Spinal Neuronal miR-124 Inhibits Microglial Activation and Contributes to Preventive Effect of Electroacupuncture on Chemotherapy-Induced Peripheral Neuropathy in Mice.

Chemotherapy-induced peripheral neuropathy (CIPN) is a persistent and irreversible side effect of antineoplastic agents. Patients with CIPN usually show chronic pain and sensory deficits with glove-and-stocking distribution. However, whether spinal neuronal microRNA (miR)-124 is involved in cisplatin-induced peripheral neuropathy remains to be studied. In this study, miR-124 was significantly reduced in the spinal dorsal horn in CIPN mice. Overexpression of neuronal miR-124 induced by injecting adeno-associated virus with neuron-specific promoter into the spinal cord of mice prevented the development of mechanical allodynia, sensory deficits, and the loss of intraepidermal nerve fibers induced by cisplatin. Meanwhile, cisplatin-induced M1 microglia activation and the release of proinflammatory cytokines were significantly inhibited by overexpression of neuronal miR-124. Furthermore, electroacupuncture (EA) treatment upregulated miR-124 expression in the spinal dorsal horn of CIPN mice. Interestingly, downregulation of spinal neuronal miR-124 significantly inhibited the regulatory effect of EA on CIPN and microglia activity as well as spinal neuroinflammation induced by cisplatin. These results demonstrate that spinal neuronal miR-124 is involved in the prevention and treatment of EA on cisplatin-induced peripheral neuropathy in mice. Our findings suggest that spinal neuronal miR-124 might be a potential target for EA effect, and we provide, to our knowledge, a new experimental basis for EA prevention of CIPN.

A regulatory role of the medial septum in the chloroquine-induced acute itch through local GABAergic system and GABAergic pathway to the anterior cingulate cortex.

Itch, a common somatic sensation, serves as a crucial protective system. Recent studies have unraveled the neural mechanisms of itch at peripheral, spinal cord as well as cerebral levels. However, a comprehensive understanding of the central mechanism governing itch transmission and regulation remains elusive. Here, we report the role of the medial septum (MS), an integral component of the basal forebrain, in modulating the acute itch processing. The increases in c-Fos+ neurons and calcium signals within the MS during acute itch processing were observed. Pharmacogenetic activation manipulation of global MS neurons suppressed the scratching behaviors induced by chloroquine or compound 48/80. Microinjection of GABA into the MS or pharmacogenetic inhibition of non-GABAergic neurons markedly suppressed chloroquine-induced scratching behaviors. Pharmacogenetic activation of the MS-ACC GABAergic pathway attenuated chloroquine-induced acute itch. Hence, our findings reveal that MS has a regulatory role in the chloroquine-induced acute itch through local increased GABA to inhibit non-GABAergic neurons and the activation of MS-ACC GABAergic pathway.

Resting-state fMRI reveals changes within the anxiety and social avoidance circuitry of the brain in mice with psoriasis-like skin lesions.

Psoriasis is an autoimmune skin disease that often co-occurs with psychological morbidities such as anxiety and depression, and psychosocial issues also lead psoriasis patients to avoid other people. However, the precise mechanism underlying the comorbidity of psoriasis and anxiety is unknown. Also, whether the social avoidance phenomenon seen in human patients also exists in psoriasis-like animal models remains unknown. In the present study, anxiety-like behaviours and social avoidance-like behaviours were observed in an imiquimod-induced psoriasis-like C57-BL6 mouse model along with typical psoriasis-like dermatitis and itch-like behaviours. The 11.7T resting-state functional magnetic resonance imaging showed differences in brain regions between the model and control group, and voxel-based morphometry showed that the grey matter volume changed in the basal forebrain region, anterior commissure intrabulbar and striatum in the psoriasis-like mice. Seed-based resting state functional connectivity analysis revealed connectivity changes in the amygdala, periaqueductal gray, raphe nuclei and lateral septum. We conclude that the imiquimod-induced psoriasis-like C57-BL6 mouse model is well suited for mechanistic studies and for performing preclinical therapeutic trials for treating anxiety and pathological social avoidance in psoriasis patients.

Spinal Neuronal GRK2 Contributes to Preventive Effect by Electroacupuncture on Cisplatin-Induced Peripheral Neuropathy in Mice.

