Interleukin‐6 induces microglial CX3CR1 expression in the spinal cord after peripheral nerve injury through the activation of p38 MAPK

Peripheral nerve injury leading to neuropathic pain induces the upregulation of interleukin (IL)‐6 and microglial CX3CR1 expression, and activation of p38 mitogen‐activated protein kinase (MAPK) in the spinal cord. Here, we investigated whether IL‐6 regulates CX3CR1 expression through p38 MAPK activation in the spinal cord in rats with chronic constriction injury (CCI) of the sciatic nerve. Similar temporal changes in the expression of IL‐6, phosphorylated p38 MAPK and CX3CR1 were observed following CCI. The increases in CX3CR1 expression, p38 MAPK activation and pain behavior after CCI were suppressed by blocking IL‐6 action with a neutralizing antibody, while they were enhanced by supplying exogenous recombinant rat IL‐6 (rrIL‐6). rrIL‐6 also induced increases in spinal CX3CR1 expression, p38 MAPK activation and pain behavior in naïve rats without nerve injury. Furthermore, treatment with the p38 MAPK‐specific inhibitor, SB203580, suppressed the increase in CX3CR1 expression induced by CCI or rrIL‐6 treatment. Finally, blocking CX3CR1 or p38 MAPK activation prevented the development of mechanical allodynia and thermal hyperalgesia induced by CCI or rrIL‐6 treatment. These results suggest a new mechanism of neuropathic pain, in which IL‐6 induces microglial CX3CR1 expression in the spinal cord through p38 MAPK activation, enhancing the responsiveness of microglia to fractalkine in the spinal cord, thus playing an important role in neuropathic pain after peripheral nerve injury.     

Interleukin-1 alpha has antiallodynic and antihyperalgesic activities in a rat neuropathic pain model

Nerve injury and the consequent release of interleukins (ILs) are processes implicated in pain transmission. To study the potential role of IL-1 in the pathogenesis of allodynia and hyperalgesia, IL-1alpha and comparative IL-1beta, IL-6, and IL-10 mRNA levels were quantified using competitive RT-PCR of the lumbar spinal cord and dorsal root ganglia (DRG; L5-L6) three and seven days after chronic constriction injury (CCI) in rats. Microglial and astroglial activation in the ipsilateral spinal cord and DRG were observed after injury. In naive and CCI-exposed rats, IL-1alpha mRNA and protein were not detected in the spinal cord. IL-1beta and IL-6 mRNAs were strongly ipsilaterally elevated on day seven after CCI. In the ipsilateral DRG, IL-1alpha, IL-6, and IL-10 mRNA levels were increased on days three and seven; IL-1beta was elevated only on day seven. Western blot analysis revealed both the presence of IL-1alpha proteins (45 and 31 kDa) in the DRG and the down-regulation of these proteins after CCI. Intrathecal administration of IL-1alpha (50-500 ng) in naive rats did not influence nociceptive transmission, but IL-1beta (50-500 ng) induced hyperalgesia. In rats exposed to CCI, an IL-1alpha or IL-1 receptor antagonist dose-dependently attenuated symptoms of neuropathic pain; however, no effect of IL-1beta was observed. In sum, the first days after CCI showed a high abundance of IL-1alpha in the DRG. Together with the antiallodynic and antihyperalgesic effects observed after IL-1alpha administration, this finding indicates an important role for IL-1alpha in the development of neuropathic pain symptoms.     

Spinal administration of a delta opioid receptor agonist attenuates hyperalgesia and allodynia in a rat model of neuropathic pain.

Neuropathic (NP) pain is a debilitating chronic pain disorder considered by some to be inherently resistant to therapy with traditional analgesics. Indeed, micro opioid receptor (OR) agonists show reduced therapeutic benefit and their long term use is hindered by the high incidence of adverse effects. However, pharmacological and physiological evidence increasingly suggests a role for deltaOR agonists in modulating NP pain symptoms. In this study, we examined the antihyperalgesic and antiallodynic effects of the spinally administered deltaOR agonist, d-[Ala(2), Glu(4)]deltorphin II (deltorphin II), as well as the changes in deltaOR expression, in rats following chronic constriction injury (CCI) of the sciatic nerve. Rats with CCI exhibited cold hyperalgesia and mechanical allodynia over a 14-day testing period. Intrathecal administration of deltorphin II reversed cold hyperalgesia on day 14 and dose-dependently attenuated mechanical allodynia. The effects of deltorphin II were mediated via activation of the deltaOR as the effect was antagonized by co-treatment with the delta-selective antagonist, naltrindole. Western blotting experiments revealed no changes in deltaOR protein in the dorsal spinal cord following CCI. Taken together, these data demonstrate the antihyperalgesic and antiallodynic effectiveness of a spinally administered deltaOR agonist following peripheral nerve injury and support further investigation of deltaORs as potential therapeutic targets in the treatment of NP pain.     

