Papers: 27 Apr 2019 - 3 May 2019

Hyperexcitability of the anterior cingulate cortex (ACC) is thought to drive aversion associated with chronic neuropathic pain. Here, we studied the contribution of input from the mediodorsal thalamus (MD) to ACC, using sciatic nerve injury and chemotherapy-induced mouse models of neuropathic pain. Activating MD inputs elicited pain-related aversion in both models. Unexpectedly, excitatory responses of layer V ACC neurons to MD inputs were significantly weaker in pain models compared to controls. This caused the ratio between excitation and feedforward inhibition elicited by MD input to shift toward inhibition, specifically for subcortically projecting (SC) layer V neurons. Furthermore, direct inhibition of SC neurons reproduced the pain-related aversion elicited by activating MD inputs. Finally, both the ability to elicit pain-related aversion and the decrease in excitation were specific to MD inputs; activating basolateral amygdala inputs produced opposite effects. Thus, chronic pain-related aversion may reflect activity changes in specific pathways, rather than generalized ACC hyperactivity.

AbstarctPreviously, distinct sex differences were observed in the pronociceptive role of spinal immune cells in neuropathic and inflammatory mouse pain models. Both peripheral and central innate and adaptive immune changes contribute to sensitization in the tibia fracture rodent model of complex regional pain syndrome (CRPS), and the current study evaluated sex differences in the development of pronociceptive immune responses after fracture. At 4 and 7 weeks post fracture the analgesic effects of a microglia inhibitor were tested in male and female mice and PCR was used to measure inflammatory mediator expression in skin and spinal cord. The temporal progression of CRPS-like changes in male and female wild-type and muMT fracture mice lacking B cells and antibodies were evaluated and IgM antibody deposition measured. Pronociceptive effects of injecting wild-type fracture mouse serum into muMT fracture mice were also tested in both sexes and the role of sex hormones was evaluated in the post fracture development of pronociceptive immune responses. Long lasting immune changes developed in the fracture limb and corresponding spinal cord of both male and female mice, including upregulated neuropeptide and cytokine signaling, microglial activation, and pronociceptive autoimmunity. These complex post fracture immune responses were sexually dichotomous and interacted in temporally evolving patterns that generated post traumatic nociceptive sensitization in both sexes lasting for up to 5 months. Unfortunately, the redundancy and plasticity of these chronic post traumatic immune responses suggest that clinical interventions focusing on any single specific pronociceptive immune change are likely to be ineffectual.

Amongst the regulators of voltage-gated ion channels is the collapsin response mediator protein 2 (CRMP2). CRMP2 regulation of the activity and trafficking of NaV1.7 voltage-gated sodium channels as well as the N-type (CaV2.2) voltage-gated calcium channel (VGCC) has been reported. On the other hand, CRMP2 does not appear to regulate L- (CaV1.x), P/Q- (CaV2.1), and R- (CaV2.3) type high VGCCs. Whether CRMP2 regulates low VGCCs remains an open question. Here, we asked if CRMP2 could regulate the low voltage-gated (T-type/CaV3.x) channels in sensory neurons. Reducing CRMP2 protein levels with short interfering RNAs yielded no change in macroscopic currents carried by T-type channels. No change in biophysical properties of the T-type currents was noted. Future studies pursuing CRMP2 druggability in neuropathic pain will benefit from the findings that CRMP2 regulates only the N-type (CaV2.2) calcium channels.