BACKGROUND: The main symptoms of chemotherapy-induced peripheral neuropathy (CIPN) include pain and numbness. Neuronal G protein-coupled receptor kinase 2 (GRK2) plays an important role in various pain models. Cisplatin treatment can induce the activation of proinflammatory microglia in spinal cord. The purpose of this study was to investigate the role of spinal neuronal GRK2 in cisplatin-induced CIPN and in the prevention of CIPN by electroacupuncture (EA). METHODS: The pain and sensory deficit behaviors of mice were examined by von Frey test and adhesive removal test. The expression of neuronal GRK2 in the spinal cord is regulated by intraspinal injection of adeno-associated virus (AAV) containing neuron-specific promoters. The protein levels of GRK2, triggering receptor expressed on myeloid cells 2 (TREM2), and DNAX-activating protein of 12 kDa (DAP12) in spinal dorsal horn were detected by Western blot, the density of intraepidermal nerve fibers (IENFs) was detected by immunofluorescence, and microglia activation were evaluated by real-time polymerase chain reaction (PCR). RESULTS: In this study, cisplatin treatment led to the decrease of GRK2 expression in the dorsal horn of spinal cord. Overexpression of neuronal GRK2 in spinal cord by intraspinal injection of an AAV vector expressing GRK2 with human synapsin (hSyn) promotor significantly inhibited the loss of IENFs and alleviated the mechanical pain and sensory deficits induced by cisplatin. Real-time PCR analysis showed that the overexpression of neuronal GRK2 significantly inhibited the messenger RNA (mRNA) upregulation of proinflammatory cytokine interleukin (IL)-1ß, IL-6, inducible nitric oxide synthase (iNOS), and M1 microglia marker cluster of differentiation (CD)16 induced by cisplatin. Furthermore, the TREM2 and DAP12, which has been demonstrated to play a role in microglia activation and in the development of CIPN, were also downregulated by overexpression of neuronal GRK2 in this study. Interestingly, preventive treatment with EA completely mimics the effect of overexpression of neuronal GRK2 in the spinal cord in this mouse model of cisplatin-induced CIPN. EA increased GRK2 level in spinal dorsal horn after cisplatin treatment. Intraspinal injection of AAV vector specifically downregulated neuronal GRK2, completely reversed the regulatory effect of EA on CIPN and microglia activation. All these indicated that the neuronal GRK2 mediated microglial activation contributed to the process of CIPN. CONCLUSIONS: Neuronal GRK2 in the spinal cord contributed to the preventive effect of EA on CIPN. The neuronal GRK2 may be a potential target for CIPN intervention.

Neuronal GRK2 regulates microglial activation and contributes to electroacupuncture analgesia on inflammatory pain in mice.

BACKGROUND: G protein coupled receptor kinase 2 (GRK2) has been demonstrated to play a crucial role in the development of chronic pain. Acupuncture is an alternative therapy widely used for pain management. In this study, we investigated the role of spinal neuronal GRK2 in electroacupuncture (EA) analgesia. METHODS: The mice model of inflammatory pain was built by subcutaneous injection of Complete Freund's Adjuvant (CFA) into the plantar surface of the hind paws. The mechanical allodynia of mice was examined by von Frey test. The mice were subjected to EA treatment (BL60 and ST36 acupuncture points) for 1 week. Overexpression and downregulation of spinal neuronal GRK2 were achieved by intraspinal injection of adeno associated virus (AAV) containing neuron-specific promoters, and microglial activation and neuroinflammation were evaluated by real-time PCR. RESULTS: Intraplantar injection with CFA in mice induced the decrease of GRK2 and microglial activation along with neuroinflammation in spinal cord. EA treatment increased the spinal GRK2, reduced neuroinflammation, and significantly decreased CFA-induced mechanical allodynia. The effects of EA were markedly weakened by non-cell-specific downregulation of spinal GRK2. Further, intraspinal injection of AAV containing neuron-specific promoters specifically downregulated neuronal GRK2, and weakened the regulatory effect of EA on CFA-induced mechanical allodynia and microglial activation. Meanwhile, overexpression of spinal neuronal GRK2 decreased mechanical allodynia. All these indicated that the neuronal GRK2 mediated microglial activation and neuroinflammation, and subsequently contributed to CFA-induced inflammatory pain. CONCLUSION: The restoration of the spinal GRK2 and subsequent suppression of microglial activation and neuroinflammation might be an important mechanism for EA analgesia. Our findings further suggested that the spinal GRK2, especially neuronal GRK2, might be the potential target for EA analgesia and pain management, and we provided a new experimental basis for the EA treatment of pain.

Current Understanding of the Neural Circuitry in the Comorbidity of Chronic Pain and Anxiety.

Chronic pain patients often develop mental disorders, and anxiety disorders are common. We hypothesize that the comorbid anxiety results from an imbalance between the reward and antireward system due to persistent pain, which leads to the dysfunction of the pain and anxiety regulatory system. In this review, we will focus on changes in neuroplasticity, especially in neural circuits, during chronic pain and anxiety as observed in animal studies. Several neural circuits within specific regions of the brain, including the nucleus accumbens, lateral habenular, parabrachial nucleus, medial septum, anterior cingulate cortex, amygdala, hippocampus, medial prefrontal cortex, and bed nucleus of the stria terminalis, will be discussed based on novel findings after chemogenetic or optogenetic manipulation. We believe that these animal studies provide novel insights into human conditions and can guide clinical practice.

