Cytochrome P45026A1 Contributes to the Maintenance of Neuropathic Pain

The cytochrome P450 proteins(CYP450s)have been implicated in catalyzing numerous important biological reactions and contribute to a variety of diseases.CYP26A1,a member of the CYP450 family,carries out the oxidative metabolism of retinoic acid(RA),the active metabolite of vitamin A.Here we report that CYP26A1 was dramatically upregulated in the spinal cord after spinal nerve ligation(SNL).CYP26A1 was mainly expressed in spinal neurons and astrocytes.HPLC analysis displayed that the content of all-trans-RA(at-RA),the substrate of CYP26A1,was reduced in the spinal cord on day 7 after SNL.Inhibition of CYP26A1 by siRNA or inhibition of CYP26A1-mediated at-RA catabolism by talarozole relieved the SNL-induced mechanical allodynia during the maintenance phase of neuropathic pain.Talarozole also reduced SNL-induced glial activation and proinflammatory cytokine production but increased anti-inflammatory cytokine(IL-10)production.The RA receptors RARα,RXRβ,and RXRγwere expressed in spinal neurons and glial cells.The promoter of Il-10 has several binding sites for RA receptors,and at-RA directly increased Il-10 mRNA expression in vitro.Finally,intrathecal IL-10 attenuated SNL-induced neuropathic pain and reduced the activation of astrocytes and microglia.Collectively,the inhibition of CYP26A1-mediated at-RA catabolism alleviates SNL-induced neuropathic pain by promoting the expression of IL-10 and suppressing glial activation.CYP26A1 may be a potential therapeutic target for the treatment of neuropathic pain.

Astrocytes in Chronic Pain:Cellular and Molecular Mechanisms

Chronic pain is challenging to treat due to the limited therapeutic options and adverse side-effects of therapies.Astrocytes are the most abundant glial cells in the central nervous system and play important roles in different pathological conditions,including chronic pain.Astrocytes regulate nociceptive synaptic transmission and network function via neuron–glia and glia–glia interactions to exaggerate pain signals under chronic pain conditions.It is also becoming clear that astrocytes play active roles in brain regions important for the emotional and memory-related aspects of chronic pain.Therefore,this review presents our current understanding of the roles of astrocytes in chronic pain,how they regulate nociceptive responses,and their cellular and molecular mechanisms of action.

Emerging role of Toll-like receptors in the control of pain and itch

Toll-like receptors （TLRs） are germline-encoded pattern-recognition receptors that initiate innate immune re- sponses by recognizing molecular structures shared by a wide range of pathogens, known as pathogen-associated molecular patterns （PAMPs）. After tissue injury or cellular stress, TLRs also detect endogenous ligands known as danger-associated molecular patterns （DAMPs）. TLRs are expressed in both non-neuronal and neuronal cell types in the central nervous system （CNS） and contribute to both infectious and non-infectious disorders in the CNS. Following tissue insult and nerve injury, TLRs （such as TLR2, TLR3, and TLR4） induce the activation of microglia and astrocytes and the production of the proinflammatory cytokines in the spinal cord, leading to the development and maintenance of inflammatory pain and neu- ropathic pain. In particular, primary sensory neurons, such as nociceptors, express TLRs （e.g., TLR4 and TLR7） to sense exogenous PAMPs and endogenous DAMPs released after tissue injury and cellular stress. These neuronal TLRs are new players in the processing of pain and itch by increasing the excitability of primary sensory neurons. Given the prevalence of chronic pain and itch and the suffering of affected people, insights into TLR signaling in the nervous system will open a new avenue for the management of clinical pain and itch.

Increased CXCL13 and CXCR5 in Anterior Cingulate Cortex Contributes to Neuropathic Pain-Related Conditioned Place Aversion

Pain consists of sensory-discriminative and emotional-affective components.The anterior cingulate cortex(ACC)is a critical brain area in mediating the affective pain.However,the molecular mechanisms involved remain largely unknown.Our recent study indicated that C-X-C motif chemokine 13(CXCL13)and its sole receptor CXCR5 are involved in sensory sensitization in the spinal cord after spinal nerve ligation(SNL).Whether CXCL13/CXCR5 signaling in the ACC contributes to the pathogenesis of pain-related aversion remains unknown.Here,we showed that SNL increased the CXCL13 level and CXCR5 expression in the ACC after SNL.Knockdown of CXCR5 by microinjection of Cxcr5 shRNA into the ACC did not affect SNL-induced mechanical allodynia but effectively alleviated neuropathic painrelated place avoidance behavior.Furthermore,electrophysiological recording from layer Ⅱ-Ⅲ neurons in the ACC showed that SNL increased the frequency and amplitude of spontaneous excitatory postsynaptic currents(sEPSCs),decreased the EPSC paired-pulse ratio,and increased the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor/N-methyl-D-aspartate receptor ratio,indicating enhanced glutamatergic synaptic transmission.Finally,superfusion of CXCL13 onto ACC slices increased the frequency and amplitude of spontaneous EPSCs.Pre-injection of Cxcr5 shRNA into the ACC reduced the increase in glutamatergic synaptic transmis sion induced by SNL.Collectively,these results suggest that CXCL13/CXCR5 signaling in the ACC is involved in neuropathic pain-related aversion via synaptic potentiation.

