Ongoing/spontaneous pain behavior is associated with ongoing/spontaneous firing (SF) in adult DRG C-fiber nociceptors (Djouhri et al., 2006). Causes of this SF are not understood. We show here that conducting (sometimes called uninjured) C-nociceptors in neuropathic pain models with more hyperpolarized resting membrane potentials (Ems) have lower SF rates. Understanding the control of their Ems may therefore be important for limiting pathological pain. We report that TREK2, a leak K ϩ channel, is selectively expressed in IB4 binding rat C-nociceptors. These IB4 ϩ C-neurons are ϳ10 mV more hyperpolarized than IB4 Ϫ C-neurons in vivo (Fang et al., 2006). TREK2 knockdown by siRNA in these neurons in culture depolarized them by ϳ10 mV, suggesting that TREK2 is responsible for this ϳ10 mV difference. In vivo, more hyperpolarized C-nociceptor Ems were associated with higher cytoplasmic edge-TREK2 expression (edge-TREK2). Edge-TREK2 decreased in C-neurons 7 d after axotomy, and their Ems depolarized by ϳ10 mV. This again supports a contribution of TREK2 to their Ems. These relationships between (1) Em and TREK2, (2) SF rate and Em, and (3) spontaneous pain behavior and C-nociceptor SF rate suggested that TREK2 knockdown might increase spontaneous pain. After CFA-induced inflammation, spontaneous foot lifting (a measure of spontaneous pain) was (1) greater in rats with naturally lower TREK2 in ipsilateral small DRG neurons and (2) increased by siRNA-induced TREK2 knockdown in vivo. We conclude that TREK2 hyperpolarizes IB4 binding C-nociceptors and limits pathological spontaneous pain. Similar TREK2 distributions in small DRG neurons of several species suggest that these role(s) of TREK2 may be widespread.

Two pore domain potassium (K2P) channels (KCNKx.x) cause K + leak currents and are major contributors to resting membrane potential. Their roles in dorsal root ganglion (DRG) neurons normally, and in pathological pain models, are poorly understood. Therefore, we examined mRNA levels for 10 K2P channels in L4 and L5 rat DRGs normally, and 1 day and 4 days after unilateral cutaneous inflammation, induced by intradermal complete Freund's adjuvant (CFA) injections. Spontaneous foot lifting (SFL) duration (spontaneous pain behaviour) was measured in 1 day and 4 day rats < 1 h before DRG harvest. mRNA levels for KCNK channels and Kv1.4 relative to GAPDH (n = 4–6 rats/group) were determined with real-time RT-PCR. This study is the first to demonstrate expression of THIK1, THIK2 and TWIK2 mRNA in DRGs. Abundance in normal DRGs was, in descending order: Kv1.4 > TRESK(KCNK18) > TRAAK(KCNK4) > TREK2(KCNK10) = TWIK2(KCNK6) > TREK1 (KCNK2) = THIK2(KCNK12) > TASK1(KCNK3) > TASK2(KCNK5) > THIK1(KCNK13) = TASK3(KCNK9). During inflammation, the main differences from normal in DRG mRNA levels were bilateral, suggesting systemic regulation, although some channels showed evidence of ipsilateral modulation. By 1 day, bilateral K2P mRNA levels had decreased (THIK1) or increased (TASK1, THIK2) but by 4 days they were consistently decreased (TASK2, TASK3) or tended to decrease (excluding TRAAK). The decreased TASK2 mRNA was mirrored by decreased protein (TASK2-immunoreactivity) at 4 days. Ipsilateral mRNA levels at 4 days compared with 1 day were lower (TRESK, TASK1, TASK3, TASK2 and THIK2) or higher (THIK1). Ipsilateral SFL duration during inflammation was positively correlated with ipsilateral TASK1 and TASK3 mRNAs, and contralateral TASK1, TRESK and TASK2 mRNAs. Thus changes in K2P mRNA levels occurred during inflammation and for 4 K2P channels were associated with spontaneous pain behaviour (SFL). K2P channels and their altered expression are therefore associated with inflammation-induced pain.

Diabetic neuropathy (DN) is one of the most frequent and troublesome complications of diabetes mellitus. Evidence from diabetic animal models and diabetic patients suggests that reduced availability of neuroprotective and pro-angiogenic factors in the nerves in combination with a chronic pro-inflammatory microenvironment and high level of oxidative stress, contribute to the pathogenesis of DN. Mesenchymal stem cells (MSCs) are of great interest as therapeutic agents for regenerative purposes, since they can secrete a broad range of cytoprotective and anti-inflammatory factors. Therefore, the use of the MSC secretome may represent a promising approach for DN treatment. Recent data indicate that the paracrine potential of MSCs could be boosted by preconditioning these cells with an environmental or pharmacological stimulus, enhancing their therapeutic efficacy. In the present study, we observed that the preconditioning of human adipose tissue-derived MSCs (AD-MSCs) with 150μM or 400μM of the iron chelator deferoxamine (DFX) for 48 hours, increased the abundance of the hypoxia inducible factor 1 alpha (HIF-1α) in a concentration dependent manner, without affecting MSC morphology and survival. Activation of HIF-1α led to the up-regulation of the mRNA levels of pro-angiogenic factors like vascular endothelial growth factor alpha and angiopoietin 1. Furthermore this preconditioning increased the expression of potent neuroprotective factors, including nerve growth factor, glial cell-derived neurotrophic factor and neurotrophin-3, and cytokines with anti-inflammatory activity like IL4 and IL5. Additionally, we observed that these molecules, which could also be used as therapeutics, were also increased in the secretome of MSCs preconditioned with DFX compared to the secretome obtained from non-preconditioned cells. Moreover, DFX preconditioning significantly increased the total antioxidant capacity of the MSC secretome and they showed neuroprotective effects when evaluated in an in vitro model of DN. Altogether, our findings suggest that DFX preconditioning of AD-MSCs improves their therapeutic potential and should be considered as a potential strategy for the generation of new alternatives for DN treatment.

