Different classes of antidepressants, such as tricyclic antidepressants, selective serotonin reuptake inhibitor (SSRI), and serotonin and norepinephrine reuptake inhibitor (SNRI), have been shown to increase GDNF production in astrocytes, which could be a key mechanism of the psychotropic effect of antidepressants. The antidepressant mirtazapine is a noradrenaline and specific serotonergic antidepressant (NaSSA) and does not block reuptake of catecholamines and serotonin. The present study examined the effect of mirtazapine on GDNF expression in rat C6 astroglial cells (C6 cells) and rat primary cultured cortical astrocytes (primary astrocytes). Mirtazapine treatment significantly increased GDNF mRNA expression and GDNF release in both C6 cells and primary astrocytes. In primary astrocytes, mirtazapine also increased the expressions of brain-derived neurotrophic factor mRNA. To mimic mirtazapine's putative mechanism of action, cells were treated with either a α2-adrenoceptor antagonist (yohimbine), 5-HT2 receptor antagonist (ketanserin), 5-HT3 receptor antagonist (ondansetron), or a mixture of these--no effect on GDNF mRNA expression was observed. Mirtazapine treatment increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, and the mirtazapine-induced GDNF and BDNF expression were blocked by MAPK/ERK kinase (MEK) inhibitor (U0126). Furthermore, the effect of mirtazapine on ERK phosphorylation and expressions of GDNF and BDNF was antagonized by Gi/o inhibitor (pertussis toxin), lysophosphatidic acid-1 (LPA1) receptor antagonist (AM966), and LPA1/LPA3 receptors antagonist (Ki16425). The current findings demonstrate that the NaSSA mirtazapine, similar to other classes of antidepressants, increases GDNF expression through a Gi/o coupled LPA1 receptor-mediated ERK pathway. The current findings suggest a general mechanism underlying the psychotropic effect antidepressants.

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.

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.

Clinical evidence indicates that cognitive impairment is a common comorbidity of chronic pain, including neuropathic pain, but the mechanism underlying cognitive impairment remains unclear. Neuroinflammation plays a critical role in the development of both neuropathic pain and cognitive impairment. High-mobility group box 1 (HMGB1) is a proinflammatory molecule and could be involved in neuroinflammation-mediated cognitive impairment in the neuropathic pain state. Hippocampal microglial activation in mice has been associated with cognitive impairment. Thus, the current study examined a potential role of HMGB1 and microglial activation in cognitive impairment in mice with neuropathic pain due to a partial sciatic nerve ligation (PSNL). Mice developed cognitive impairment over two weeks, but not one week, after nerve injury. Nerve-injured mice demonstrated decreased nuclear fraction HMGB1, suggesting increased extracellular release of HMGB1. Furthermore, two weeks after PSNL, significant microglia activation was observed in hippocampus. Inhibition of microglial activation with minocycline, local hippocampal microglia depletion with clodronate liposome, or blockade of HMGB1 with either glycyrrhizic acid (GZA) or anti-HMGB1 antibody in PSNL mice reduced hippocampal microglia activation and ameliorated cognitive impairment. Other changes in the hippocampus of PSNL mice potentially related to cognitive impairment, including decreased hippocampal neuron dendrite length and spine densities and decreased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor (AMPAR) subunits, were prevented with anti-HMGB1 antibody treatment. The current findings suggest that neuro-inflammation involves a number of cellular-level changes and microglial activation. Blocking neuro-inflammation, particularly through blocking HMGB1 could be a novel approach to reducing co-morbidities such as cognitive impairment associated with neuropathic pain.

Connexin 43 (Cx43) expression is decreased in the prefrontal cortex of patients with depression, but its significance is still unknown. Neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), are involved in the effects of antidepressant. However, the relationship between Cx43 expression and induction of brain-derived neurotrophic factor production by antidepressants is unknown. On the basis of our previous study, which showed that adrenergic receptors stimulation results in potentiation of BDNF expression in astrocytes with downregulated Cx43 expression, we investigated the induction of BDNF expression by amitriptyline, a tricyclic antidepressant, in Cx43-knockdown astrocytes. Amitriptyline treatment potentiated BDNF expression in Cx43-knockdown astrocytes compared with those treated with non-targeting small interfering RNA (siRNA). Using a pharmacological approach, we revealed that the potentiating effect of amitriptyline on BDNF expression was mediated by lysophosphatidic acid (LPA) receptor1/3 (LPA1/3) stimulation and subsequent activation of Src-extracellular signal-regulated kinase (ERK) signaling. These findings suggest that downregulation of Cx43 in patients with depression might contribute to the therapeutic efficacy of antidepressants rather than the pathogenesis of depression.

