Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury

Background: Peripheral nerve inflammation or lesion can affect contralateral healthy structures, and thus results in mirror-image pain. Supraspinal structures play important roles in the occurrence of mirror pain. The anterior cingulate cortex (ACC) is a first order cortical region that responds to painful stimuli. In the present study, we systematically investigate and compare the neuroimmune changes in the bilateral ACC region using unilateral- (spared nerve injury, SNI) and mirror-(L5 ventral root transection, L5-VRT) pain models, aiming to explore the potential supraspinal neuroimmune mechanism underlying the mirror-image pain. Methods: The up-and-down method with von Frey hairs was used to measure the mechanical allodynia. Viral injections for the designer receptors exclusively activated by designer drugs (DREADD) were used to modulate ACC pyramidal neurons. Immunohistochemistry, immunofluorescence, western blotting, protein microarray were used to detect the regulation of inflammatory signaling.Results: Increased expressions of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and chemokine CX3CL1 in ACC induced by unilateral nerve injury were observed on the contralateral side in the SNI group but on the bilateral side in the L5-VRT group, representing a stronger immune response to L5-VRT surgery. In remote ACC, both SNI and L5-VRT induced robust bilateral increase in the protein level of Nav1.6 (SCN8A), a major voltage-gated sodium channel (VGSC) that regulates neuronal activity in the mammalian nervous system. However, the L5-VRT-induced Nav1.6 response occurred at PO 3d, earlier than the SNI-induced one, 7 days after surgery. Modulating ACC pyramidal neurons via DREADD-Gq or DREADD-Gi greatly changed the ACC CX3CL1 levels and the mechanical paw withdrawal threshold. Neutralization of endogenous ACC CX3CL1 by contralateral anti-CX3CL1 antibody attenuated the induction and the maintenance of mechanical allodynia and eliminated the upregulation of CX3CL1, TNF-α and Nav1.6 protein levels in ACC induced by SNI. Furthermore, contralateral ACC anti-CX3CL1 also inhibited the expression of ipsilateral spinal c-Fos, Iba1, CD11b, TNF-α and IL-6. Conclusions: The descending facilitation function mediated by CX3CL1 and its downstream cascade may play a pivotal role, leading to enhanced pain sensitization and even mirror-image pain. Strategies that target chemokine-mediated ACC hyperexcitability may lead to novel therapies for the treatment of neuropathic pain.

Spinal cord homeostatic plasticity gates mechanical allodynia in chronic pain

Central sensitization caused by disinhibition of spinal cord circuits is a key mechanism of mechanical allodynia in neuropathic pain. Despite intense efforts, the molecular mechanisms that drive disinhibition and induce allodynia after peripheral nerve injury remain unclear. Using the spared-nerve injury (SNI) model of allodynia, we here demonstrate that SNI induces disinhibition of spinal nociceptive circuits by triggering homeostatic synaptic plasticity. Specifically, SNI-triggered homeostatic plasticity suppresses the inhibitory outputs of parvalbumin-positive (PV+) interneurons that form synapses on both primary afferent terminals and excitatory interneurons, causing hyperactivation of the nociceptive pathway. Using genetic manipulations, we identified the retinoic acid receptor RARα as the key mediator of the homeostatic synaptic plasticity underlying this synaptic disinhibition. Deletion of RARα in PV+ neurons blocked SNI-induced spinal disinhibition, central sensitization, and allodynia. Moreover, deletion of RARα in spinal PV+ neurons or application of an RARα antagonist in the spinal cord prevented the development of SNI-induced mechanical allodynia. Together, our results reveal a molecular mechanism of neuropathic pain whereby homeostatic plasticity causes the mis-direction of tactile information flow to ascending nociceptive pathways following peripheral nerve injury.

TNF-α-Mediated RIPK1 Pathway Participates in the Development of Trigeminal Neuropathic Pain in Rats

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) participates in the regulation of cellular stress and inflammatory responses, but its function in neuropathic pain remains poorly understood. This study evaluated the role of RIPK1 in neuropathic pain following inferior alveolar nerve injury. We developed a model using malpositioned dental implants in male Sprague Dawley rats. This model resulted in significant mechanical allodynia and upregulated RIPK1 expression in the trigeminal subnucleus caudalis (TSC). The intracisternal administration of Necrosatin-1 (Nec-1), an RIPK1 inhibitor, blocked the mechanical allodynia produced by inferior alveolar nerve injury The intracisternal administration of recombinant rat tumor necrosis factor-α (rrTNF-α) protein in naive rats produced mechanical allodynia and upregulated RIPK1 expression in the TSC. Moreover, an intracisternal pretreatment with Nec-1 inhibited the mechanical allodynia produced by rrTNF-α protein. Nerve injury caused elevated TNF-α concentration in the TSC and a TNF-α block had anti-allodynic effects, thereby attenuating RIPK1 expression in the TSC. Finally, double immunofluorescence analyses revealed the colocalization of TNF receptor and RIPK1 with astrocytes. Hence, we have identified that astroglial RIPK1, activated by the TNF-α pathway, is a central driver of neuropathic pain and that the TNF-α-mediated RIPK1 pathway is a potential therapeutic target for reducing neuropathic pain following nerve injury.