5‐HT2C receptor agonists attenuate pain‐related behaviour in a rat model of trigeminal neuropathic pain

Peripheral branches of the trigeminal nerve may be damaged during maxillofacial injury or surgical procedures and trigeminal trauma may induce severe pain that is very challenging to treat. Chronic constriction injury to the infraorbital nerve (ION‐CCI) by loose ligatures has proven a useful model for some types of trigeminal neuropathic pain disorder. Using ION‐CCI rats, we examined the antiallodynic effects of intrathecally administered agents which are selective for 5‐HT2C receptors. Allodynia was evaluated by applying von Frey filaments to skin innervated by the injured ION. Dose‐dependent antiallodynic effects followed administration of three 5‐HT2C receptor agonists, 6‐chloro‐2‐(1‐piperazinyl)‐pyrazine (MK212: 10, 30, and 100 μg); (S)‐2‐(chloro‐5‐fluoro‐indol‐l‐yl)‐1‐methyamine fumarate (RO 60‐0175: 10, 30, and 100 μg); (AaR)‐8,9‐dichloro‐2,3,4,4a‐tetrahydro‐1H‐pyrazino[1,2‐a]quinoxalin‐5(6H)‐one (WAY‐161503: 10, 30, and 100 μg). ED50 values for antiallodynic effects of MK212, RO 60‐0175, and WAY‐161503 were 39.62, 46.67, and 51.22 μg, respectively. Intrathecal administration of the 5‐HT2C receptor antagonist, 8‐[5‐2,4‐dimethoxy‐5‐(4‐trifluoromethylphenylsulphonamido)phenyl‐5‐oxopentyl]‐1,3,8‐triazaspiro[4,5]decane‐2,4‐dione (RS‐102221: 30 μg) did not alter the mechanical threshold. Intrathecal pretreatment with RS‐102221 (10 and 30 μg) reduced the antiallodynic effects of the highest dose of 5‐HT2C agonists. These results indicated that, in this rat model, the 5‐HT2C receptor plays a role in spinal inhibition of trigeminal neuropathic pain.     

Involvement of neural cell adhesion molecule signaling in glial cell line-derived neurotrophic factor-induced analgesia in a rat model of neuropathic pain

Since neuropathic pain is resistant to conventional analgesics such as opiates and non-steroidal anti-inflammatory drugs, the development of new types of drugs for its treatment has been awaited. Several key molecules associated with nociception have been suggested as potential targets for new analgesics. Glial cell line-derived neurotrophic factor (GDNF) has a variety of functions affecting the survival and development of specified neural cell populations, mediated via transmission of intracellular signals through binding to its high-affinity receptor, GFR*1, and subsequent activation of a tyrosine receptor kinase, RET, neural cell adhesion molecule (NCAM), or other signaling molecules. GDNF also exhibits analgesic effects in rodent models of neuropathic pain, although the underlying mechanisms are still largely unknown, including the intracellular signal transduction involved. We report here that NCAM signaling plays a role in mediating the analgesic effect of GDNF in rats with chronic constrictive injury (CCI). We found that NCAM was expressed in intrinsic neurons in the spinal dorsal horn and in dorsal root ganglion neurons with small cell bodies. Reduction of NCAM expression by NCAM antisense oligodeoxynucleotide administration to CCI rats abolished the analgesic effect of GDNF without affecting RET signaling activation. An NCAM mimetic peptide, C3d, partially reduced the chronic pain induced by CCI. These findings suggest that NCAM signaling plays a critical role in the analgesic effect of GDNF and that development of new drugs activating GDNF-NCAM signaling may represent a new strategy for the relief of intractable pain.     

Novel histamine H3 receptor antagonists GSK189254 and GSK334429 are efficacious in surgically-induced and virally-induced rat models of neuropathic pain

Several studies have implicated a potential role for histamine H₃ receptors in pain processing, although the data are somewhat conflicting. In the present study we investigated the effects of the novel potent and highly selective H₃ receptor antagonists GSK189254 (6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride) and GSK334429 (1-(1-methylethyl)-4-({1-[6-(trifluoromethyl)-3-pyridinyl]-4-piperidinyl}carbonyl)hexahydro-1H-1,4-diazepine) in two rat models of neuropathic pain, namely the chronic constriction injury (CCI) model and the varicella-zoster virus (VZV) model. Both GSK189254 (0.3, 3 and/or 10 mg/kg p.o.) and GSK334429 (1, 3 and 10 mg/kg p.o.) significantly reversed the CCI-induced decrease in paw withdrawal threshold (PWT) measured using an analgesymeter and/or von Frey hairs. In addition, GSK189254 (3 mg/kg p.o.) and GSK334429 (10 mg/kg p.o.) both reversed the VZV-induced decrease in PWT using von Frey hairs. We also investigated the potential site of action of this analgesic effect of H₃ antagonists using autoradiography. Specific binding to H₃ receptors was demonstrated with [³H]-GSK189254 in the dorsal horn of the human and rat spinal cord, and in human dorsal root ganglion (DRG), consistent with the potential involvement of H₃ receptors in pain processing. In conclusion, we have shown for the first time that chronic oral administration of selective H₃ antagonists is effective in reversing neuropathic hypersensitivity in disease-related models, and that specific H₃ receptor binding sites are present in the human DRG and dorsal horn of the spinal cord. These data suggest that H₃ antagonists such as GSK189254 and GSK334429 may be useful for the treatment of neuropathic pain.     