P2X receptors constitute a gene family of cation channels gated by extracellular ATP. They mediate fast ionotropic purinergic signaling in neurons and non-excitable cell types in vertebrates. The highly calcium-permeable P2X4 subtype has been shown to play a significant role in cardiovascular physiology, inflammatory responses and neuro-immune communication. We previously reported the discovery of a P2X4-selective antagonist, the small organic compound BX430, with submicromolar potency for human P2X4 receptors and marked species-dependence (Ase et al., 2015). The present study investigates the molecular basis of P2X4 inhibition by the non-competitive blocker BX430 using a structural and functional approach relying on mutagenesis and electrophysiology. We provide evidence for the critical contribution of a single hydrophobic residue located in the ectodomain of P2X4 channel subunits, Ile312 in human P2X4, which determines blockade by BX430. We also show that the nature of this extracellular residue in various vertebrate P2X4 orthologs underlies their specific sensitivity or resistance to the inhibitory effects of BX430. Taking advantage of high-resolution crystallographic data available on zebrafish P2X4, we used molecular dynamics simulation to model the docking of BX430 on an allosteric binding site around Ile315 (zebrafish numbering) in the ectodomain of P2X4. We also observed that the only substitution I312D (human numbering) that renders P2X4 silent by itself has also a profound silencing effect on all other P2X subtypes tested when introduced at homologous positions. The generic impact of this aspartate mutation on P2X function indicates that the pre-TM2 subregion involved is conserved functionally and defines a novel allosteric inhibitory site present in all P2X receptor channels. This conserved structure-channel activity relationship might be exploited for the rational design of potent P2X subtype-selective antagonists of therapeutic value.

Neuropathic pain is a worldwide health concern with poor treatment outcomes. Accumulating evidence suggests that histone hypoacetylation is involved in development and maintenance of neuropathic pain. Thus, many natural and synthetic histone deacetylase (HDACs) inhibitors were tested and exhibited a remarkable analgesic effect against neuropathic pain in animals. However, studies evaluating specific subtypes of HDACs contributing to neuropathic pain are limited. In this study, using the chronic constriction injury (CCI) rat model, we found that mRNA and protein levels of HDAC2 were increased in the lumbar spinal cord of rats after sciatic nerve injury. Intrathecal injection of TSA, a pan-HDAC inhibitor, suppressed the increase in HDAC2 protein but not mRNA, and showed a dose-dependent pain-relieving effect. By introducing HDAC2-specific shRNA into the spinal cord via a lentivirus vector, we confirmed that HDAC2 mediates mechanical and thermal hyperalgesia after nerve injury. Further examination found two essential participants in neuropathic pain in the inhibitory circuit of the central nervous system: GAD65 and KCC2 were increased in the spinal cord of CCI rats after HDAC2 knockdown. Thus, our research confirmed that HDAC2 was involved in mechanical and thermal hyperalgesia induced by peripheral nerve injury. Furthermore, GAD65 and KCC2 were the possible downstream targets of HDAC2 in pain modulation pathways.

The neurosteroid dehydroepiandrosterone sulphate (DHEAS) activates the sigma-1 receptor, inhibits gamma-aminobutyric acid A (GABA) and glycine receptors, and induces hyperalgesic effects. Although its effects have been studied in various tissues of the nervous system, its synaptic mechanisms in nociceptive pathways remain to be elucidated.

Pain is a common nonmotor symptom of Parkinson's disease (PD) that remains neglected and misunderstood. Elucidating the nondopaminergic circuitry may be key to better understanding PD and improving current treatments. We investigated the role of monoamines in nociceptive behavior and descending analgesic circuitry in a rat 6-hydroxydopamine (6-OHDA)-induced PD model and explored the resulting motor dysfunctions and inflammatory responses. Rats pretreated with noradrenaline and serotonin reuptake inhibitors were given unilateral striatal 6-OHDA injections and evaluated for mechanical hyperalgesia and motor impairments. Through immunohistochemistry, the number and activation of neurons, and the staining for astrocytes, microglia and enkephalin were evaluated in specific brain structures and the dorsal horn of the spinal cord. The PD model induced bilateral mechanical hyperalgesia that was prevented by reuptake inhibitors in the paw contralateral to the lesion. Reuptake inhibitors also prevented postural immobility and asymmetric rotational behavior in PD rats without interfering with dopaminergic neuron loss or glial activation in the substantia nigra. However, the inhibitors changed the periaqueductal gray circuitry, protected against neuronal impairment in the locus coeruleus and nucleus raphe magnus, and normalized spinal enkephalin and glial staining in lesioned rats. These data indicate that the preservation of noradrenergic and serotonergic systems regulates motor responses and nociceptive circuitry during PD not by interfering directly with nigral lesions but by modulating the opioid system and glial response in the spinal cord. Taken together, these results suggest that nondopaminergic circuitry is essential to the motor and nonmotor symptoms of PD and must be further investigated.

