Perineural Treatment with High Mobility Group Box-1 Monoclonal Antibody Prevents Initiation of Pain-Like Behaviors in Female Mice with Trigeminal Neuropathy.

Post-traumatic trigeminal neuropathy (PTTN) is a type of chronic pain caused by damage to the trigeminal nerve. A previous study reported that pretreatment with anti-high mobility group box-1 (HMGB1) neutralizing antibodies (nAb) prevented the onset of PTTN following distal infraorbital nerve chronic constriction injury (dIoN-CCI) in male mice. Clinical evidence indicates a high incidence of PTTN in females. Although our previous study found that perineural HMGB1 is crucial in initiation of PTTN in male mice, it is currently unknown whether HMGB1 is also involved in the pathogenesis of PTTN in female mice. Therefore, in the current study, we examined the effect of anti-HMGB1 nAb on pain-like behavior in female mice following dIoN-CCI surgery. We found that dIoN-CCI surgery enhanced reactivity to mechanical and cold stimuli in female mice, which was suppressed by treatment with anti-HMGB1 nAb. Moreover, the increase in macrophages after dIoN-CCI was significantly attenuated by pretreatment with anti-HMGB1 nAb. Furthermore, anti-HMGB1 nAb treatment inhibited microglial activation in the trigeminal spinal tract nucleus. These data suggest that HMGB1 also plays a crucial role in the onset of PTTN after nerve injury in female mice. Thus, anti-HMGB1 nAb could be a novel therapeutic agent for inhibiting the onset of PTTN in female and male mice.

Evaluation of intracellular signal molecules that regulate TLR4-stimulated inflammatory mediator expression in cultured rat chondrocytes.

Osteoarthritis (OA) is characterized by inflammation of joints and degradation of articular cartilage matrix. As involvement of damage-associated molecular patterns (DAMPs) in the pathogenesis of OA has been reported, the present study comprehensively investigated the regulation of inflammatory mediator expression in chondrocytes mediated by Toll-like receptor 4 (TLR4), a receptor for DAMPs. Treatment of cultured rat chondrocytes with lipopolysaccharide (LPS) induced the mRNA expression of proinflammatory cytokines (interleukin [IL]-1ß, IL-6, tumor necrosis factor [TNF]), matrix degradation enzymes (metalloproteinase [MMP] 3, MMP13), and inducible nitric oxide synthase (iNOS) through TLR4. Transforming growth factor ß-activated kinase-1 (TAK1) and nuclear factor-κB (NF-κB) were crucial for the upregulated expression of these inflammatory mediators. The induction of IL-1ß and TNF was regulated by extracellular signal-regulated kinase (ERK), while the induction of IL-6 was mediated by Tank-binding kinase 1 (TBK1) and c-Jun N-terminal kinase (JNK). The induction of MMP3 and MMP13 was regulated by TBK1, ERK, and JNK, while the induction of iNOS was mediated by ERK and JNK. In summary, some of the regulatory mechanisms underlying the expression of key inflammatory mediators for OA pathogenesis have been demonstrated. Further clarification may allow these signaling molecules to become new therapeutic targets for OA treatment strategies.

Downregulation of connexin43 potentiates noradrenaline-induced expression of brain-derived neurotrophic factor in primary cultured cortical astrocytes.

Decreased expression of brain-derived neurotrophic factor (BDNF) is involved in the pathology of depressive disorders. Astrocytes produce BDNF following antidepressant treatment or stimulation of adrenergic receptors. Connexin43 (Cx43) is mainly expressed in central nervous system astrocytes and its expression is downregulated in patients with major depression. How changes in Cx43 expression affect astrocyte function, including BDNF production, is poorly understood. The current study examined the effect of Cx43 knockdown on BDNF expression in cultured cortical astrocytes after stimulation of adrenergic receptors. The expression of Cx43 in rat primary cultured cortical astrocytes was downregulated with RNA interference. Levels of messenger RNAs (mRNAs) or proteins were measured by real-time PCR and western blotting, respectively. Knockdown of Cx43 potentiated noradrenaline (NA)-induced expression of BDNF mRNA in cultured astrocytes. NA treatment induced proBDNF protein expression in astrocytes transfected with small interfering RNA (siRNA) targeting Cx43, but not with control siRNA. This potentiation was mediated by the Src tyrosine kinase-extracellular signal-regulated kinase (ERK) pathway through stimulation of adrenergic α1 and ß receptors. Furthermore, the Gq/11 protein-Src-ERK pathway and the G-protein coupled receptor kinase 2-Src-ERK pathway were involved in α1 and ß adrenergic receptor-mediated potentiation of BDNF mRNA expression, respectively. The current studies demonstrate a novel mechanism of BDNF expression in cortical astrocytes mediated by Cx43, in which downregulation of Cx43 increases, through adrenergic receptors, the expression of BDNF. The current findings indicate a potentially novel mechanism of action of antidepressants, via regulation of astrocytic Cx43 expression and subsequent BDNF expression.