P2X3 receptor upregulation in trigeminal ganglion neurons through TNFα production in macrophages contributes to trigeminal neuropathic pain in rats

BackgroundTrigeminal neuralgia is a characteristic disease that manifests as orofacial phasic or continuous severe pain triggered by innocuous orofacial stimulation; its mechanisms are not fully understood. In this study, we established a new animal model of trigeminal neuralgia and investigated the role of P2X₃ receptor (P2X₃R) alteration in the trigeminal ganglion (TG) via tumor necrosis factor alpha (TNFα) signaling in persistent orofacial pain.MethodsTrigeminal nerve root compression (TNC) was performed in male Sprague-Dawley rats. Changes in the mechanical sensitivity of whisker pad skin, amount of TNFα in the TG, and number of P2X₃R and TNF receptor-2 (TNFR2)-positive TG neurons were assessed following TNC. The effects of TNFR2 antagonism in TG and subcutaneous P2X₃R antagonism on mechanical hypersensitivity following TNC were examined.ResultsTNC induced unilateral continuous orofacial mechanical allodynia, which was depressed by carbamazepine. The accumulation of macrophages showing amoeboid-like morphological changes and expression of TNFα in the TG was remarkably increased following TNC treatment. The number of P2X₃R- and TNFR2-positive TG neurons innervating the orofacial skin was significantly increased following TNC. TNFα was released from activated macrophages that occurred in the TG following TNC, and TNFR2 antagonism in the TG significantly diminished the TNC-induced increase in P2X₃R-immunoreactive TG neurons. Moreover, subcutaneous P2X₃R antagonism in the whisker pad skin significantly depressed TNC-induced mechanical allodynia.ConclusionsTherefore, it can be concluded that the signaling of TNFα released from activated macrophages in the TG induces the upregulation of P2X₃R expression in TG neurons innervating the orofacial region, resulting in orofacial mechanical allodynia following TNC.     

Tumor necrosis factor-α and interleukin-6 are not correlated with the characteristics of Complex Regional Pain Syndrome type 1 in 66 patients

It was previously shown in a group of 9 patients with complex regional pain syndrome type 1 (CRPS1) that levels of the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are higher in blister fluid from the involved side. We hypothesize that local inflammation is responsible for the characteristics of CRPS1. The aim of this study was to confirm the previous observation in a large group of CRPS1 patients, repeating the measurement of TNF-α and IL-6 in blister fluid. Furthermore, we sought to determine whether these cytokines are responsible for the characteristics of CRPS1 and characterize the relationship between cytokine levels and duration of the disease. Sixty-six patients with CRPS1 participated. Skin blisters were artificially induced for measurement of cytokines in both extremities. The following disease characteristics were assessed: pain and differences in temperature, volume, and mobility between the extremities. TNF-α and IL-6 levels were significantly higher in blister fluid from the involved side. However, cytokine levels did not correlate with the characteristics or duration of the disease. Our findings confirm the presence of local inflammation in a population of 66 patients in the first 2 years of CRPS1. Proinflammatory cytokines seem to be only partly involved in the pathophysiology of CRPS1, as indicated by the lack of coherence between TNF-α and IL-6 levels and the signs and symptoms of inflammation and disease duration. Other inflammatory mediators and mechanisms, such as central sensitization, are probably involved in the early stages of CRPS1.     

Triptolide reduces salivary gland damage in a non-obese diabetic mice model of Sjögren’s syndrome via JAK/STAT and NF-κB signaling pathways

Triptolide (TP) has anti-inflammatory and immunosuppressive effects. However, the effect of triptolide on Sjögren’s syndrome (SS) is rarely reported. In this paper, we studied the effects of triptolide on non-obese diabetes mice model of SS. In this study, salivary flow rate was measured every two weeks, and autoantibodies levels in the serum were detected. Salivary gland index and spleen index were detected, pathological changes of salivary gland were detected by hematoxylin-eosin staining, inflammatory factors were detected by enzyme linked immunosorbent assay, lymphocytes were detected by flow cytometry, proliferation of T cells and B cells were detected, and related proteins were detected by Western blot. Triptolide increased salivary flow rate and salivary gland index, and decreased spleen gland index. Moreover, triptolide reduced the infiltration of lymphocytes to salivary glands, decreased the level of autoantibodies in serum, and reduced the inflammatory factors in salivary glands and IFN-γ induced salivary gland epithelial cells. Further, triptolide inhibited activator of JAK/STAT pathway and NF-κB pathway. In conclusion, triptolide could inhibit the infiltration of lymphocytes and the expression of inflammatory factors through JAK/STAT pathway and NF-κB pathway. Thus, triptolide may be used as a potential drug to treat SS.     