The Role of Prostaglandin E1 as a Pain Mediator through Facilitation of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 via the EP2 Receptor in Trigeminal Ganglion Neurons of Mice

Cyclooxygenase metabolizes dihomo-γ-linolenic acid and arachidonic acid to form prostaglandin (PG) E, including PGE1 and PGE2, respectively. Although PGE2 is well known to play an important role in the development and maintenance of hyperalgesia and allodynia, the role of PGE1 in pain is unknown. We confirm whether PGE1 induced pain using orofacial pain behavioral test in mice and determine the target molecule of PGE1 in TG neurons with whole-cell patch-clamp and immunohistochemistry. Intradermal injection of PGE1 to the whisker pads of mice induced a reduced threshold, enhancing the excitability of HCN channel-expressing trigeminal ganglion (TG) neurons. The HCN channel-generated inward current (Ih) was increased by 135.3 ± 4.8% at 100 nM of PGE1 in small- or medium-sized TG, and the action of PGE1 on Ih showed a concentration-dependent effect, with a median effective dose (ED50) of 29.3 nM. Adenylyl cyclase inhibitor (MDL12330A), 8-bromo-cAMP, and the EP2 receptor antagonist AH6809 inhibited PGE1-induced Ih. Additionally, PGE1-induced mechanical allodynia was blocked by CsCl and AH6809. PGE1 plays a role in mechanical allodynia through HCN2 channel facilitation via the EP2 receptor in nociceptive neurons, suggesting a potential therapeutic target in that PGE1 could be involved in pain as endogenous substances under inflammatory conditions.

Investigating the antinociceptive effects of N-docosahexaenoyl ethanolamine and novel kappa opioid receptor agonists

<p>Chronic pain causes patients to endure prolonged suffering and discomfort, often having profound effects on quality of life. In New Zealand, one in five people currently suffer from chronic pain. To treat chronic pain, patients are typically prescribed drugs that activate the mu opioid receptor (MOPr), such as morphine, codeine and oxycodone. In recent years in the United States of America, there has been a rapid increase in the use of prescription and non-prescription opioid drugs, with opioid overdoses now the leading cause of accidental death. In New Zealand, daily doses of prescription opioids quadrupled in the ten year period from 2001-2011. Clearly, there is a need for the development of more effective and safe medications. This thesis evaluated two classes of non-addictive compounds: bioactive lipids and kappa opioid receptor (KOPr) agonists. N-docosahexaenoyl ethanolamine (DHEA) is an N-acyl ethanolamine class lipid that is structurally similar to the endocannabinoid anandamide. DHEA has previously been shown to have immune-modulatory effects in vitro, however, the in vivo effects have not previously been tested. Using the intraplantar 2% formaldehyde model in mice, DHEA reduced inflammatory and nociceptive pain via both intraperitoneal (i.p.) and local intraplantar (i.pl.) administration. DHEA significantly reduced formaldehyde-induced footpad oedema and reduced the infiltration of neutrophils into the inflamed tissue. The antinociceptive and anti-oedematous effects were not modulated by pre-treatment with either cannabinoid 1- or 2-type receptor antagonists. DHEA did not have any effect in a thermal nociceptive pain model and did not show any motor coordination impairment or changes in thermoregulation. In the search for non-addictive analgesics, KOPr agonists are a promising alternative. In contrast to MOPr agonists, KOPr agonists play a critical role in regulating the reward system. Salvinorin A (SalA) is a selective KOPr agonist that has antinociceptive and anti-inflammatory effects in vivo, with limited abuse potential. However, the short duration of action and aversive side effects limit the clinical usefulness. The present study aimed to investigate the antinociceptive effects of acute administration of novel analogues of SalA. In the dose-response tail withdrawal assay, SalA and the novel analogues 16-Ethynyl SalA and 16-Bromo SalA were more potent than the traditional KOPr agonist U50,488, and 16-Ethynyl SalA was more efficacious. 16-Ethynyl SalA and 16-Bromo SalA both had a longer duration of action in the warm water tail withdrawal assay and the hot plate test compared to SalA. In the intraplantar 2% formaldehyde test, SalA, 16-Ethynyl SalA and 16-Bromo SalA significantly reduced nociceptive pain and inflammatory pain, effects which were reversed by the KOPr antagonist nor-binaltorphimine. SalA, 16-Ethynyl SalA and 16-Bromo SalA reduced paw oedema and reduced the infiltration of neutrophils into the inflamed tissue. However, SalA, 16-Ethynyl SalA and 16-Bromo SalA produced motor incoordination effects. However, 16-Ethynyl SalA did not alter thermoregulation. The KOPr agonists were further assessed in a model of paclitaxel-induced neuropathic pain. In the acute dose-response experiment, 16-Ethynyl SalA was significantly more potent at reducing mechanical allodynia compared to morphine in both male and female mice. SalA and 16-Ethynyl SalA were more potent at reducing cold allodynia than morphine. In a chronic administration model over 22 days, for the treatment of cold and mechanical allodynia, all of the opioid treatments reduced pain, however, the traditional KOPr agonist U50,488, was the most potent, by reducing the male mechanical allodynia and cold allodynia in both sexes back to baseline levels. The ultrastructure of the sciatic nerves were studied, however, it was found that U50,488 did not reverse the effects of paclitaxel on myelin degeneration and mitochondrial damage. Overall, this study has identified DHEA as a modest treatment for inflammatory pain, with reduced side effects and a mechanism of action in contrast to other compounds with a similar structure. The novel KOPr agonists had significant effects in acute pain models with longer duration of action than the parent compound SalA. This is the first known study to investigate the effects of KOPr agonists in a paclitaxel-induced neuropathic pain model, showing that KOPr agonists are a potential therapeutic avenue for this debilitating condition.</p>

