Molecular Pain

There is evidence for augmented processing of pain and impaired endogenous pain inhibition in Fibromyalgia syndrome (FM). In order to fully understand the mechanisms involved in FM pathology, there is a need for closer investigation of endogenous pain modulation. In the present study, we compared the functional connectivity of the descending pain inhibitory network in age-matched FM patients and healthy controls (HC). We performed functional magnetic resonance imaging (fMRI) in 42 subjects; 14 healthy and 28 age-matched FM patients (2 patients per HC), during randomly presented, subjectively calibrated pressure pain stimuli. A seed-based functional connectivity analysis of brain activity was performed. The seed coordinates were based on the findings from our previous study, comparing the fMRI signal during calibrated pressure pain in FM and HC: the rostral anterior cingulate cortex (rACC) and thalamus.

FM patients required significantly less pressure (kPa) to reach calibrated pain at 50 mm on a 0–100 visual analogue scale ( p<.001, two-tailed). During fMRI scanning, the rACC displayed significantly higher connectivity to the amygdala, hippocampus, and brainstem in healthy controls, compared to FM patients. There were no regions where FM patients showed higher rACC connectivity. Thalamus showed significantly higher connectivity to the orbitofrontal cortex in healthy controls but no regions showed higher thalamic connectivity in FM patients.

Patients with FM displayed less connectivity within the brain's pain inhibitory network during calibrated pressure pain, compared to healthy controls. The present study provides brain-imaging evidence on how brain regions involved in homeostatic control of pain are less connected in FM patients. It is possible that the dysfunction of the descending pain modulatory network plays an important role in maintenance of FM pain and our results may translate into clinical implications by using the functional connectivity of the pain modulatory network as an objective measure of pain dysregulation.

Oxaliplatin, the third-generation platinum compound, has evolved as one of the most important therapeutic agents in colorectal cancer chemotherapy. The main limiting factor in oxaliplatin treatment is painful neuropathy that is difficult to treat. This side effect has been studied for several years, but its full mechanism is still inconclusive, and effective treatment does not exist. Data suggest that oxaliplatin's initial neurotoxic effect is peripheral and oxidative stress-dependent. A spinal target is also suggested in its mechanism of action. The flavonoids rutin and quercetin have been described as cell-protecting agents because of their antioxidant, antinociceptive, and anti-inflammatory actions. We proposed a preventive effect of these agents on oxaliplatin-induced painful peripheral neuropathy based on their antioxidant properties.

Oxaliplatin (1 mg/kg, i.v.) was injected in male Swiss mice, twice a week (total of nine injections). The development of sensory alterations, such as thermal and mechanical allodynia, was evaluated using the tail immersion test in cold water (10°C) and the von Frey test. Rutin and quercetin (25–100 mg/kg, i.p.) were injected 30 min before each oxaliplatin injection. The animals' spinal cords were removed for histopathological and immunohistochemical evaluation and malondialdehyde assay.

Oxaliplatin significantly increased thermal and mechanical nociceptive response, effects prevented by quercetin and rutin at all doses. Fos immunostaining in the dorsal horn of the spinal cord confirmed these results. The oxidative stress assays mainly showed that oxaliplatin induced peroxidation in the spinal cord and that rutin and quercetin decreased this effect. The flavonoids also decreased inducible nitric oxide synthase and nitrotyrosine immunostaining in the dorsal horn of the spinal cord. These results suggest that nitric oxide and peroxynitrite are also involved in the neurotoxic effect of oxaliplatin and that rutin and quercetin can inhibit their effect in the spinal cord. We also observed the preservation of dorsal horn structure using histopathological analyses.

Oxaliplatin induced painful peripheral neuropathy in mice, an effect that was prevented by rutin and quercetin. The mechanism of action of oxaliplatin appears to be, at least, partially oxidative stress-induced damage in dorsal horn neurons, with the involvement of lipid peroxidation and protein nitrosylation.

Cisplatin is primarily used for treatment of ovarian and testicular cancer. Oxaliplatin is the only effective treatment for metastatic colorectal cancer. Both are known to cause dose related, cumulative toxic effects on the peripheral nervous system and thirty to forty percent of cancer patients receiving these agents experience painful peripheral neuropathy. The mechanisms underlying painful platinum-induced neuropathy remain poorly understood. Previous studies have demonstrated important roles for TRPV1, TRPM8, and TRPA1 in inflammation and nerve injury induced pain.

In this study, using real-time, reverse transcriptase, polymerase chain reaction (RT-PCR), we analyzed the expression of TRPV1, TRPM8, and TRPA1 induced by cisplatin or oxaliplatin in vitro and in vivo. For in vitro studies, cultured E15 rat dorsal root ganglion (DRG) neurons were treated for up to 48 hours with cisplatin or oxaliplatin. For in vivo studies, trigeminal ganglia (TG) were isolated from mice treated with platinum drugs for three weeks. We show that cisplatin and oxaliplatin-treated DRG neurons had significantly increased in TRPV1, TRPA1, and TRPM8 mRNA expression. TG neurons from cisplatin treated mice had significant increases in TRPV1 and TRPA1 mRNA expression while oxaliplatin strongly induced only TRPA1. Furthermore, compared to the cisplatin-treated wild-type mice, cisplatin-treated TRPV1-null mice developed mechanical allodynia but did not exhibit enhancement of noxious heat- evoked pain responses. Immunohistochemistry studies showed that cisplatin-treated mice had no change in the proportion of the TRPV1 immunopositive TG neurons.

These results indicate that TRPV1 and TRPA1 could contribute to the development of thermal hyperalgesia and mechanical allodynia following cisplatin-induced painful neuropathy but that TRPV1 has a crucial role in cisplatin-induced thermal hyperalgesia in vivo.