The inflammatory response related to surgery is considered surgical inflammation. Most anesthetic agents directly or indirectly suppress the immune response. However, the intravenous anesthetics pentobarbital and ketamine were reported to inhibit the lipopolysaccharide-induced inflammatory response such as cytokines formation. Neurogenic inflammation is inflammation originating from the local release of inflammatory mediators, such as substance P (SP), by primary afferent neurons after noxious stimuli like surgery. Thus, in this study, we examined whether pentobarbital and ketamine suppress SP release from cultured dorsal root ganglion (DRG) neurons. DRG cells were dissected from male Wistar rats. Released SP was measured by radioimmunoassay. We demonstrated that higher concentrations of pentobarbital (100-1,000 μM) significantly inhibited capsaicin (100 nM)-induced, but not high K+ (50 mM)-induced, SP release from DRG cells, although a high concentration of ketamine (1 mM) did not. This study revealed that pentobarbital functions between the activation of vanilloid receptor subtype 1 (TRPV1) receptors, to which capsaicin selectively binds, and the opening of voltage-operated Ca2+ channels (VOCC) in the nerve endings. Therefore, the anti-inflammatory action of pentobarbital is mediated through different mechanisms than those of ketamine. Thus, the inhibitory effect of pentobarbital on SP release from peripheral terminals may protect against neurogenic inflammation after surgery.

Cocaine is an addictive drug that acts in brain reward areas. Recent evidence suggests that cocaine stimulates synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in midbrain, increasing dopamine neuron activity via disinhibition. Although a mechanism for cocaine-stimulated 2-AG synthesis is known, our understanding of 2-AG release is limited. In NG108 cells and mouse midbrain tissue, we find that 2-AG is localized in non-synaptic extracellular vesicles (EVs) that are secreted in the presence of cocaine via interaction with the chaperone protein sigma-1 receptor (Sig-1R). The release of EVs occurs when cocaine causes dissociation of the Sig-1R from ADP-ribosylation factor (ARF6), a G-protein regulating EV trafficking, leading to activation of myosin light chain kinase (MLCK). Blockade of Sig-1R function, or inhibition of ARF6 or MLCK also prevented cocaine-induced EV release and cocaine-stimulated 2-AG-modulation of inhibitory synapses in DA neurons. Our results implicate the Sig-1R-ARF6 complex in control of EV release and demonstrate that cocaine-mediated 2-AG release can occur via EVs.

Many patients suffer from serious adverse effects including respiratory distress and pulmonary edema during and after chemotherapy with paclitaxel or vinorelbine. These effects appear to be due to the activation of neurokinin-1 receptors. The present study investigated the influences of paclitaxel and vinorelbine on the substance P (sP) release from cultured dorsal root ganglion (DRG) cells using a radioimmunoassay. Both paclitaxel and vinorelbine evoked sP release in a dose- and time-dependent manner within 60 min at a concentration range of 0.1-10 microM. The sP release levels induced by the two drugs were attenuated by pretreatment with the protein kinase Cs (PKCs) inhibitors (bisindolylmaleimide I and Gö6976). Moreover, the paclitaxel- or vinorelbine-induced sP release was diminished in the absence of extracellular Ca2+ or the presence of LaCl3 (an extracellular Ca2+ influx blocker). A Ca2+ imaging assay further indicated that both paclitaxel and vinorelbine gradually increased the intracellular Ca2+ concentration, and these increases lasted for at least 15 min and were suppressed by Gö6976. Paclitaxel caused the membrane translocation of only PKCbeta within 10 min after stimulation, whereas vinorelbine induced the translocation of both PKCalpha and beta. The paclitaxel- and vinorelbine-induced sP release levels were separately inhibited by ruthenium red (a transient receptor potential (TRP) channel blocker) and gabapentin (an inhibitor of voltage-gated Ca2+ channels (VGCCs)). These findings suggest that paclitaxel and vinorelbine evoke the sP release from cultured DRG cells by the extracellular Ca2+ influx through TRP channels activated by PKCbeta and VGCCs activated by both PKCalpha and beta, respectively.

