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.

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 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 4days 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.

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⁺channel, is selectively expressed in IB4 binding rat C-nociceptors. These IB4⁺C-neurons are ∼10 mV more hyperpolarized than IB4⁻C-neuronsin vivo(Fang et al., 2006). TREK2 knockdown by siRNA in these neurons in culture depolarized them by ∼10 mV, suggesting that TREK2 is responsible for this ∼10 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 ∼10 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 knockdownin 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.

I_h, 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 I_h 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 I_h, 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 I_h in MSAs may relate to their very high HCN2. In normal C-nociceptors, low HCN1 and HCN2 were consistent with their low/undetectable I_h. In some C-LTMs HCN2-intensities were higher than in C-nociceptors. Together, HCN1 and HCN2 expressions reflect previously reported I_h magnitudes and properties in neuronal subgroups, suggesting these isoforms underlie I_h 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.

Key point: I _h is a hyperpolarisation‐activated current that influences neuronal excitability and is present in some sensory neurons. The magnitude and properties of I_h in different groups of sensory neurons that respond to painful stimuli (nociceptors) or to non‐painful stimuli, such as low threshold mechanoreceptors (LTMs), were unknown. We found that neurons with the greatest I_h were the nociceptors and LTMs with the fastest conducting fibres. The highest I_h of all was present in LTM neurons that sense muscle stretch and length (muscle spindle afferents). The high levels of I_h could fundamentally influence excitability of fast conducting sensory neurons which detect muscle stretch/length, touch and pressure, and painful stimuli. I_h could thus influence sensations associated with all these. The properties of I_h are similar to those of HCN1‐ and HCN2‐related I_h, suggesting that these channels underlie the current. Abstract  The hyperpolarization‐activated current (I_h) has been implicated in nociception/pain, but its expression levels in nociceptors remained unknown. We recorded I_h 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, I_h amplitude and I_h density (at −100 mV) were significantly positively correlated with CV. Median I_h magnitudes and I_h 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. I_h 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 I_h; others had τ values closer to HCN1+HCN2 channels, or HCN2 in the presence of cAMP. In contrast, median half‐activation voltages (V_0.5) of −80 to −86 mV for neuronal subgroups suggest contributions of HCN2 to I_h. τ values were unrelated to CV but were inversely correlated with I_h and I_h density for all non‐MSA LTMs, and for Aδ‐nociceptors. From activation curves ∼2–7% of I_h would be activated at normal membrane potentials. The high I_h 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 I_h expression and/or properties (e.g. in chronic/pathological pain states) may influence both nociceptor and LTM excitability.