Investigating the antinociceptive effects of N-docosahexaenoyl ethanolamine and novel kappa opioid receptor agonists

<p>Chronic pain causes patients to endure prolonged suffering and discomfort, often having profound effects on quality of life. In New Zealand, one in five people currently suffer from chronic pain. To treat chronic pain, patients are typically prescribed drugs that activate the mu opioid receptor (MOPr), such as morphine, codeine and oxycodone. In recent years in the United States of America, there has been a rapid increase in the use of prescription and non-prescription opioid drugs, with opioid overdoses now the leading cause of accidental death. In New Zealand, daily doses of prescription opioids quadrupled in the ten year period from 2001-2011. Clearly, there is a need for the development of more effective and safe medications. This thesis evaluated two classes of non-addictive compounds: bioactive lipids and kappa opioid receptor (KOPr) agonists. N-docosahexaenoyl ethanolamine (DHEA) is an N-acyl ethanolamine class lipid that is structurally similar to the endocannabinoid anandamide. DHEA has previously been shown to have immune-modulatory effects in vitro, however, the in vivo effects have not previously been tested. Using the intraplantar 2% formaldehyde model in mice, DHEA reduced inflammatory and nociceptive pain via both intraperitoneal (i.p.) and local intraplantar (i.pl.) administration. DHEA significantly reduced formaldehyde-induced footpad oedema and reduced the infiltration of neutrophils into the inflamed tissue. The antinociceptive and anti-oedematous effects were not modulated by pre-treatment with either cannabinoid 1- or 2-type receptor antagonists. DHEA did not have any effect in a thermal nociceptive pain model and did not show any motor coordination impairment or changes in thermoregulation. In the search for non-addictive analgesics, KOPr agonists are a promising alternative. In contrast to MOPr agonists, KOPr agonists play a critical role in regulating the reward system. Salvinorin A (SalA) is a selective KOPr agonist that has antinociceptive and anti-inflammatory effects in vivo, with limited abuse potential. However, the short duration of action and aversive side effects limit the clinical usefulness. The present study aimed to investigate the antinociceptive effects of acute administration of novel analogues of SalA. In the dose-response tail withdrawal assay, SalA and the novel analogues 16-Ethynyl SalA and 16-Bromo SalA were more potent than the traditional KOPr agonist U50,488, and 16-Ethynyl SalA was more efficacious. 16-Ethynyl SalA and 16-Bromo SalA both had a longer duration of action in the warm water tail withdrawal assay and the hot plate test compared to SalA. In the intraplantar 2% formaldehyde test, SalA, 16-Ethynyl SalA and 16-Bromo SalA significantly reduced nociceptive pain and inflammatory pain, effects which were reversed by the KOPr antagonist nor-binaltorphimine. SalA, 16-Ethynyl SalA and 16-Bromo SalA reduced paw oedema and reduced the infiltration of neutrophils into the inflamed tissue. However, SalA, 16-Ethynyl SalA and 16-Bromo SalA produced motor incoordination effects. However, 16-Ethynyl SalA did not alter thermoregulation. The KOPr agonists were further assessed in a model of paclitaxel-induced neuropathic pain. In the acute dose-response experiment, 16-Ethynyl SalA was significantly more potent at reducing mechanical allodynia compared to morphine in both male and female mice. SalA and 16-Ethynyl SalA were more potent at reducing cold allodynia than morphine. In a chronic administration model over 22 days, for the treatment of cold and mechanical allodynia, all of the opioid treatments reduced pain, however, the traditional KOPr agonist U50,488, was the most potent, by reducing the male mechanical allodynia and cold allodynia in both sexes back to baseline levels. The ultrastructure of the sciatic nerves were studied, however, it was found that U50,488 did not reverse the effects of paclitaxel on myelin degeneration and mitochondrial damage. Overall, this study has identified DHEA as a modest treatment for inflammatory pain, with reduced side effects and a mechanism of action in contrast to other compounds with a similar structure. The novel KOPr agonists had significant effects in acute pain models with longer duration of action than the parent compound SalA. This is the first known study to investigate the effects of KOPr agonists in a paclitaxel-induced neuropathic pain model, showing that KOPr agonists are a potential therapeutic avenue for this debilitating condition.</p>