High mobility group box-1 (HMGB1) is associated with the pathogenesis of inflammatory diseases. A previous study reported that intravenous injection of anti-HMGB1 monoclonal antibody significantly attenuated brain edema in a rat model of stroke, possibly by attenuating glial activation. Peripheral nerve injury leads to increased activity of glia in the spinal cord dorsal horn. Thus, it is possible that the anti-HMGB1 antibody could also be efficacious in attenuating peripheral nerve injury-induced pain. Following partial sciatic nerve ligation (PSNL), rats were treated with either anti-HMGB1 or control IgG. Intravenous treatment with anti-HMGB1 monoclonal antibody (2 mg/kg) significantly ameliorated PSNL-induced hind paw tactile hypersensitivity at 7, 14 and 21 days, but not 3 days, after ligation, whereas control IgG had no effect on tactile hypersensitivity. The expression of HMGB1 protein in the spinal dorsal horn was significantly increased 7, 14 and 21 days after PSNL; the efficacy of the anti-HMGB1 antibody is likely related to the presence of HMGB1 protein. Also, the injury-induced translocation of HMGB1 from the nucleus to the cytosol occurred mainly in dorsal horn neurons and not in astrocytes and microglia, indicating a neuronal source of HMGB1. Markers of astrocyte (glial fibrillary acidic protein (GFAP)), microglia (ionized calcium binding adaptor molecule 1 (Iba1)) and spinal neuron (cFos) activity were greatly increased in the ipsilateral dorsal horn side compared to the sham-operated side 21 days after PSNL. Anti-HMGB1 monoclonal antibody treatment significantly decreased the injury-induced expression of cFos and Iba1, but not GFAP. The results demonstrate that nerve injury evokes the synthesis and release of HMGB1 from spinal neurons, facilitating the activity of both microglia and neurons, which in turn leads to symptoms of neuropathic pain. Thus, the targeting of HMGB1 could be a useful therapeutic strategy in the treatment of chronic pain.

Post-traumatic trigeminal neuropathy (PTTN) is a chronic sensory disorder that afflicts patients with nerve injury caused by orofacial and dental surgery or cervicofacial trauma. Currently, effective treatment strategies for PTTN are lacking, and patients treated with conventional drugs for PTTN experience adverse effects such as drowsiness and drug addiction. In the present study, we investigated whether mirogabalin, a novel gabapentinoid, could be an effective treatment for PTTN induced by distal infraorbital nerve chronic constriction injury (dIoN-CCI) in the mouse. Increased facial grooming time and hyper-responsiveness to acetone were observed in dIoN-CCI mice. These pain-related behaviors were attenuated by intraperitoneal injection of mirogabalin. In particular, mirogabalin significantly diminished the increase in facial grooming time. The analgesic effect of mirogabalin injection started 45 min after the injection and persisted for 6 h. Additionally, 10 mg/kg mirogabalin did not affect locomotor activity in the open field test, suggesting that it does not cause sedation. Together, the current findings suggest that mirogabalin could be a valuable therapeutic drug for PTTN following orofacial surgeries without sedative side effects.

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.

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.

Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER) chaperones implicated in neuropathic pain. Here we examine if the Sig-1R may relate to neuropathic pain at the level of dorsal root ganglia (DRG). We focus on the neuronal excitability of DRG in a "spare nerve injury" (SNI) model of neuropathic pain in rats and find that Sig-1Rs likely contribute to the genesis of DRG neuronal excitability by decreasing the protein level of voltage-gated Cav2.2 as a translational inhibitor of mRNA. Specifically, during SNI, Sig-1Rs translocate from ER to the nuclear envelope via a trafficking protein Sec61β. At the nucleus, the Sig-1R interacts with cFos and binds to the promoter of 4E-BP1, leading to an upregulation of 4E-BP1 that binds and prevents eIF4E from initiating the mRNA translation for Cav2.2. Interestingly, in Sig-1R knockout HEK cells, Cav2.2 is upregulated. In accordance with those findings, we find that intra-DRG injection of Sig-1R agonist (+)pentazocine increases frequency of action potentials via regulation of voltage-gated Ca2+ channels. Conversely, intra-DRG injection of Sig-1R antagonist BD1047 attenuates neuropathic pain. Hence, we discover that the Sig-1R chaperone causes neuropathic pain indirectly as a translational inhibitor.