Lysophosphatidic acid induces thrombospondin-1 production in primary cultured rat cortical astrocytes.

Lysophosphatidic acid (LPA), a brain membrane-derived lipid mediator, plays important roles including neural development, function, and behavior. In the present study, the effects of LPA on astrocyte-derived synaptogenesis factor thrombospondins (TSPs) production were examined by real-time PCR and western blotting, and the mechanism underlying this event was examined by pharmacological approaches in primary cultured rat cortical astrocytes. Treatment of astrocytes with LPA increased TSP-1 mRNA, and TSP-2 mRNA, but not TSP-4 mRNA expression. TSP-1 protein expression and release were also increased by LPA. LPA-induced TSP-1 production were inhibited by AM966 a LPA1 receptor antagonist, and Ki16425, LPA1/3 receptors antagonist, but not by H2L5146303, LPA2 receptor antagonist. Pertussis toxin, Gi/o inhibitor, but not YM-254890, Gq inhibitor, and NF499, Gs inhibitor, inhibited LPA-induced TSP-1 production, indicating that LPA increases TSP-1 production through Gi/o-coupled LPA1 and LPA3 receptors. LPA treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). LPA-induced TSP-1 mRNA expression was inhibited by U0126, MAPK/ERK kinase (MEK) inhibitor, but not SB202190, p38 MAPK inhibitor, or SP600125, JNK inhibitor. However, LPA-induced TSP-1 protein expression was diminished with inhibition of all three MAPKs, indicating that these signaling molecules are involved in TSP-1 protein production. Treatment with antidepressants, which bind to astrocytic LPA1 receptors, increased TSP-1 mRNA and protein production. The current findings show that LPA/LPA1/3 receptors signaling increases TSP-1 production in astrocytes, which could be important in the pathogenesis of affective disorders and could potentially be a target for the treatment of affective disorders.

Decreased connexin43 expression in the hippocampus is related to the antidepressant effect of amitriptyline in neuropathic pain mice.

Downregulation of astrocytic connexin43 (Cx43) has been observed in several brain regions in rodents and patients with depression. However, its specific role in this effect remains unknown. Moreover, chronic pain can induce depressive disorders. Therefore, the current study examined the relationship between Cx43 expression and depressive-like behavior in a neuropathic pain model. Neuropathic pain was induced by spared nerve injury (SNI) in mice. Depressive-like behavior was evaluated using the forced swim test. Expression of Cx43 in the hippocampus was evaluated using Western blotting and real-time PCR. SNI downregulated Cx43 protein in the contralateral hippocampus of mice, whereas expression of hippocampal Cx43 mRNA was not altered following SNI. Although SNI mice showed longer immobility time compared with sham mice during the forced swim test, duration of depressive-like behavior was not correlated with the expression of Cx43 in the hippocampus of SNI mice. Repeated intraperitoneal administration of amitriptyline ameliorated SNI-induced depressive-like behavior. Furthermore, the antidepressant effect of amitriptyline was correlated with decreased hippocampal Cx43 expression in SNI mice. The current findings suggest that the alteration of Cx43 expression in the hippocampus may not be involved in the induction of depressive disorder but may influence the efficacy of antidepressants. Therefore, the level of Cx43 expression in the hippocampus could be a key parameter to evaluate individual differences in antidepressant effects in patients with depressive disorder.

Treatment with Histone Deacetylase Inhibitor Attenuates Peripheral Inflammation-Induced Cognitive Dysfunction and Microglial Activation: The Effect of SAHA as a Peripheral HDAC Inhibitor.