The primary somatosensory cortex (S1) comprises a number of functionally distinct regions, reflecting the diversity of somatosensory receptor submodalities innervating the body. In particular, two spatially and functionally distinct nociceptive regions have been described in primate S1 (Vierck et al., 2013; Whitsel et al., 2018). One region is located mostly in Brodmann cytoarchitectonic area 1, where a subset of neurons exhibit functional characteristics associated with myelinated Aδ nociceptors and perception of 1/sharp, discriminative pain. The second region is located at the transition between S1 and primary motor cortex (M1) in area 3a, where neurons exhibit functional characteristics associated with unmyelinated C nociceptors and perception of 2/slow, burning pain. To test the hypothesis that in rats the transitional zone (TZ) – which is a dysgranular region at the transition between M1 and S1 – is the functional equivalent of the nociresponsive region of area 3a in primates, extracellular spike discharge activity was recorded from TZ neurons in rats under general isoflurane anesthesia. Thermonoxious stimuli were applied by lowering the contralateral forepaw or hindpaw into a 48-51˚C heated water bath for 5-10s. Neurons in TZ were found to be minimally affected by non-noxious somatosensory stimuli, but highly responsive to thermonoxious skin stimuli in a slow temporal summation manner closely resembling that of nociresponsive neurons in primate area 3a. Selective inactivation of TZ by topical lidocaine application suppressed or delayed the nociceptive withdrawal reflex, suggesting that TZ exerts a tonic facilitatory influence over spinal cord neurons producing this reflex. In conclusion, TZ appears to be a rat homolog of the nociresponsive part of monkey area 3a. A possibility is considered that this region might be primarily engaged in autonomic aspects of nociception.

Axons in the central nervous system (CNS) typically fail to regenerate after injury. This failure is multi-factorial and caused in part by disruption of the axonal cytoskeleton. The cytoskeleton, in particular microtubules (MT), plays a critical role in axonal transport and axon growth during development. In this regard, members of the kinesin superfamily of proteins (KIFs) regulate the extension of primary axons toward their targets and control the growth of collateral branches. KIF2A negatively regulates axon growth through MT depolymerization. Using three different injury models to induce SCI in adult rats, we examined the temporal and cellular expression of KIF2A in the injured spinal cord. We observed a progressive increase of KIF2A expression with maximal levels at 10 days to 8 weeks post-injury as determined by Western blot analysis. KIF2A immunoreactivity was present in axons, spinal neurons and mature oligodendrocytes adjacent to the injury site. Results from the present study suggest that KIF2A at the injured axonal tips may contribute to neurite outgrowth inhibition after injury, and that its increased expression in inhibitory spinal neurons adjacent to the injury site might contribute to an intrinsic wiring-control mechanism associated with neuropathic pain. Further studies will determine whether KIF2A may be a potential target for the development of regeneration-promoting or pain-preventing therapies.

Orofacial inflammation could activate satellite glial cells (SGCs) in the trigeminal ganglion (TG) to produce interleukin 1β (IL-1β) which plays crucial roles in the development of inflammatory pain. Recent studies have shown that gamma-amino butyric acid-B (GABA) receptor could modulate the expression of inflammatory cytokines in microglia and astrocytes in the spinal cord. The objective of this study was to investigate whether GABA receptors in TG SGCs attenuate inflammatory facial pain via mediating IL-1β following inflammation and its mechanisms. Complete Freund's adjuvant (CFA) was injected into the whisker pad of rats to induce inflammation in vivo. Lipopolysaccharide (LPS) was added to culture medium to activate SGCs in vitro. Behavioral measures showed that microinjection of baclofen (a selective GABA receptor agonist) into the TG ameliorated the mechanical allodynia of CFA-treated rats. Interestingly, baclofen pretreatment inhibited SGC activation and IL-1β production, however, preserved the decreased expression of GABA receptors in SGCs activated by CFA in vivo and LPS in vitro. In addition, baclofen suppressed the increased expression of p-NF- κ B p65, p-I κ Bα, and p-p38 MAPK, while reversed the decreased production of I κ Bα, and further enhanced the increased expression of p-ERK(1/2) in LPS-treated SGCs in vitro. Finally, those effects of baclofen were abolished by saclofen (a specific GABA receptor antagonist) co-administration. Altogether, these results demonstrated for the first time that activation of GABA receptor might inhibit IL-1β production by suppressing NF- κ B and p38 MAPK signaling pathway activation and restore GABA receptor expression in SGCs to attenuate inflammatory facial pain.