Previous studies have demonstrated that continuous substance P (SP) infusion into the rat striatum attenuated hind paw formalin-induced nociceptive behaviors and mechanical hypersensitivity via a neurokinin-1 (NK1) receptor dependent mechanism. However, whether there is a role of striatal infusion of SP on chronic, neuropathic pain has yet to be demonstrated. The present study investigated the effect of continuous SP infusion into the rat striatum using a reverse microdialysis method is antinociceptive in a rat model of chronic, mononeuropathic pain. Two weeks after partial sciatic nerve injury, the ipsilateral hind paw demonstrated mechanical hypersensitivity. Infusion of SP (0.2, 0.4, or 0.8 μg/mL, 1 μL/min) for 120 min into the contralateral striatum dose-dependently relieved mechanical hypersensitivity. The antinociceptive effect of SP infusion was inhibited by co-infusion with the NK1 receptor antagonist CP96345 (10 μM). Neither ipsilateral continuous infusion nor acute microinjection of SP (10 ng) into the contralateral striatum was antinociceptive. A role of striatal muscarinic cholinergic neurons is suggested since co-infusion of SP with atropine (10 μM), but not the nicotinic receptor mecamylamine (10 μM), blocked antinociception. The current study suggests that activation of striatal muscarinic receptors through NK1 receptors could be a novel approach to managing chronic pain.

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-1β (IL-1β) was up-regulated within activated astrocytes and block of TLR4 prevented the upregulation of IL-1β. Interleukin-1β appears to be secreted by activated astrocytes, as IL-1β 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-1β in spinal astrocytes through a TLR4-dependent pathway and increases glutamatergic nociceptive transduction. These spinal mechanisms could be key steps that maintain neuropathic pain.

Clinical evidence indicates that major depression is a common comorbidity of chronic pain, including neuropathic pain; however, the cellular basis for chronic pain-mediated major depression remains unclear. Mitochondrial dysfunction induces neuroinflammation and has been implicated in various neurological diseases, including depression. Nevertheless, the relationship between mitochondrial dysfunction and anxiodepressive-like behaviors in the neuropathic pain state remains unclear. The current study examined whether hippocampal mitochondrial dysfunction and downstream neuroinflammation are involved in anxiodepressive-like behaviors in mice with neuropathic pain, which was induced by partial sciatic nerve ligation (PSNL). At 8 weeks after surgery, there was decreased levels of mitochondrial damage-associated molecular patterns, such as cytochrome c and mitochondrial transcription factor A, and increased level of cytosolic mitochondrial DNA in the contralateral hippocampus, suggesting the development of mitochondrial dysfunction. Type I interferon (IFN) mRNA expression in the hippocampus was also increased at 8 weeks after PSNL surgery. The restoration of mitochondrial function by curcumin blocked the increased cytosolic mitochondrial DNA and type I IFN expression in PSNL mice and improved anxiodepressive-like behaviors. Blockade of type I IFN signaling by anti-IFN alpha/beta receptor 1 antibody also improved anxiodepressive-like behaviors in PSNL mice. Together, these findings suggest that neuropathic pain induces hippocampal mitochondrial dysfunction followed by neuroinflammation, which may contribute to anxiodepressive-behaviors in the neuropathic pain state. Improving mitochondrial dysfunction and inhibiting type I IFN signaling in the hippocampus might be a novel approach to reducing comorbidities associated with neuropathic pain, such as depression and anxiety.

High mobility box 1 (HMGB1), a damage-associated molecular pattern, has crucial roles in induction of neuropathic pain. Upregulation of HMGB1 around the injured sciatic nerve contributes to mechanical hypersensitivity following partial sciatic nerve ligation (PSNL) of mice. However, central mechanisms mediating perineural HMGB1-induced nociceptive hypersensitivity, especially within the spinal dorsal horn, have not been determined. The current study shows that perineural treatment of naïve mice with recombinant HMGB1, which mimics increased HMGB1 around the injured sciatic nerve of PSNL mice, significantly induced activation of microglia, but not astrocytes, in the spinal dorsal horn. Intraperitoneal injection of minocycline, a microglial inhibitor, ameliorated perineural rHMGB1-induced mechanical hypersensitivity. In addition, blockade of spinal N-methyl-D-aspartate (NMDA) receptors significantly prevented perineural rHMGB1-induced mechanical hypersensitivity and microglial activation. In contrast, non-NMDA receptors, neurokinin 1 receptor, colony-stimulating factor 1 receptor and P2Y12 receptor were not involved in perineural rHMGB1-induced mechanical hypersensitivity. Furthermore, repeated perineural treatment with an anti-HMGB1 antibody blocked activation of spinal microglia in PSNL mice. Collectively, the current findings demonstrate that increased HMGB1 around injured sciatic nerve might induce nociceptive hypersensitivity through activation of spinal microglia. Thus, HMGB1-dependent mechanisms between the injured sciatic nerve and spinal dorsal horn could be crucial in induction of neuropathic pain.