It has been demonstrated that peripheral inflammation induces cognitive dysfunction. Several histone deacetylase (HDAC) inhibitors ameliorate cognitive dysfunction in animal models of not only peripheral inflammation but also Alzheimer's disease. However, it is not clear which HDAC expressed in the central nervous system or peripheral tissues is involved in the therapeutic effect of HDAC inhibition on cognitive dysfunction. Hence, the present study investigated the effect of peripheral HDAC inhibition on peripheral inflammation-induced cognitive dysfunction. Suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor that is mainly distributed in peripheral tissues after intraperitoneal administration, was found to prevent peripheral inflammation-induced cognitive dysfunction. Moreover, pretreatment with SAHA dramatically increased mRNA expression of interleukin-10, an anti-inflammatory cytokine, in peripheral and central tissues and attenuated peripheral inflammation-induced microglial activation in the CA3 region of the hippocampus. Minocycline, a macrophage/microglia inhibitor, also ameliorated cognitive dysfunction. Furthermore, as a result of treatment with liposomal clodronate, depletion of peripheral macrophages partially ameliorated the peripheral inflammation-evoked cognitive dysfunction. Taken together, these findings demonstrate that inhibition of peripheral HDAC plays a critical role in preventing cognitive dysfunction induced by peripheral inflammation via the regulation of anti-inflammatory cytokine production and the inhibition of microglial functions in the hippocampus. Thus, these findings could provide support for inhibition of peripheral HDAC as a novel therapeutic strategy for inflammation-induced cognitive dysfunction.

Pretreatment with High Mobility Group Box-1 Monoclonal Antibody Prevents the Onset of Trigeminal Neuropathy in Mice with a Distal Infraorbital Nerve Chronic Constriction Injury.

Persistent pain following orofacial surgery is not uncommon. High mobility group box 1 (HMGB1), an alarmin, is released by peripheral immune cells following nerve injury and could be related to pain associated with trigeminal nerve injury. Distal infraorbital nerve chronic constriction injury (dIoN-CCI) evokes pain-related behaviors including increased facial grooming and hyper-responsiveness to acetone (cutaneous cooling) after dIoN-CCI surgery in mice. In addition, dIoN-CCI mice developed conditioned place preference to mirogabalin, suggesting increased neuropathic pain-related aversion. Treatment of the infraorbital nerve with neutralizing antibody HMGB1 (anti-HMGB1 nAb) before dIoN-CCI prevented both facial grooming and hyper-responsiveness to cooling. Pretreatment with anti-HMGB1 nAb also blocked immune cell activation associated with trigeminal nerve injury including the accumulation of macrophage around the injured IoN and increased microglia activation in the ipsilateral spinal trigeminal nucleus caudalis. The current findings demonstrated that blocking of HMGB1 prior to nerve injury prevents the onset of pain-related behaviors, possibly through blocking the activation of immune cells associated with the nerve injury, both within the CNS and on peripheral nerves. The current findings further suggest that blocking HMGB1 before tissue injury could be a novel strategy to prevent the induction of chronic pain following orofacial surgeries.

Continuous infusion of substance P inhibits acute, but not subacute, inflammatory pain induced by complete Freund's adjuvant.

Previous studies have reported that continuous infusion with substance P (SP) into rat dorsal striatum ameliorated both mechanical allodynia in both formalin-evoked transient inflammatory pain and neuropathic pain models. However, a role of striatal SP in persistent inflammatory pain has not been demonstrated. The current study examined the effect of continuous infusion of SP into the rat dorsal striatum by reverse microdialysis on persistent inflammatory pain induced by complete Freund's adjuvant (CFA). Intraplantar injection of CFA evoked both mechanical allodynia and paw edema 3 and 7 days post-injection. The continuous infusion of SP ameliorated the CFA-evoked mechanical allodynia, but not paw edema, 3 days after the CFA injection. This antinociceptive effect of SP was partially inhibited by co-infusion with the neurokinin-1 (NK1) receptor antagonist CP96345. Conversely, at 7 days both CFA-evoked mechanical allodynia and paw edema were not affected by SP treatment. To clarify why the effect of SP treatment on CFA-induced pain changed, we evaluated NK1 receptor protein levels at both time points. The NK1 receptor protein level was decreased at 7, but not 3, days post CFA injection. These data suggest that persistent inflammatory pain can downregulate the striatal NK1 receptor. The current study demonstrates that striatal SP-NK1 receptor pathway can exert antinociceptive effect only on the third days of inflammatory pain phase defined as an acute but not the 7 days defined as a subacute.

TLR4-TAK1-p38 MAPK pathway and HDAC6 regulate the expression of sigma-1 receptors in rat primary cultured microglia.

