Potassium channel modulation in macrophages sensitizes dorsal root ganglion neurons after nerve injury.

Macrophages and satellite glial cells are found between injured and uninjured neurons in the lumbar dorsal root ganglia (DRG). We explored the mechanism of neuro-immune and neuron-glia crosstalk leading to hyperexcitability of DRG neurons. After spared nerve injury (SNI), CX3CR1+ resident macrophages became activated, proliferated, and increased inward-rectifying potassium channel Kir 2.1 currents. Conditioned medium (CM) by macrophages, obtained from DRG of SNI mice, sensitized small DRG neurons from naïve mice. However, treatment with CM from GFAP+ glial cells did not affect neuronal excitability. When subjected to this macrophage-derived CM, DRG neurons had increased spontaneous activity, current-evoked responses and voltage-gated NaV 1.7 and NaV 1.8 currents. Silencing Kir 2.1 in macrophages after SNI prevented the induction of neuronal hyperexcitability from their CM. Blocking vesicular exocytosis or soluble tumor necrosis factor in CM or interfering with the downstream intracellular p38 pathway in neurons, also prevented neuronal hyperexcitability. Blocking protein trafficking in neurons reduced the effect of CM, suggesting that the hyperexcitable state resulted from changes in NaV channel trafficking. These results suggest that DRG macrophages, primed by peripheral nerve injury, contribute to neuron-glia crosstalk, NaV channel dysregulation and neuronal hyperexcitability implicated in the development of neuropathic pain.

Characterisation of GFAP-Expressing Glial Cells in the Dorsal Root Ganglion after Spared Nerve Injury.

Satellite glial cells (SGCs), enveloping primary sensory neurons' somas in the dorsal root ganglion (DRG), contribute to neuropathic pain upon nerve injury. Glial fibrillary acidic protein (GFAP) serves as an SGC activation marker, though its DRG satellite cell specificity is debated. We employed the hGFAP-CFP transgenic mouse line, designed for astrocyte studies, to explore its expression within the peripheral nervous system (PNS) after spared nerve injury (SNI). We used diverse immunostaining techniques, Western blot analysis, and electrophysiology to evaluate GFAP+ cell changes. Post-SNI, GFAP+ cell numbers increased without proliferation, and were found near injured ATF3+ neurons. GFAP+ FABP7+ SGCs increased, yet 75.5% of DRG GFAP+ cells lacked FABP7 expression. This suggests a significant subset of GFAP+ cells are non-myelinating Schwann cells (nmSC), indicated by their presence in the dorsal root but not in the ventral root which lacks unmyelinated fibres. Additionally, patch clamp recordings from GFAP+ FABP7-cells lacked SGC-specific Kir4.1 currents, instead displaying outward Kv currents expressing Kv1.1 and Kv1.6 channels specific to nmSCs. In conclusion, this study demonstrates increased GFAP expression in two DRG glial cell subpopulations post-SNI: GFAP+ FABP7+ SGCs and GFAP+ FABP7- nmSCs, shedding light on GFAP's specificity as an SGC marker after SNI.

Reversing anterior insular cortex neuronal hypoexcitability attenuates compulsive behavior in adolescent rats.

Development of self-regulatory competencies during adolescence is partially dependent on normative brain maturation. Here, we report that adolescent rats as compared to adults exhibit impulsive and compulsive-like behavioral traits, the latter being associated with lower expression of mRNA levels of the immediate early gene zif268 in the anterior insula cortex (AIC). This suggests that underdeveloped AIC function in adolescent rats could contribute to an immature pattern of interoceptive cue integration in decision making and a compulsive phenotype. In support of this, we report that layer 5 pyramidal neurons in the adolescent rat AIC are hypoexcitable and receive fewer glutamatergic synaptic inputs compared to adults. Chemogenetic activation of the AIC attenuated compulsive traits in adolescent rats supporting the idea that in early stages of AIC maturity there exists a suboptimal integration of sensory and cognitive information that contributes to inflexible behaviors in specific conditions of reward availability.

The Antidiabetic Drug Metformin Regulates Voltage-Gated Sodium Channel NaV1.7 via the Ubiquitin-Ligase NEDD4-2.

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 (NaVs). 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 NaV1.7 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 NaV1.7 current density when cotransfected with NEDD4-2. In mouse dorsal root ganglion (DRG) neurons, without changing the biophysical properties of NaV1.7, metformin significantly decreased NaV1.7 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 Nedd4Lfl/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.

Spinal Cord T-Cell Infiltration in the Rat Spared Nerve Injury Model: A Time Course Study.