Spinal CCL2 Promotes Central Sensitization, Long-Term Potentiation, and Inflammatory Pain via CCR2： Further Insights into Molecular, Synaptic, and Cellular Mechanisms

Mounting evidence supports an important role of chemokines, produced by spinal cord astrocytes, in promoting central sensitization and chronic pain. In particular, CCL2 （C-C motif chemokine ligand 2） has been shown to enhance N-methyl-D-aspartate （NMDA）-induced currents in spinal outer lamina II （Iio） neurons. However, the exact molecular, synaptic, and cellular mechanisms by which CCL2 modulates central sensitization are still unclear. We found that spinal injection of the CCR2 antagonist RS504393 attenuated CCL2- and inflammation-induced hyperalgesia. Single-cell RT-PCR revealed CCR2 expres- sion in excitatory vesicular glutamate transporter subtype 2-positive （VGLUT2＋） neurons. CCL2 increased NMDA- induced currents in CCR2＋/VGLUT2＋ neurons in lamina IIo; it also enhanced the synaptic NMDA currents evoked by dorsal root stimulation; and furthermore, it increased the total and synaptic NMDA currents in somatostatin- expressing excitatory neurons. Finally, intrathecal RS504393 reversed the long-term potentiation evoked in the spinal cord by C-fiber stimulation. Our findings suggest that CCL2 directly modulates synaptic plasticity in CCR2- expressing excitatory neurons in spinal lamina Iio, and this underlies the generation of central sensitization in patho- logical pain.

Chemokine Receptor CXCR3 in the Spinal Cord Contributes to Chronic Itch in Mice

Recent studies have shown that the chemokine receptor CXCR3 and its ligand CXCL10 in the dorsal root ganglion mediate itch in experimental allergic contact dermatitis （ACD）. CXCR3 in the spinal cord also con- tributes to the maintenance of neuropathic pain. However, whether spinal CXCR3 is involved in acute or chronic itch remains unclear. Here, we report that Cxcr3-/- mice showed normal scratching in acute itch models but reduced scratching in chronic itch models of dry skin and ACD. In contrast, both formalin-induced acute pain and complete Freund＇s adjuvant-induced chronic inflammatory pain were reduced in Cxcr3-/- mice. In addition, the expression of CXCR3 and CXCL10 was increased in the spinal cord in the dry skin model induced by acetone and diethyl ether followed by water （AEW）. Intrathecal injection of a CXCR3 antagonist alleviated AEW-induced itch. Further- more, touch-elicited itch （alloknesis） after compound 48/80 or AEW treatment was suppressed in Cxcr3-/- mice. Finally, AEW-induced astrocyte activation was inhibited in Cxcr3-/- mice. Taken together, these data suggest that spinal CXCR3 mediates chronic itch and alloknesis, and targeting CXCR3 may provide effective treatment for chronic pruritus.

Antioxidants Attenuate Acute and Chronic Itch:Peripheral and Central Mechanisms of Oxidative Stress in Pruritus

Itch（pruritus） is one of the most disabling syndromes in patients suffering from skin, liver, or kidney diseases. Our previous study highlighted a key role of oxidative stress in acute itch. Here, we evaluated the effects of antioxidants in mouse models of acute and chronic itch and explored the potential mechanisms. The effects of systemic administration of the antioxidants N-acetyl-Lcysteine（NAC） and N-tert-butyl-a-phenylnitrone（PBN）were determined by behavioral tests in mouse models of acute itch induced by compound 48/80 or chloroquine, and chronic itch by treatment with a mixture of acetonediethyl-ether-water. We found that systemic administration of NAC or PBN significantly alleviated compound 48/80-and chloroquine-induced acute itch in a dose-dependent manner, attenuated dry skin-induced chronic itch, and suppressed oxidative stress in the affected skin.Antioxidants significantly decreased the accumulation of intracellular reactive oxygen species directly induced by compound 48/80 and chloroquine in the cultured dorsal root ganglia-derived cell line ND7-23. Finally, the antioxidants remarkably inhibited the compound 48/80-induced phosphorylation of extracellular signal-regulated kinase in the spinal cord. These results indicated that oxidative stress plays a critical role in acute and chronic itch in the periphery and spinal cord and antioxidant treatment may be a promising strategy for anti-itch therapy.

