorotoxin was injected for 4 days. Intrathecal Cltx to OVX and OVX + SNI rats was administered for 4 consecutive days (days 7-10 after SNI) to further determine the contribution of ClC-3 to neuropathic pain. Patch clamp technology in current clamp mode was used to measure the current threshold (rheobase) dorsal root ganglion (DRG) neurons and the minimal current that evoked action potentials (APs) as excitability parameters. The mean number of APs at double-strength rheobase verified neuronal excitability. There was no difference in behaviors and ClC-3 expression after OVX. Compared with OVX + SNI rats, OVX + SNI + E2 rats showed a lower paw withdrawal threshold to the acetone stimulus, but the PWTL was not significantly different, indicating increased sensitivity to cold but not to thermal pain. Co-immunofluorescent data revealed that ClC-3 was mainly distributed in A- and C-type nociceptive neurons, especially in medium/small-sized neurons. 17β-estradiol administration was associated with increased expression of ClC-3. 17β-estradiol-induced increase in ClC-3 expression was blocked by co-administration of Cltx. Cltx causes hyperalgesia and decreased expression of ClC-3 in OVX rats. Patch clamp results suggested that 17β-estradiol attenuated the excitability of neurons induced by SNI by up-regulating the expression of ClC-3 in the DRG of OVX rats. 17β-estradiol administration significantly improved cold allodynia thresholds in OVX rats with SNI. The mechanism for this decreased sensitivity may be related to the upregulation of ClC-3 expression in the DRG.

Project description:BackgroundDorsal root ganglia (DRG) somata from rodents have provided an excellent model system to study ion channel properties and modulation using electrophysiological investigation. As in other vertebrates, zebrafish (Danio rerio) DRG are organized segmentally and possess peripheral axons that bifurcate into each body segment. However, the electrical properties of zebrafish DRG sensory neurons, as compared with their mammalian counterparts, are relatively unexplored because a preparation suitable for electrophysiological studies has not been available.Methodology/principal findingsWe show enzymatically dissociated DRG neurons from juvenile zebrafish expressing Isl2b-promoter driven EGFP were easily identified with fluorescence microscopy and amenable to conventional whole-cell patch-clamp studies. Two kinetically distinct TTX-sensitive Na(+) currents (rapidly- and slowly-inactivating) were discovered. Rapidly-inactivating I(Na) were preferentially expressed in relatively large neurons, while slowly-inactivating I(Na) was more prevalent in smaller DRG neurons. RT-PCR analysis suggests zscn1aa/ab, zscn8aa/ab, zscn4ab and zscn5Laa are possible candidates for these I(Na) components. Voltage-gated Ca(2+) currents (I(Ca)) were primarily (87%) comprised of a high-voltage activated component arising from ω-conotoxin GVIA-sensitive Ca(V)2.2 (N-type) Ca(2+) channels. A few DRG neurons (8%) displayed a miniscule low-voltage-activated component. I(Ca) in zebrafish DRG neurons were modulated by neurotransmitters via either voltage-dependent or -independent G-protein signaling pathway with large cell-to-cell response variability.Conclusions/significanceOur present results indicate that, as in higher vertebrates, zebrafish DRG neurons are heterogeneous being composed of functionally distinct subpopulations that may correlate with different sensory modalities. These findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and thus provide a basis for future studies.

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Virus-mediated shRNA knockdown of Na(v)1.3 in rat dorsal root ganglion attenuates nerve injury-induced neuropathic pain.

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