Satellite glial cells (SGCs) activation in the trigeminal ganglia (TG) is critical in various abnormal orofacial sensation in nerve injury and inflammatory conditions. SGCs express several subtypes of P2 purinergic receptors contributing to the initiation and maintenance of neuropathic pain. The P2Y receptor, a G-protein-coupled receptor activated by uridine diphosphate (UDP)-glucose and other UDP sugars, mediates various physiologic events such as immune, inflammation, and pain. However, the expression, distribution, and function of P2Y receptor in SGCs remains largely unexplored. Our study reported the expression and functional identification of P2Y receptor in SGCs. SGCs were isolated from TG of rat, and the P2Y receptor expression was examined using immunofluorescence technique. Cell proliferation and viability were examined via cell counting kit-8 experiment. Immunofluorescence demonstrated the presence of P2Y receptor in SGCs. Immunofluorescence and western blot showed that UDP-glucose treatment upregulated glial fibrillary acid protein, a common marker for glial activation. Extracellular UDP-glucose enhanced the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, which were both abolished by the P2Y receptor inhibitor (PPTN). Furthermore, quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay demonstrated that extracellular UDP-glucose significantly enhanced interleukin-1β (IL-1β) and chemokine CCL2 (CCL2) release, which was abolished by PPTN and significantly decreased by inhibitors of MEK/ERK (U0126) and p38 (SB202190). Our findings directly proved the functional presence of P2Y receptor in SGCs. It was also verified that P2Y receptor activation was involved in activating SGCs, phosphorylating MAPKs, and promoting the secretion of IL-1β and CCL2 via ERK and p38 pathway.

We examined the effect of immobilization, low-intensity muscle contraction exercise, and transcutaneous electrical nerve stimulation (TENS) on tissue inflammation and acute pain following the onset of arthritis in a rat model. Sixty Wistar rats were divided into five groups: (1) Arthritis group, (2) arthritis and immobilization (Immobilization group), (3) arthritis and low intensity muscle contraction (Exercise group), (4) arthritis and TENS (TENS group), and (5) sham arthritis (Sham group). Arthritis was induced in the right knee joints by single injection of 3% kaolin and carrageenan. Immobilization of the right hindlimb was conducted by full extension of the right knee joints and full plantar flexion of the ankle joints using a plaster cast for 7 days after injection. The right quadriceps muscles were subjected to electrical stimulation (frequency: 50 Hz; intensity: 2-3 mA) for 20 min/day as contraction exercise for one week. TENS was delivered at 20 min/day for one week (frequency: 50 Hz; intensity: 1 mA). The pressure pain threshold (PPT) and paw withdrawal response (PWR) were evaluated at 1 and 7 days after injection. We also analyzed the number of CD68-positive cells in the synovium by immunohistochemistry and determined the expression level of calcitonin gene-related peptide (CGRP) in the spinal dorsal horn with immunofluorescence. Improvements of both PPT and PWR were observed in the Exercise group at 7 days after injection compared to those of the Arthritis and Immobilization groups, although only improvement of PPT was observed in the TENS group. The number of CD68-positive cells in the synovium and CGRP expression in the dorsal horn decreased only in the Exercise group. These results suggested that low-intensity muscle contraction exercise might be a better treatment for reduction of arthritis-induced inflammation and acute pain compared to immobilization and TENS.