Ih, which influences neuronal excitability, has recently been measured in vivo in sensory neuron subtypes in dorsal root ganglia (DRGs). However, expression levels of HCN (hyperpolarization-activated cyclic nucleotide-gated) channel proteins that underlie Ih were unknown. We therefore examined immunostaining of the most abundant isoforms in DRGs, HCN1 and HCN2 in these neuron subtypes. This immunostaining was cytoplasmic and membrane-associated (ring). Ring-staining for both isoforms was in neurofilament-rich A-fiber neurons, but not in small neurofilament-poor C-fiber neurons, although some C-neurons showed cytoplasmic HCN2 staining. We recorded intracellularly from DRG neurons in vivo, determined their sensory properties (nociceptive or low-threshold-mechanoreceptive, LTM) and conduction velocities (CVs). We then injected fluorescent dye enabling subsequent immunostaining. For each dye-injected neuron, ring- and cytoplasmic-immunointensities were determined relative to maximum ring-immunointensity. Both HCN1- and HCN2-ring-immunointensities were positively correlated with CV in both nociceptors and LTMs; they were high in Aβ-nociceptors and Aα/β-LTMs. High HCN1 and HCN2 levels in Aα/β-neurons may, via Ih, influence normal non-painful (e.g. touch and proprioceptive) sensations as well as nociception and pain. HCN2-, not HCN1-, ring-intensities were higher in muscle spindle afferents (MSAs) than in all other neurons. The previously reported very high Ih in MSAs may relate to their very high HCN2. In normal C-nociceptors, low HCN1 and HCN2 were consistent with their low/undetectable Ih. In some C-LTMs HCN2-intensities were higher than in C-nociceptors. Together, HCN1 and HCN2 expressions reflect previously reported Ih magnitudes and properties in neuronal subgroups, suggesting these isoforms underlie Ih in DRG neurons. Expression of both isoforms was NT3-dependent in cultured DRG neurons. HCN2-immunostaining in small neurons increased 1 day after cutaneous inflammation (CFA-induced) and recovered by 4 days. This could contribute to acute inflammatory pain. HCN2-immunostaining in large neurons decreased 4 days after CFA, when NT3 was decreased in the DRG. Thus HCN2-expression control differs between large and small neurons.

The hyperpolarization-activated current (Ih) has been implicated in nociception/pain, but its expression levels in nociceptors remained unknown. We recorded Ih magnitude and properties by voltage clamp from dorsal root ganglion (DRG) neurons in vivo, after classifying them as nociceptive or low-threshold-mechanoreceptors (LTMs) and as having C-, Aδ- or Aα/β-conduction velocities (CVs). For both nociceptors and LTMs, Ih amplitude and Ih density (at −100 mV) were significantly positively correlated with CV. Median Ih magnitudes and Ih density in neuronal subgroups were respectively: muscle spindle afferents (MSAs): −4.6 nA, −33 pA pF−1; cutaneous Aα/β LTMs: −2.2 nA, −20 pA pF−1; Aβ-nociceptors: −2.6 nA, −21 pA pF−1; both Aδ-LTMs and nociceptors: −1.3 nA, ~−14 pA pF−1; C-LTMs: −0.4 nA, −7.6 pA pF−1; and C-nociceptors: −0.26 nA, −5 pA pF−1. Ih activation slow time constants (slow τ values) were strongly correlated with fast τ values; both were shortest in MSAs. Most neurons had τ values consistent with HCN1-related Ih; others had τ values closer to HCN1+HCN2 channels, or HCN2 in the presence of cAMP. In contrast, median half-activation voltages (V0.5) of −80 to −86 mV for neuronal subgroups suggest contributions of HCN2 to Ih. τ values were unrelated to CV but were inversely correlated with Ih and Ih density for all non-MSA LTMs, and for Aδ-nociceptors. From activation curves ~2–7% of Ih would be activated at normal membrane potentials. The high Ih may be important for excitability of A-nociceptors (responsible for sharp/pricking-type pain) and Aα/β-LTMs (tactile sensations and proprioception). Underlying HCN expression in these subgroups therefore needs to be determined. Altered Ih expression and/or properties (e.g. in chronic/pathological pain states) may influence both nociceptor and LTM excitability.