N‐methyl‐d‐aspartate receptor‐mediated modulations of the anti‐allodynic effects of 5‐HT1B/1D receptor stimulation in a rat model of trigeminal neuropathic pain

Previous studies showed that triptans and other 5‐HT_1B/1D‐receptor agonists attenuate hyper‐responsiveness to mechanical stimulation of the face in a rat model of trigeminal neuropathic pain, probably by activating 5‐HT_1B/1D‐receptors on primary afferent nociceptive fibers. We now tested whether blockade of post‐synaptic receptors for the excitatory amino acid glutamate released by these fibers would increase this action. We thus evaluated whether (±)1‐hydroxy‐3‐aminopyrrolidine‐2‐one (HA‐966), an antagonist at the glycine/d ‐serine site of N‐methyl‐d ‐aspartate (NMDA)‐receptors, would potentiate the anti‐allodynic action of dihydroergotamine and zolmitriptan in rats with chronic constriction injury to the infraorbital nerve (CCI‐ION). Complementary studies were performed with other NMDA‐receptor ligands and in rats with chronic constriction injury to the sciatic nerve (CCI‐SN) for comparison. Injury was produced by loose ligatures of the nerves. Responsiveness to mechanical stimulation (vibrissae or hindpaw territories) with von Frey filaments was used to evaluate allodynia 2 weeks after nerve ligature. Rats received NMDA‐receptor ligands or saline 20 min before dihydroergotamine (25–100μg/kg, i.v.) or zolmitriptan (25–100μg/kg, s.c.). HA‐966 (2.5 mg/kg, s.c.), inactive on its own, enhanced the anti‐allodynic effects of dihydroergotamine (eightfold increase) and zolmitriptan (threefold increase) in CCI‐ION rats, but these drugs exerted no effects in allodynic CCI‐SN rats. NMDA‐receptor blockade by memantine (5 mg/kg, i.p.) also enhanced, whereas activation at glycine/NMDA site by d ‐cycloserine (3 mg/kg, i.p.) reduced the anti‐allodynic properties of zolmitriptan in CCI‐ION rats. Combined administration of NMDA‐receptor antagonist and 5‐HT_1B/1D‐receptor agonist may be a promising approach for alleviating trigeminal neuropathic pain.     

Regulatory mechanisms of C4b‐binding protein (C4BP)α and β expression in rat hepatocytes by lipopolysaccharide and interleukin‐6

Summary. Background: C4b‐binding protein (C4BP), a multimeric protein structurally composed of α chains (C4BPα) and a β chain (C4BPβ), regulates the anticoagulant activity of protein S (PS). Patients with sepsis have increased levels of plasma C4BP, which appears to be induced by interleukin (IL)‐6. However, it is not fully understood how lipopolysaccharide (LPS) and IL‐6 affect the plasma C4BP antigen level and C4BPα and C4BPβ expression in hepatocytes. Objectives: To assess the effect of LPS and IL‐6 on plasma C4BP, PS–C4BP complex levels, PS activity, and C4BP expression by rat liver in vivo and on C4BP expression by isolated rat hepatocytes in vitro. Results: Plasma C4BP antigen level transiently decreased from 2 to 12 h after LPS (2 mg kg^?1) injection, and then it abruptly increased up to 24 h after LPS injection. Plasma C4BP antigen level increased until 8 h after IL‐6 (10 μg kg^?1) injection, and then gradually decreased up to 24 h after IL‐6 injection. LPS significantly decreased the protein and mRNA expression of both C4BPα and C4BPβ in rat hepatocytes, and this effect was inhibited by NFκB and MEK/ERK inhibitors. IL‐6 mediated increase in C4BPβ expression in rat hepatocytes, which leads to increased plasma PS–C4BP complex level and to decreased plasma PS activity, was inhibited by inhibition of STAT‐3. Conclusion: LPS decreases both C4BPα and C4BPβ expression via the NFκB and MEK/ERK pathways, whereas IL‐6 specifically increases C4BPβ expression via the STAT‐3 pathway, causing an increase in plasma PS–C4BP complex, and thus decreasing the anticoagulant activity of PS.