Microglia maintain brain homeostasis as the main immune cells in the central nervous system. Activation of sigma-1 receptor (Sig1R) plays neuroprotective and anti-inflammatory roles in microglia. Recent studies showed that Sig1R expression level has been reduced in the brain of the patients with neurodegenerative diseases including Alzheimer's disease. However, the mechanisms underlying the down regulation of the Sig1R has not been clear. Treatment of rat primary cultured microglia with the inflammogen lipopolysaccharide (LPS) significantly decreased the expression of Sig1R mRNA in a concentration and time-dependent manner. The effects of LPS were blocked by pretreatment with TAK-242, a toll-like receptor 4 (TLR4) antagonist. Furthermore, inhibitors of transforming growth factor beta-activated kinase 1 (TAK1), p38 mitogen-activated protein kinase (MAPK) and histone deacetylase 6 (HDAC6) restored the LPS-induced downregulation of Sig1R. Thus, the current findings demonstrate that TLR4 activation leads to the downregulation of the Sig1R expression via TLR4-TAK1-p38 MAPK pathway and the inhibition of HDAC6 can increase Sig1R expression in microglia. The current findings suggest that downregulation of Sig1R may contribute to neuroinflammation-induced microglial dysfunction, regulation of microglial Sig1R may be novel therapeutic drug candidates for neurodegenerative and neuroinflammatory diseases.

Spinal high-mobility group box-1 induces long-lasting mechanical hypersensitivity through the toll-like receptor 4 and upregulation of interleukin-1ß in activated astrocytes.

Intrathecal treatment with recombinant high-mobility group box-1 (rHMGB1) in naïve mice leads to a persistent and significantly decreased hind paw withdrawal threshold to mechanical stimuli, suggesting that spinal HMGB1 evokes abnormal pain processing. By contrast, repeated intrathecal treatment with anti-HMGB1 antibody significantly reverses hind paw mechano-hypersensitivity in mice with a partial sciatic nerve ligation (PSNL). By contrast, the cellular mechanism by which spinal HMGB1 induces neuropathic pain has yet to be fully elaborated. The current study tested the hypothesis that spinal HMGB1 could induce mechanical hypersensitivity through the activation of specific receptor in glial cells. Intrathecal pretreatment with toll-like receptor (TLR) 4 inhibitors, but not TLR5, receptor for advanced glycation end-products and C-X-C chemokine receptor type 4 inhibitors, prevented rHMGB1-evoked mechanical hypersensitivity. Activation of spinal astrocytes appears to be crucial for the mechanism of action of rHMGB1 in naïve mice, as intrathecal pretreatment with astrocytic inhibitors prevented the rHMGB1-induced mechanical hypersensitivity. Interleukin-1ß (IL-1ß) was up-regulated within activated astrocytes and block of TLR4 prevented the upregulation of IL-1ß. Interleukin-1ß appears to be secreted by activated astrocytes, as IL-1ß neutralizing antibody prevented rHMGB1-induced mechanical hypersensitivity. Furthermore, intrathecal pretreatment with either MK801 or gabapentin prevented the rHMGB1-induced mechanical hypersensitivity, suggesting roles for spinal glutamate and the N-methyl-d-aspartate receptor in the mediation of rHMGB1-induced mechanical hypersensitivity. Thus, the current findings suggest that spinal HMGB1 upregulates IL-1ß in spinal astrocytes through a TLR4-dependent pathway and increases glutamatergic nociceptive transduction. These spinal mechanisms could be key steps that maintain neuropathic pain.

Stimulation of nuclear receptor REV-ERBs suppresses production of pronociceptive molecules in cultured spinal astrocytes and ameliorates mechanical hypersensitivity of inflammatory and neuropathic pain of mice.

The orphan nuclear receptors REV-ERBα and REV-ERBß (REV-ERBs) are crucial in the regulation of inflammatory-related gene transcription in astroglioma cells, but their role in nociceptive transduction has yet to be elaborated. Spinal dorsal horn astrocytes contribute to the maintenance of chronic pain. Treatment of cultured spinal astrocytes with specific REV-ERBs agonists SR9009 or GSK4112 significantly prevented lipopolysaccharide (LPS)-induced mRNA upregulation of pronociceptive molecules interleukin-1ß (IL-1ß) mRNA, interleukin-6 (IL-6) mRNA and matrix metalloprotease-9 (MMP-9) mRNA, but not CCL2 mRNA expression. Treatment with SR9009 also blocked tumor necrosis factor-induced IL-1ß mRNA, IL-6 mRNA and MMP-9 mRNA. In addition, treatment with SR9009 significantly blocked LPS-induced upregulation of IL-1ß protein, IL-6 protein and MMP-9 activity. The inhibitory effects of SR9009 on LPS-induced expression of pronociceptive molecules were blocked by knockdown of REV-ERBs expression with short interference RNA, confirming that SR9009 exerts its effect through REV-ERBs. Intrathecal LPS treatment in male mice induces hind paw mechanical hypersensitivity, and upregulation of IL-1ß mRNA, IL-6 mRNA and glial fibrillary acidic protein (GFAP) expression in spinal dorsal horn. Intrathecal pretreatment of SR9009 prevented the onset of LPS-induced mechanical hypersensitivity, cytokine expression and GFAP expression. Intrathecal injection of SR9009 also ameliorated mechanical hypersensitivity during the maintenance phase of complete Freund's adjuvant-induced inflammatory pain and partial sciatic nerve ligation-, paclitaxel-, and streptozotocin-induced neuropathy in mice. The current findings suggest that spinal astrocytic REV-ERBs could be critical in the regulation of nociceptive transduction through downregulation of pronociceptive molecule expression. Thus, spinal REV-ERBs could be an effective therapeutic target in the treatment of chronic pain.