Nociceptin/orphanin FQ (N/OFQ) is an opioid-related peptide that is markedly up-regulated in sensory neurons in vivo following peripheral inflammation and plays a key role in pain physiology. To identify substances that up-regulate N/OFQ expression in sensory neurons, we carried out an in vitro screen using purified adult mouse dorsal root ganglion (DRG) neurons and identified the potent proinflammatory agent bacterial lipopolysaccharide (LPS) as a very effective inducer of N/OFQ. The robust response of these neurons to LPS enabled us to identify the components of a putative neuronal LPS receptor complex. In contrast to the immune system, where the functional LPS receptor complex is composed of CD-14 together with either MD-2 and TLR4 on myeloid cells or the homologous receptors MD-1 and RP105 on mature B cells, DRG neurons express the unusual combination of CD-14, TLR4, and MD-1. Blocking antibodies against TLR4 and MD-1 prevented induction of N/OFQ by LPS, and, in immunoprecipitation experiments, MD-1 coprecipitated with TLR4. Our findings suggest that LPS regulates N/OFN expression in sensory neurons via a novel combination of LPS receptor components and demonstrate for the first time a direct action of a key initiator of innate immune responses on neurons.

Endogenous enkephalins and ␦ opiates affect sensory function and pain sensation by inhibiting synaptic transmission in sensory circuits via delta opioid receptors (DORs). DORs have long been suspected of mediating these effects by modulating voltage-dependent Ca 2ϩ entry in primary sensory neurons. However, not only has this hypothesis never been validated in these cells, but in fact several previous studies have only turned up negative results. By using whole-cell current recordings, we show that the ␦ enkephalin analog [D-Ala 2 , D-Leu 5 ]-enkephalin (DADLE) inhibits, via DORs, L-, N-, P-, and Q-high voltageactivated Ca 2ϩ channel currents in cultured rat dorsal root ganglion (DRG) neurons. The percentage of responding cells was remarkably high (75%) within a novel subpopulation of substance P-containing neurons compared with the other cells (18-35%). DADLE (1 M) inhibited 32% of the total barium current through calcium channels (I Ba ). A ␦ (naltrindole, 1 M), but not a (␤-funaltrexamine, 5 M), antagonist prevented the DADLE response, whereas a DOR-2 subtype (deltorphin-II, 100 nM), but not a DOR-1 (DPDPE, 1 M), agonist mimicked the response. L-, N-, P-, and Q-type currents contributed, on average, 18, 48, 14, and 16% to the total I Ba and 19, 50, 26, and 20% to the DADLE-sensitive current, respectively. The druginsensitive R-type current component was not affected by the agonist. This work represents the first demonstration that DORs modulate Ca 2ϩ entry in sensory neurons and suggests that ␦ opioids could affect diverse Ca 2ϩ -dependent processes linked to Ca 2ϩ influx through different high-voltage-activated channel types.

Background Diabetic polyneuropathy (DPN) is the most common and early developing complication of diabetes mellitus, and the key contributor for foot ulcers development, with no specific therapies available. Different studies have shown that mesenchymal stem cell (MSC) administration is able to ameliorate DPN; however, limited cell survival and safety reasons hinder its transfer from bench to bedside. MSCs secrete a broad range of antioxidant, neuroprotective, angiogenic, and immunomodulatory factors (known as conditioned medium), which are all decreased in the peripheral nerves of diabetic patients. Furthermore, the abundance of these factors can be boosted in vitro by incubating MSCs with a preconditioning stimulus, enhancing their therapeutic efficacy. We hypothesize that systemic administration of conditioned medium derived from preconditioned MSCs could reverse DPN and prevent foot ulcer formation in a mouse model of type II diabetes mellitus. Methods Diabetic BKS db/db mice were treated with systemic administration of conditioned medium derived from preconditioned human MSCs; conditioned medium derived from non-preconditioned MSCs or vehicle after behavioral signs of DPN was already present. Conditioned medium or vehicle administration was repeated every 2 weeks for a total of four administrations, and several functional and structural parameters characteristic of DPN were evaluated. Finally, a wound was made in the dorsal surface of both feet, and the kinetics of wound closure, re-epithelialization, angiogenesis, and cell proliferation were evaluated. Results Our molecular, electrophysiological, and histological analysis demonstrated that the administration of conditioned medium derived from non-preconditioned MSCs or from preconditioned MSCs to diabetic BKS db/db mice strongly reverts the established DPN, improving thermal and mechanical sensitivity, restoring intraepidermal nerve fiber density, reducing neuron and Schwann cell apoptosis, improving angiogenesis, and reducing chronic inflammation of peripheral nerves. Furthermore, DPN reversion induced by conditioned medium administration enhances the wound healing process by accelerating wound closure, improving the re-epithelialization of the injured skin and increasing blood vessels in the wound bed in a skin injury model that mimics a foot ulcer. Conclusions Studies conducted indicate that MSC-conditioned medium administration could be a novel cell-free therapeutic approach to reverse the initial stages of DPN, avoiding the risk of lower limb amputation triggered by foot ulcer formation and accelerating the wound healing process in case it occurs.