Opening KATP Channels Induces Inflammatory Tolerance and Prevents Chronic Pain

Background: Current treatments for chronic pain are not satisfactory, prompting a frantic search for new therapeutics and new therapeutic targets. Our previous study indicates KATP channel opener has analgesic effect, but the mechanism has not been elucidated. We speculated that KATP channel opener may increase suppressor of cytokine signaling (SOCS)-3 expression to induce inflammatory tolerance and attenuate chronic pain. Methods: The plantar incision (PI) surgery-induced postoperative pain was performed to establish chronic pain model. Growth arrest–specific 6 (Gas6)-/- and Axl-/- mice were used for signaling research. The microglia cell line BV-2 was cultured for in vitro experiments.Results: KATP channel opener significantly attenuated incision-induced mechanical allodynia in mice, associated with the up-regulated expression of SOCS3. Opening KATP channels induced the expression of SOCS3 dependent on Gas6/Axl signaling pathway in microglia. Opening KATP channels inhibits incision-induced mechanical allodynia by activating Gas6/Axl-SOCS3 signaling pathway. Opening KATP channels induces inflammatory tolerance to relieve neuroinflammation and postoperative pain.Conclusions: We demonstrated that KATP channel opening activated Gas6/Axl/SOCS3 signaling to induce inflammatory tolerance and relief chronic pain. We explored a new target for anti-inflammatory and analgesia by regulating the innate immune system, and provide a theoretical basis for clinical preemptive analgesia.

The Up-regulation of TNF-α Maintains Trigeminal Neuralgia by Modulating MAPKs Phosphorylation and BKCa Channels in Trigeminal Nucleus Caudalis

Trigeminal neuralgia (TN) is a severe chronic neuropathic pain. Despite numerous available medical interventions, the therapeutic effects are not ideal. To control the pain attacks, the need for more contemporary drugs continues to be a real challenge. Our previous study reported that Ca2+-activated K+ channels (BKCa) channels modulated by mitogen-activated protein kinases (MAPKs) in the trigeminal ganglia (TG) neurons play crucial roles in regulating TN, and some research studies demonstrated that inflammatory cytokine tumor necrosis factor alpha (TNF-α) could promote neuropathic pain. Meanwhile, the trigeminal nucleus caudalis (TNC), the first central site of the trigeminal nociceptive pathway, is responsible for processing sensory and pain signals from the peripheral orofacial area. Thus, this study is aimed to further investigate whether TNF-α and MAPKs phosphorylation in the TNC could mediate the pathogenesis of TN by modulating BKCa channels. The results showed that TNF-α of the TNC region is upregulated significantly in the chronic constriction injury of infraorbital nerve (ION-CCI) rats model, which displayed persistent facial mechanical allodynia. The normal rats with target injection of exogenous TNF-α to the fourth brain ventricle behaved just like the ION-CCI model rats, the orofacial mechanical pain threshold decreased clearly. Meanwhile, the exogenous TNF-α increased the action potential frequency and reduced the BKCa currents of TNC neurons significantly, which could be reversed by U0126 and SB203580, the inhibitors of MAPK. In addition, U0126, SB203580, and another MAPK inhibitor SP600125 could relieve the facial mechanical allodynia by being injected into the fourth brain ventricle of ION-CCI model rats, respectively. Taken together, our work suggests that the upregulation of TNF-α in the TNC region would cause the increase of MAPKs phosphorylation and then the negative regulation of BKCa channels, resulting in the TN.