Role of Connexins in Chronic Pain and Their Potential as Therapeutic Targets for Next-Generation Analgesics.

Chronic pain, including inflammatory, neuropathic pain, is a serious clinical issue. There are increasing numbers of patients with chronic pain due to the growing number of elderly and it is estimated that about 25% of the global population will develop chronic pain. Chronic pain patients are refractory to medications used to treat acute pain such as opioids and non-steroidal anti-inflammatory drugs. Furthermore, the complexity and diversity of chronic pain mechanisms hinder the development of new analgesics. Thus, a better understanding of the mechanism of chronic pain is needed, which would facilitate the development of novel analgesics based on novel mechanisms. With this goal, connexins (Cxs) could be targeted for the development of new analgesics. Connexins are proteins with 20 subtypes, and function as channels, gap junctions between cells, and hemichannels that sample the extracellular space and release molecules such as neurotransmitters. Furthermore, Cxs could have functions independent of channel activity. Recent studies have shown that Cxs could be crucial in the induction and maintenance of chronic pain, and modulation of the activity or the expression of Cxs ameliorates nociceptive hypersensitivity in multiple chronic pain models. This review will cite novel findings on the role of of Cxs in the nociceptive transduction pathway under the chronic pain state and antinociceptive effects of various molecules modulating activity or expression of Cxs. Also, the potential of Cx modulation as a therapeutic strategy for intractable chronic pain will be discussed.

Downregulation of spinal astrocytic connexin43 leads to upregulation of interleukin-6 and cyclooxygenase-2 and mechanical hypersensitivity in mice.

Connexin43 (Cx43), involved in intercellular signaling, is expressed in spinal dorsal horn astrocytes and crucial in the maintenance of neuropathic pain. Downregulation of spinal astrocytic Cx43 in mice enhances glutamatergic neurotransmission by decreasing glutamate transporter GLT-1 expression, resulting in cutaneous hypersensitivity. Decreased expression of astrocytic Cx43 could lead to altered expression of other nociceptive molecules. Transfection of Cx43-targeting siRNA in cultured spinal astrocytes increased expression of the pronociceptive cytokine interleukin-6 (IL-6) and the prostaglandin synthesizing enzyme cyclooxygenase-2 (COX-2). Increased expression of IL-6 and COX-2 was due to decreased Cx43 expression rather than due to diminished Cx43 channel function. In mice, downregulation of spinal Cx43 expression by intrathecal treatment with Cx43-targeting siRNA increased IL-6 and COX-2 expression and induced hind paw mechanical hypersensitivity. Cx43 siRNA-induced mechanical hypersensitivity was attenuated by intrathecal treatment with anti-IL-6 neutralizing antibody and intraperitoneal treatment of selective COX-2 inhibitor celecoxib, demonstrating that these molecules play a role in nociceptive processing following Cx43 downregulation. Restoring spinal Cx43 by intrathecal injection of an adenovirus vector expressing Cx43 in mice with a partial sciatic nerve ligation reduced spinal IL-6 and COX-2 expression. Suppression of glycogen synthase kinase-3ß (GSK-3ß), a serine/threonine protein kinase, prevented upregulation of IL-6 and COX-2 expression induced by Cx43 downregulation in both cultured astrocytes and in mouse spinal dorsal horn. Inhibition of spinal GSK-3ß also ameliorated Cx43 siRNA-induced mechanical hypersensitivity. The current findings indicate that downregulation of spinal astrocytic Cx43 leads to changes in spinal expression of pronociceptive molecules underlying the maintenance of pain following nerve injury.

Downregulation of the spinal dorsal horn clock gene Per1 expression leads to mechanical hypersensitivity via c-jun N-terminal kinase and CCL2 production in mice.