A major risk factor for major depressive disorder (MDD) is stress. Stress leads to the release of high-mobility group box-1 (HMGB1), which in turn leads to neuroinflammation, a potential pathophysiological basis of MDD. The mechanism underlying stress-induced HMGB1 release is not known, but stress-associated glucocorticoids could be involved. To test this, rat primary cultured cortical astrocytes, the most abundant cell type in the central nervous system (CNS), were treated with corticosterone and HMGB1 release was assessed by Western blotting and ELISA. Significant HMGB1 was released with treatment with either corticosterone or dexamethasone, a synthetic glucocorticoid. HMGB1 translocated from the nucleus to the cytoplasm following corticosterone treatment. HMGB1 release was significantly attenuated with glucocorticoid receptor blocking. In addition, inhibition of pannexin-1, and P2X7 receptors led to a significant decrease in corticosterone-induced HMGB1 release. Taken together, corticosterone stimulates astrocytic glucocorticoid receptors and triggers cytoplasmic translocation and extracellular release of nuclear HMGB1 through a mechanism involving pannexin-1 and P2X7 receptors. Thus, under conditions of stress, glucocorticoids induce astrocytic HMGB1 release, leading to a neuroinflammatory state that could mediate neurological disorders such as MDD.

Microglia have both protective and degenerative roles in the central nervous system. The α7 nicotinic acetylcholine receptor (nAChR) is crucial in the regulation of the neuroprotective role in microglia. Recent studies have demonstrated decreased expression of α7 nAChR in brain in response to neuroinflammation, but the mechanism mediating the downregulation of the α7 nAChR has yet to be elaborated. Treatment of microglial cell line BV2 cells or rat primary cultured microglia with the inflammogen lipopolysaccharide (LPS) significantly decreased the expression of α7 nAChR mRNA in a time and concentration-dependent manner. The effects of LPS were prevented by pretreatment with TAK-242, a toll-like receptor 4 (TLR4) blocker. The LPS-induced downregulation of α7 nAChR was also prevented with trichostatin A, a histone deacetylase (HDAC) inhibitor, but not 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor. Further pharmacological probing revealed that HDAC2 and HDAC3 were involved in the effects of LPS. Treatment of BV2 cells with LPS significantly reduced acetylation of histone H3 at lysine 9 of the α7 nAChR promoter. The current findings demonstrate that inflammation-evoked activation of TLR4 leads to the reduction of the neuroprotective function of microglia through the downregulation of the α7 nAChR. Also, histone modification could be crucial in the regulation of the neuroprotective role of microglia during neuroinflammatory states.

As spinal microglia have a critical role in the development of chronic pain, regulation of their activity is essential for pain relief. Previous study has shown that stimulation of the REV-ERB nuclear receptors in the spinal dorsal horn produces antinociception in animal models of both inflammatory and neuropathic pain. However, the involvement of spinal microglia in the antinociceptive action of REV-ERBs remains to be elucidated. In the current study, we found that intrathecal treatment with the REV-ERB agonist SR9009 significantly blocked the increase in ionized calcium-binding adaptor molecule immunoreactivity in the spinal dorsal horn of mice following intrathecal administration of lipopolysaccharide and peripheral sciatic nerve ligation. Furthermore, both Rev-erbα and Rev-erbβ mRNAs were expressed in cultured rat spinal microglia. Treatment of cultured rat spinal microglia with SR9009 significantly blocked the lipopolysaccharide-induced increase in interleukin (IL)-1β and IL-6 mRNA expression. In conclusion, the current findings suggest that REV-ERBs negatively regulate spinal microglial activity and might contribute to the REV-ERB-mediated antinociceptive effect in the spinal dorsal horn.