Neuropathic pain is characterized by pain hypersensitivity to innocuous stimuli (tactile allodynia) that is nearly always resistant to known treatments such as non-steroidal anti-inflammatory drugs or even opioids. It has been reported that some antidepressants are effective for treating neuropathic pain. However, the underlying molecular mechanisms are not well understood. We have recently demonstrated that blocking P2X₄ receptors in the spinal cord reverses tactile allodynia after peripheral nerve injury in rats, implying that P2X₄ receptors are a key molecule in neuropathic pain. We investigated a possible role of antidepressants as inhibitors of P2X₄ receptors and analysed their analgesic mechanism using an animal model of neuropathic pain.

Antidepressants strongly inhibited ATP-mediated Ca²⁺ responses in P2X₄ receptor-expressing 1321N1 cells, which are known to have no endogenous ATP receptors. Paroxetine exhibited the most powerful inhibition of calcium influx via rat and human P2X₄ receptors, with IC₅₀ values of 2.45 μM and 1.87 μM, respectively. Intrathecal administration of paroxetine produced a striking antiallodynic effect in an animal model of neuropathic pain. Co-administration of WAY100635, ketanserin or ondansetron with paroxetine induced no significant change in the antiallodynic effect of paroxetine. Furthermore, the antiallodynic effect of paroxetine was observed even in rats that had received intrathecal pretreatment with 5,7-dihydroxytryptamine, which dramatically depletes spinal 5-hydroxytryptamine.

These results suggest that paroxetine acts as a potent analgesic in the spinal cord via a mechanism independent of its inhibitory effect on serotonin transporters. Powerful inhibition on P2X₄ receptors may underlie the analgesic effect of paroxetine, and it is possible that some antidepressants clinically used in patients with neuropathic pain show antiallodynic effects, at least in part via their inhibitory effects on P2X₄ receptors.

The cooling agents menthol and icilin act as agonists at TRPM8 and TRPA1. In vitro, activation of TRPM8 by icilin and cold, but not menthol, is dependent on the activity of a sub-type of phospholipase A2, iPLA2. Lysophospholipids (e.g. LPC) produced by PLA2 activity can also activate TRPM8. The role of TRPA1 as a primary cold sensor in vitro is controversial, although there is evidence that TRPA1 plays a role in behavioural responses to noxious cold stimuli. In this study, we have investigated the roles of TRPM8 and TRPA1 and the influence of iPLA2 on noxious cold sensitivities in naïve animals and after local administration of menthol, icilin and LPC. The roles of the channels in cold sensitivity were investigated in mice lacking either TRPM8 ( Trpm8^−/−) or TRPA1 ( Trpa1^−/−).

Intraplantar administration of icilin evoked a dose-dependent increase in sensitivity to a 10°C stimulus that was inhibited by iPLA2 inhibition with BEL. In contrast the cold hypersensitivities elicited by intraplantar menthol and LPC were not inhibited by BEL treatment. BEL had no effect on basal cold sensitivity and mechanical hypersensitivities induced by the TRPV1 agonist, capsaicin, and the P2X3 agonist α,β-methylene ATP. Both Trpm8^−/− and Trpa1^−/− mice showed longer latencies for paw withdrawal from a 10°C stimulus than wild-type littermates. Cold hypersensitivities induced by either icilin or LPC were absent in Trpm8^−/− mice but were retained in Trpa1^−/− mice. In contrast, cold hypersensitivity evoked by menthol was present in Trpm8^−/− mice but was lost in Trpa1^−/− mice.

The findings that iPLA2 inhibition blocked the development of cold hypersensitivity after administration of icilin but failed to affect menthol-induced hypersensitivity agree well with our earlier in vitro data showing a differential effect of iPLA2 inhibition on the agonist activities of these agents. The ability of LPC to induce cold hypersensitivity supports a role for iPLA2 in modulating TRPM8 activity in vivo. Studies on genetically modified mice demonstrated that the effects of icilin and LPC were mediated by TRPM8 and not TRPA1. In contrast, menthol-induced cold hypersensitivity was dependent on expression of TRPA1 and not TRPM8.

Paroxysmal extreme pain disorder (PEPD) is an autosomal dominant painful neuropathy with many, but not all, cases linked to gain-of-function mutations in SCN9A which encodes voltage-gated sodium channel Na_v1.7. Severe pain episodes and skin flushing start in infancy and are induced by perianal probing or bowl movement, and pain progresses to ocular and mandibular areas with age. Carbamazepine has been effective in relieving symptoms, while other drugs including other anti-epileptics are less effective.

Sequencing of SCN9A coding exons from an English patient, diagnosed with PEPD, has identified a methionine 1627 to lysine (M1627K) substitution in the linker joining segments S4 and S5 in domain IV. We confirm that M1627K depolarizes the voltage-dependence of fast-inactivation without substantially altering activation or slow-inactivation, and inactivates from the open state with slower kinetics. We show here that M1627K does not alter development of closed-state inactivation, and that M1627K channels recover from fast-inactivation faster than wild type channels, and produce larger currents in response to a slow ramp stimulus. Using current-clamp recordings, we also show that the M1627K mutant channel reduces the threshold for single action potentials in DRG neurons and increases the number of action potentials in response to graded stimuli.

M1627K mutation was previously identified in a sporadic case of PEPD from France, and we now report it in an English family. We confirm the initial characterization of mutant M1627K effect on fast-inactivation of Na_v1.7 and extend the analysis to other gating properties of the channel. We also show that M1627K mutant channels render DRG neurons hyperexcitable. Our new data provide a link between altered channel biophysics and pain in PEPD patients.