The immune system is involved in the development of neuropathic pain. In particular, the infiltration of T-lymphocytes into the spinal cord following peripheral nerve injury has been described as a contributor to sensory hypersensitivity. We used the spared nerve injury (SNI) model of neuropathic pain in Sprague Dawley adult male rats to assess proliferation, and/or protein/gene expression levels for microglia (Iba1), T-lymphocytes (CD2) and cytotoxic T-lymphocytes (CD8). In the dorsal horn ipsilateral to SNI, Iba1 and BrdU stainings revealed microglial reactivity and proliferation, respectively, with different durations. Iba1 expression peaked at D4 and D7 at the mRNA and protein level, respectively, and was long-lasting. Proliferation occurred almost exclusively in Iba1 positive cells and peaked at D2. Gene expression observation by RT-qPCR array suggested that T-lymphocytes attracting chemokines were upregulated after SNI in rat spinal cord but only a few CD2/CD8 positive cells were found. A pronounced infiltration of CD2/CD8 positive T-cells was seen in the spinal cord injury (SCI) model used as a positive control for lymphocyte infiltration. Under these experimental conditions, we show early and long-lasting microglia reactivity in the spinal cord after SNI, but no lymphocyte infiltration was found.

Neuropathic Pain Phenotype Does Not Involve the NLRP3 Inflammasome and Its End Product Interleukin-1ß in the Mice Spared Nerve Injury Model.

The NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is one of the main sources of interleukin-1ß (IL-1ß) and is involved in several inflammatory-related pathologies. To date, its relationship with pain has not been studied in depth. The aim of our study was to elucidate the role of NLRP3 inflammasome and IL-1ß production on neuropathic pain. Results showed that basal pain sensitivity is unaltered in NLRP3-/- mice as well as responses to formalin test. Spared nerve injury (SNI) surgery induced the development of mechanical allodynia and thermal hyperalgesia in a similar way in both genotypes and did not modify mRNA levels of the NLRP3 inflammasome components in the spinal cord. Intrathecal lipopolysaccharide (LPS) injection increases apoptosis-associated speck like protein (ASC), caspase-1 and IL-1ß expression in both wildtype and NLRP3-/- mice. Those data suggest that NLRP3 is not involved in neuropathic pain and also that other sources of IL-1ß are implicated in neuroinflammatory responses induced by LPS.

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain.

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Navs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na(v)s remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Navs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Na(v)1.7-specific inhibitor, ProTxII, allowed the isolation of Na(v)1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Na(v)1.7 and Na(v)1.8 currents. The redistribution of Na(v)1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L(-/-)). SNS-Nedd4L(-/-) mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Na(v)1.7 and Na(v)1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Na(v)s and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

Rufinamide attenuates mechanical allodynia in a model of neuropathic pain in the mouse and stabilizes voltage-gated sodium channel inactivated state.

BACKGROUND: Voltage-gated sodium channels dysregulation is important for hyperexcitability leading to pain persistence. Sodium channel blockers currently used to treat neuropathic pain are poorly tolerated. Getting new molecules to clinical use is laborious. We here propose a drug already marketed as anticonvulsant, rufinamide. METHODS: We compared the behavioral effect of rufinamide to amitriptyline using the Spared Nerve Injury neuropathic pain model in mice. We compared the effect of rufinamide on sodium currents using in vitro patch clamp in cells expressing the voltage-gated sodium channel Nav1.7 isoform and on dissociated dorsal root ganglion neurons to amitriptyline and mexiletine. RESULTS: In naive mice, amitriptyline (20 mg/kg) increased withdrawal threshold to mechanical stimulation from 1.3 (0.6-1.9) (median [95% CI]) to 2.3 g (2.2-2.5) and latency of withdrawal to heat stimulation from 13.1 (10.4-15.5) to 30.0 s (21.8-31.9), whereas rufinamide had no effect. Rufinamide and amitriptyline alleviated injury-induced mechanical allodynia for 4 h (maximal effect: 0.10 ± 0.03 g (mean ± SD) to 1.99 ± 0.26 g for rufinamide and 0.25 ± 0.22 g to 1.92 ± 0.85 g for amitriptyline). All drugs reduced peak current and stabilized the inactivated state of voltage-gated sodium channel Nav1.7, with similar effects in dorsal root ganglion neurons. CONCLUSIONS: At doses alleviating neuropathic pain, amitriptyline showed alteration of behavioral response possibly related to either alteration of basal pain sensitivity or sedative effect or both. Side-effects and drug tolerance/compliance are major problems with drugs such as amitriptyline. Rufinamide seems to have a better tolerability profile and could be a new alternative to explore for the treatment of neuropathic pain.