Light-emitting diode phototherapy: pain relief and underlying mechanisms.

Pain is a common symptom of an illness. For decades, pain treatments such as non-steroidal anti-inflammatory drugs, opioids, and surgical nerve blocking have been widely used, but each method has its limitations. Photobiomodulation is a recently developed method for pain management, with light-emitting diodes (LEDs) being a more recent development used in pain management because of their low cost, low side effects, and high safety. Here, we reviewed the phototherapeutic effects of LEDs on different pain conditions. We also discussed possible physicochemical and neurobiological mechanisms underlying LED therapy, especially its effects on inflammatory pain.

Spinal IL-33/ST2 signaling mediates chronic itch in mice through the astrocytic JAK2-STAT3 cascade.

Interleukin-33 (IL-33) and its receptor ST2 contribute to spinal glial activation and chronic pain. A recent study showed that peripheral IL-33 plays a pivotal role in the pathogenesis of chronic itch induced by poison ivy. However, how IL-33/ST2 signaling in the spinal cord potentially mediates chronic itch remains elusive. Here, we determined that St2-/- substantially reduced scratching behaviors in 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis (ACD) as well as acetone and diethylether followed by water-induced dry skin in mice. Intrathecal administration of the neutralizing anti-ST2 or anti-IL-33 antibody remarkably decreased the scratching response in DNFB-induced ACD mice. Expression of spinal IL-33 and ST2 significantly increased in ACD mice, as evidenced by increased mRNA and protein levels. Immunofluorescence and in situ hybridization demonstrated that increased expression of spinal IL-33 was predominant in oligodendrocytes and astrocytes, whereas ST2 was mainly expressed in astrocytes. Further studies showed that in ACD mice, the activation of astrocytes and increased phosphorylation of signal transducer and activator of transcription 3 (STAT3) were markedly attenuated by St2-/- . Intrathecal injection of Janus Kinase 2 Inhibitor AG490 significantly alleviated scratching behaviors in ACD mice. rIL-33 pretreatment exacerbated gastrin-releasing peptide (GRP)-evoked scratching behaviors. This increased gastrin-releasing peptide receptor (GRPR) expression was abolished by St2-/- . Tnf-α upregulation was suppressed by St2-/- . Our results indicate that the spinal IL-33/ST2 signaling pathway contributes to chronic itch via astrocytic JAK2-STAT3 cascade activation, promoting TNF-α release to regulate the GRP/GRPR signaling-related itch response. Thus, these findings provide a potential therapeutic option for treating chronic pruritus.

Vascular Endothelial Growth Factor A Signaling Promotes Spinal Central Sensitization and Pain-related Behaviors in Female Rats with Bone Cancer.

BACKGROUND: Cancer pain is a pervasive clinical symptom impairing life quality. Vascular endothelial growth factor A has been well studied in tumor angiogenesis and is recognized as a therapeutic target for anti-cancer treatment. This study tested the hypothesis that vascular endothelial growth factor A and vascular endothelial growth factor receptor 2 contribute to bone cancer pain regulation associated with spinal central sensitization. METHODS: This study was performed on female rats using a metastatic breast cancer bone pain model. Nociceptive behaviors were evaluated by mechanical allodynia, thermal hyperalgesia, spontaneous pain, and CatWalk gait analysis. Expression levels were measured by real-time quantitative polymerase chain reaction, western blot, and immunofluorescence analysis. Excitatory synaptic transmission was detected by whole-cell patch-clamp recordings. The primary outcome was the effect of pharmacologic intervention of spinal vascular endothelial growth factor A/vascular endothelial growth factor receptor 2-signaling on bone cancer pain behaviors. RESULTS: The mRNA and protein expression of vascular endothelial growth factor A and vascular endothelial growth factor receptor 2 were upregulated in tumor-bearing rats. Spinal blocking vascular endothelial growth factor A or vascular endothelial growth factor receptor 2 significantly attenuated tumor-induced mechanical allodynia (mean ± SD: vascular endothelial growth factor A, 7.6 ± 2.6 g vs. 5.3 ± 3.3 g; vascular endothelial growth factor receptor 2, 7.8 ± 3.0 g vs. 5.2 ± 3.4 g; n = 6; P < 0.0001) and thermal hyperalgesia (mean ± SD: vascular endothelial growth factor A, 9.0 ± 2.4 s vs. 7.4 ± 2.7 s; vascular endothelial growth factor receptor 2, 9.3 ± 2.5 s vs. 7.5 ± 3.1 s; n = 6; P < 0.0001), as well as spontaneous pain and abnormal gaits. Exogenous vascular endothelial growth factor A enhanced excitatory synaptic transmission in a vascular endothelial growth factor receptor 2-dependent manner, and spinal injection of exogenous vascular endothelial growth factor A was sufficient to cause pain hypersensitivity via vascular endothelial growth factor receptor 2-mediated activation of protein kinase C and Src family kinase in naïve rats. Moreover, spinal blocking vascular endothelial growth factor A/vascular endothelial growth factor receptor 2 pathways suppressed protein kinase C-mediated N-methyl-D-aspartate receptor activation and Src family kinase-mediated proinflammatory cytokine production. CONCLUSIONS: Vascular endothelial growth factor A/vascular endothelial growth factor receptor 2 contributes to central sensitization and bone cancer pain via activation of neuronal protein kinase C and microglial Src family kinase pathways in the spinal cord.