CXCL10/CXCR3 Signaling in the DRG Exacerbates Neuropathic Pain in Mice

Chemokines and receptors have been implicated in the pathogenesis of chronic pain.Here,we report that spinal nerve ligation(SNL)increased CXCR3 expression in dorsal root ganglion(DRG)neurons,and intra-DRG injection of Cxcr3 shRNA attenuated the SNL-induced mechanical allodynia and heat hyperalgesia.SNL also increased the m RNA levels of CXCL9,CXCL10,and CXCL11,whereas only CXCL10 increased the number of action potentials(APs)in DRG neurons.Furthermore,in Cxcr3^(-/-)mice,CXCL10 did not increase the number of APs,and the SNL-induced increase of the numbers of APs in DRG neurons was reduced.Finally,CXCL10 induced the activation of p38 and ERK in ND7-23 neuronal cells and DRG neurons.Pretreatment of DRG neurons with the P38 inhibitor SB203580 decreased the number of APs induced by CXCL10.Our data indicate that CXCR3,activated by CXCL10,mediates p38 and ERK activation in DRG neurons and enhances neuronal excitability,which contributes to the maintenance of neuropathic pain.

Spinal CCL2 Promotes Pain Sensitization by Rapid Enhancement of NMDA-Induced Currents Through the ERK-GluN2B Pathway in Mouse Lamina Ⅱ Neurons

Previous studies have shown that CCL2(C-C motif chemokine ligand 2)induces chronic pain,but the exact mechanisms are still unknown.Here,we established models to explore the potential mechanisms.Behavioral experiments revealed that an antagonist of extracellular signal-regulated kinase(ERK)inhibited not only CCL2-induced inflammatory pain,but also pain responses induced by complete Freund’s adjuvant.We posed the question of the intracellular signaling cascade involved.Subsequent experiments showed that CCL2 up-regulated the expression of phosphorylated ERK(pERK)and N-methyl D-aspartate receptor[NMDAR]subtype 2B(GluN2B);meanwhile,antagonists of CCR2 and ERK effectively reversed these phenomena.Whole-cell patchclamp recordings revealed that CCL2 enhanced the NMDAR-induced currents via activating the pERK pathway,which was blocked by antagonists of GluN2B and ERK.In summary,we demonstrate that CCL2 directly interacts with CCR2 to enhance NMDAR-induced currents,eventually leading to inflammatory pain mainly through the CCL2-CCR2-pERK-GluN2B pathway.

CCL2/CCR2 Contributes to the Altered Excitatory-inhibitory Synaptic Balance in the Nucleus Accumbens Shell Following Peripheral Nerve Injury-induced Neuropathic Pain

The medium spiny neurons(MSNs)in the nucleus accumbens(NAc)integrate excitatory and inhibitory synaptic inputs and gate motivational and emotional behavior output.Here we report that the relative intensity of excitatory and inhibitory synaptic inputs to MSNs of the NAc shell was decreased in mice with neuropathic pain induced by spinal nerve ligation(SNL).SNL increased the frequency,but not the amplitude of spontaneous inhibitory postsynaptic currents(sIPSCs),and decreased both the frequency and amplitude of spontaneous excitatory postsynaptic currents(sEPSCs)in the MSNs.SNL also decreased the paired-pulse ratio(PPR)of evoked IPSCs but increased the PPR of evoked EPSCs.Moreover,acute bath application of C–C motif chemokine ligand 2(CCL2)increased the frequency and amplitude of sIPSCs and sEPSCs in the MSNs,and especially strengthened the amplitude of N-methyl-D-aspartate receptor(NMDAR)-mediated miniature EPSCs.Further Ccl2 overexpression in the NAc in vivo decreased the peak amplitude of the sEPSC/sIPSC ratio.Finally,Ccr2 knock-down improved the impaired induction of NMDAR-dependent long-term depression(LTD)in the NAc after SNL.These results suggest that CCL2/CCR2 signaling plays a role in the integration of excitatory/inhibitory synaptic transmission and leads to an increase of the LTD induction threshold at the synapses of MSNs during neuropathic pain.

Descending Modulation of Spinal Itch Transmission by Primary Somatosensory Cortex

Dear Editor,Itch(pruritus)is an unpleasant somatic sensation that is accompanied by the desire to scratch.While itch provides a warning signal that protects us from potential threats in normal conditions,severe or chronic itch associated with dermatosis or systemic diseases is often difficult to alleviate and causes severe skin and tissue damage.

TLR8 in the Trigeminal Ganglion Contributes to the Maintenance of Trigeminal Neuropathic Pain in Mice

Trigeminal neuropathic pain(TNP)is a significant health problem but the involved mechanism has not been completely elucidated.Toll-like receptors(TLRs)have recently been demonstrated to be expressed in the dorsal root ganglion and involved in chronic pain.Here,we show that TLR8 was persistently increased in the trigeminal ganglion(TG)neurons in model of TNP induced by partial infraorbital nerve ligation(pIONL).In addition,deletion or knockdown of Tlr8 in the TG attenuated pIONL-induced mechanical allodynia,reduced the activation of ERK and p38-MAPK,and decreased the expression of pro-inflammatory cytokines in the TG.Furthermore,intra-TG injection of the TLR8 agonist VTX-2337 induced pain hypersensitivity.VTX-2337 also increased the intracellular Ca^(2+)concentration,induced the activation of ERK and p38,and increased the expression of pro-inflammatory cytokines in the TG.These data indicate that TLR8 contributes to the maintenance of TNP through increasing MAPK-mediated neuroinflammation.Targeting TLR8 signaling may be effective for the treatment of TNP.