Disturbances of circadian rhythm and dysregulation of clock gene expression are involved in the induction of various neurological disorder states, including chronic pain. However, the relationship between the CNS circadian-clock gene system and nociception remains poorly defined. Significant circadian oscillations of Period (Per1, Per2), Bmal1 and Cryptochrome 1 (Cry1) mRNA expression have been observed in the lumbar spinal dorsal horn of naïve mice. The current study examined the expression of clock genes in the lumbar spinal dorsal horn of mice with neuropathic pain due to a partial sciatic nerve ligation (PSNL). Seven days after PSNL, the mice displayed a robust unilateral hind paw mechanical hypersensitivity. The normal circadian oscillations of Per1, Per2 and Cry1, but not Bmal1, mRNA expression were significantly suppressed in the ipsilateral lumbar spinal dorsal horn of PSNL mice 7days following surgery. The circadian expression of PER1 protein, in particular, was also significantly suppressed in the ipsilateral spinal dorsal horn of PSNL mice. Double-labeling immunohistochemistry revealed downregulation of PER1 in neurons and astrocytes, but not microglia. Knockdown of Per1 expression by intrathecal treatment with Per1 siRNA also induced mechanical hypersensitivity, phosphorylation of c-jun N-terminal kinase (JNK) and the upregulation of chemokine (C-C motif) ligand 2 (CCL2) production in the lumbar spinal dorsal horn. Per1 siRNA-induced mechanical hypersensitivity was attenuated with intrathecal treatment of either the JNK inhibitor SP600125 or the selective CCL2 receptor (CCR2) antagonist RS504393, indicating that these intracellular messengers are crucial in mediating the mechanical hypersensitivity following the downregulation of PER1 expression. These results suggest that the downregulation of the spinal dorsal horn clock genes such as Per1 expressed could be crucial in the induction of neuropathic pain following peripheral nerve injury. Modulating clock gene Per1 expression could be a novel therapeutic strategy in alleviating neuropathic pain.

Perineural expression of high-mobility group box-1 contributes to long-lasting mechanical hypersensitivity via matrix metalloprotease-9 up-regulation in mice with painful peripheral neuropathy.

High-mobility group box-1 (HMGB1) has been shown to be critical in the modulation of nociceptive transduction following a peripheral neuropathy. However, the precise role of peripherally expressed HMGB1 in neuropathic pain has yet to be fully elaborated. Following a partial sciatic nerve ligation (PSNL) in mice, a persistent ipsilateral up-regulation of HMGB1 was observed from 3 to 21 days after PSNL, in paralleled with a robust ipsilateral hind paw mechanical hypersensitivity. Increased HMGB1 was detected in both infiltrating macrophages and proliferating Schwann cells in the ipsilateral nerve 14 days following PSNL. Repeated perineural treatment with anti-HMGB1 antibody significantly ameliorated PSNL-induced mechanical hypersensitivity. Several pronociceptive molecules, including matrix metalloprotease-9 (MMP-9), tumor necrosis factor-α, interleukin-1ß (IL-1ß), and cyclooxygenase-2, were up-regulated in injured sciatic nerve 14 days following PSNL. Repeated perineural treatment with an anti-HMGB1 antibody significantly suppressed expression of MMP-9, but not other pronociceptive molecules. Perineural treatment with a selective MMP-9 inhibitor ameliorated PSNL-induced mechanical hypersensitivity. The current findings demonstrate that the maintenance of the neuropathic state following an injured nerve is dependent on the up-regulation of HMGB1 and MMP-9. Thus, blocking HMGB1 function in sciatic nerve could be a potent therapeutic strategy for the treatment of neuropathic pain. Increased peripheral high-mobility group box-1 (HMGB1) is involved in the modulation of nociceptive transduction following a peripheral neuropathy. Following nerve injury in mice, increased HMGB1 is detected in both infiltrating macrophages and proliferating Schwann cells in the ipsilateral nerve. Repeated perineural treatment with anti-HMGB1 antibody significantly ameliorates nerve injury-induced mechanical hypersensitivity, and suppresses expression of matrix metalloprotease-9 (MMP-9). The findings demonstrate that the maintenance of the neuropathic state following an injury nerve is dependent on the up-regulation of HMGB1 and MMP-9.

Downregulation of connexin36 in mouse spinal dorsal horn neurons leads to mechanical allodynia.

Connexin36 (Cx36), a component of neuronal gap junctions, is crucial for interneuronal communication and regulation. Gap junction dysfunction underlies neurological disorders, including chronic pain. Following a peripheral nerve injury, Cx36 expression in the ipsilateral spinal dorsal horn was markedly decreased over time, which paralleled the time course of hind paw tactile allodynia. Intrathecal (i.t.) injection of Cx36 siRNA (1 and 5 pg) significantly reduced the expression of Cx36 protein in the lumbar spinal cord, peaking 3 days after the injection, which corresponded with the onset of hind paw tactile allodynia. It is possible that some of the tactile allodynia resulting from Cx36 downregulation could be mediated through excitatory neuromodulators, such as glutamate and substance P. The Cx36 knockdown-evoked tactile allodynia was significantly attenuated by i.t. treatment with the N-methyl-D-aspartate glutamate receptor antagonist MK-801 but not the substance P receptor antagonist CP96345. Immunohistochemistry showed that Cx36 was colocalized with glycine transporter-2, a marker for inhibitory glycinergic spinal interneurons, but not with glutamate decarboxylase 67, a marker for inhibitory GABAergic spinal interneurons. The results indicate that spinal inhibition through glycinergic interneurons is reduced, leading to increased glutamatergic neurotransmission, as a result of Cx36 downregulation. The current data suggest that gap junction dysfunction underlies neuropathic pain and further suggest a novel target for the development of analgesics.

Tumor necrosis factor-mediated downregulation of spinal astrocytic connexin43 leads to increased glutamatergic neurotransmission and neuropathic pain in mice.

Spinal cord astrocytes are critical in the maintenance of neuropathic pain. Connexin 43 (Cx43) expressed on spinal dorsal horn astrocytes modulates synaptic neurotransmission, but its role in nociceptive transduction has yet to be fully elaborated. In mice, Cx43 is mainly expressed in astrocytes, not neurons or microglia, in the spinal dorsal horn. Hind paw mechanical hypersensitivity was observed beginning 3days after partial sciatic nerve ligation (PSNL), but a persistent downregulation of astrocytic Cx43 in ipsilateral lumbar spinal dorsal horn was not observed until 7days post-PSNL, suggesting that Cx43 downregulation mediates the maintenance and not the initiation of nerve injury-induced hypersensitivity. Downregulation of Cx43 expression by intrathecal treatment with Cx43 siRNA also induced mechanical hypersensitivity. Conversely, restoring Cx43 by an adenovirus vector expressing Cx43 (Ad-Cx43) ameliorated PSNL-induced mechanical hypersensitivity. The sensitized state following PSNL is likely maintained by dysfunctional glutamatergic neurotransmission, as Cx43 siRNA-induced mechanical hypersensitivity was attenuated with intrathecal treatment of glutamate receptor antagonists MK801 and CNQX, but not neurokinin-1 receptor antagonist CP96345 or the Ca(2+) channel subunit α2δ1 blocker gabapentin. The source of this dysfunctional glutamatergic neurotransmission is likely decreased clearance of glutamate from the synapse rather than increased glutamate release into the synapse. Astrocytic expression of glutamate transporter GLT-1, but not GLAST, and activity of glutamate transport were markedly decreased in mice intrathecally injected with Cx43-targeting siRNA but not non-targeting siRNA. Glutamate release from spinal synaptosomes prepared from mice treated with either Cx43-targeting siRNA or non-targeting siRNA was unchanged. Intrathecal injection of Ad-Cx43 in PSNL mice restored astrocytic GLT-1 expression. The cytokine tumor necrosis factor (TNF) has been implicated in the induction of central sensitization, particularly through its actions on astrocytes, in the spinal cord following peripheral injury. Intrathecal injection of TNF in naïve mice induced the downregulation of both Cx43 and GLT-1 in spinal dorsal horn, as well as hind paw mechanical hypersensitivity, as observed in PSNL mice. Conversely, intrathecal treatment of PSNL mice with the TNF inhibitor etanercept prevented not only mechanical hypersensitivity but also the downregulation of Cx43 and GLT-1 expression in astrocytes. The current findings indicate that spinal astrocytic Cx43 are essential for the maintenance of neuropathic pain following peripheral nerve injury and suggest modulation of Cx43 as a novel target for developing analgesics for neuropathic pain.

A ß1/2 adrenergic receptor-sensitive intracellular signaling pathway modulates CCL2 production in cultured spinal astrocytes.

The phosphorylation of c-jun N-terminal kinase (JNK) and the subsequent production of C-C chemokine CCL2 (monocyte chemoattractant protein; MCP-1) in spinal astrocytes contribute to the initiation of neurological disorders including chronic pain. Astrocytes express neurotransmitter receptors which could be targeted to ameliorate neurological disorders. In the current study, the involvement of the ß-adrenergic system in the regulation of JNK activity and CCL2 production after stimulation with tumor necrosis factor (TNF)-α, one of many initiators of neuroinflammation, was elucidated. Treatment of cultured spinal astrocytes with isoproterenol (a ß-adrenergic receptor agonist; 1 µM) reduced both TNF-α-induced JNK1 phosphorylation, as observed by Western blotting, and the subsequent increase of both CCL2 mRNA expression and CCL2 production, which were measured by real time-PCR and ELISA, respectively. The effects of isoproterenol were completely blocked by pretreatment with either propranolol (a ß-adrenoceptor antagonist) or H89 (a protein kinase A [PKA] inhibitor). The current study revealed that the regulation of glycogen synthase kinase-3ß (GSK-3ß) activity is a crucial factor in the inhibitory action of isoproterenol. The TNF-α-induced JNK1 phosphorylation was significantly blocked by treatment with GSK-3ß inhibitors (either LiCl or TWS119), and stimulation of ß-adrenergic receptors induced the inhibition of GSK-3ß through the phosphorylation of Ser(9) . Moreover, treatment with isoproterenol markedly suppressed the TNF-α-induced increase of CCL2 mRNA expression and CCL2 production through a ß-adrenergic receptor-PKA pathway mediated by GSK-3ß regulation. Thus, activation of ß1/2 adrenergic receptors expressed in spinal astrocytes could be a novel method of moderating neurological disorders with endogenous catecholamines or selective agonists.

Continuous infusion of substance P into rat striatum alleviates nociceptive behavior via phosphorylation of extracellular signal-regulated kinase 1/2.

Intraplantar injection of 0.4% formalin into the rat hind paw leads to a biphasic nociceptive response; an 'acute' phase (0-15 min) and 'tonic' phase (16-120 min), which is accompanied by significant phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the contralateral striatum at 120 min post-formalin injection. To uncover a possible relationship between the slow-onset substance P (SP) release and increased ERK1/2 phosphorylation in the striatum, continuous infusion of SP into the striatum by reverse microdialysis (0.4 µg/mL in microdialysis fiber, 1 µL/min) was performed to mimic volume neurotransmission of SP. Continuous infusion for 3 h of SP reduced the duration of 'tonic' phase nociception, and this SP effect was mediated by neurokinin 1 (NK1) receptors since pre-treatment with NK1 receptor antagonist CP96345 (10 µM) blocked the effect of SP infusion. However, formalin-induced 'tonic' phase nociception was significantly prolonged following acute injection of the MAP/ERK kinase 1/2 inhibitor PD0325901 (100 pmol) by microinjection. The coinfusion of SP and PD0325901 significantly increased the 'tonic' phase of nociception. These data demonstrate that volume transmission of striatal SP triggered by peripheral nociceptive stimulation does not lead to pain facilitation but a significant decrease of tonic nociception by the activation of the SP-NK1 receptor-ERK1/2 system. Noxious stimulation induces a slow-onset substance P (SP) release as a volume transmitter, activating extra-synaptic NK1 receptors, and evokes phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. The SP-NK1-ERK1/2 system in the striatum decreases tonic nociception.

[Investigation of Chronic Pain for the Development of Novel Analgesics Focusing on High Mobility Group Box-1].

Neuropathic pain is an infirm type of chronic pain, which results in functional and emotional impairment. There is an urgent need for novel therapeutic approaches because there is lack of effective treatment for neuropathic pain. Posttraumatic trigeminal neuropathy (PTTN), a chronic sensory disorder following trauma, for example, during dental implant surgery or third molar removal, can cause orofacial numbness, paresthesia and pain. Unlike other painful peripheral neuropathies, the chance of developing PTTN is predictable based on preoperative assessment such as X-ray of the trigeminal nerve tract and the surgery site. High mobility group box-1 (HMGB1) acts as damage associated molecular patterns (DAMPs) and contributes to the pathogenesis of neuropathic pain including diabetic and chemotherapy-induced peripheral neuropathy. Recently, we have demonstrated that HMGB1 around injured sciatic nerves is a key molecule triggering the onset of neuropathic pain. We therefore hypothesize that inhibition of HMGB1 could prevent the onset of PTTN. In a mouse PTTN model, pretreatment with anti-HMGB1 neutralizing antibody can attenuate PTTN-induced behavioral painful responses and suppress microglial activation in spinal trigeminal nucleus caudalis. In summary, perioperative inhibition of HMGB1 activity could be used to prevent the onset of PTTN. This review summarizes recent findings regarding the role of HMGB1 in the induction of neuropathic pain and may generate new translational opportunities for pain treatment.