Triggering receptor expressed on myeloid cells 2 (TREM2) dependent microglial activation promotes cisplatin-induced peripheral neuropathy in mice.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common adverse side effect of many antineoplastic agents. Patients treated with chemotherapy often report pain and paresthesias in a "glove-and-stocking" distribution. Diverse mechanisms contribute to the development and maintenance of CIPN. However, the role of spinal microglia in CIPN is not completely understood. In this study, cisplatin-treated mice displayed persistent mechanical allodynia, sensory deficits and decreased density of intraepidermal nerve fibers (IENFs). In the spinal cord, activation of microglia, but not astrocyte, was persistently observed until week five after the first cisplatin injection. Additionally, mRNA levels of inflammation related molecules including IL-1ß, IL-6, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS) and CD16, were increased after cisplatin treatment. Intraperitoneal (i.p.) or intrathecal (i.t.) injection with minocycline both alleviated cisplatin-induced mechanical allodynia and sensory deficits, and prevented IENFs loss. Furthermore, cisplatin enhanced triggering receptor expressed on myeloid cells 2 (TREM2) /DNAX-activating protein of 12 kDa (DAP12) signaling in the spinal cord microglia. The blockage of TREM2 by i.t. injecting anti-TREM2 neutralizing antibody significantly attenuated cisplatin-induced mechanical allodynia, sensory deficits and IENFs loss. Meanwhile, anti-TREM2 neutralizing antibody prominently suppressed the spinal IL-6, TNF-α, iNOS and CD16 mRNA level, but it dramatically up-regulated the anti-inflammatory cytokines IL-4 and IL-10. The data demonstrated that cisplatin triggered persistent activation of spinal cord microglia through strengthening TREM2/DAP12 signaling, which further resulted in CIPN. Functional blockage of TREM2 or inhibition of microglia both benefited for cisplatin-induced peripheral neuropathy. Microglial TREM2/DAP12 may serve as a potential target for CIPN intervention.

Further observations on the behavioral and neural effects of bone marrow stromal cells in rodent pain models.

BACKGROUND: Bone marrow stromal cells (BMSCs) have shown potential to treat chronic pain, although much still needs to be learned about their efficacy and mechanisms of action under different pain conditions. Here, we provide further convergent evidence on the effects of BMSCs in rodent pain models. RESULTS: In an orofacial pain model involving injury of a tendon of the masseter muscle, BMSCs attenuated behavioral pain conditions assessed by von Frey filaments and a conditioned place avoidance test in female Sprague-Dawley rats. The antihyperalgesia of BMSCs in females lasted for <8 weeks, which is shorter than that seen in males. To relate preclinical findings to human clinical conditions, we used human BMSCs. Human BMSCs (1.5 M cells, i.v.) attenuated mechanical and thermal hyperalgesia induced by spinal nerve ligation and suppressed spinal nerve ligation-induced aversive behavior, and the effect persisted through the 8-week observation period. In a trigeminal slice preparation, BMSC-treated and nerve-injured C57B/L mice showed reduced amplitude and frequency of spontaneous excitatory postsynaptic currents, as well as excitatory synaptic currents evoked by electrical stimulation of the trigeminal nerve root, suggesting inhibition of trigeminal neuronal hyperexcitability and primary afferent input by BMSCs. Finally, we observed that GluN2A (N-methyl-D-aspartate receptor subunit 2A) tyrosine phosphorylation and protein kinase Cgamma (PKCg) immunoreactivity in rostral ventromedial medulla was suppressed at 8 weeks after BMSC in tendon-injured rats. CONCLUSIONS: Collectively, the present work adds convergent evidence supporting the use of BMSCs in pain control. As PKCg activity related to N-methyl-D-aspartate receptor activation is critical in opioid tolerance, these results help to understand the mechanisms of BMSC-produced long-term antihyperalgesia, which requires opioid receptors in rostral ventromedial medulla and apparently lacks the development of tolerance.

Glial activation in the periaqueductal gray promotes descending facilitation of neuropathic pain through the p38 MAPK signaling pathway.

The midbrain ventrolateral periaqueductal gray (VL-PAG) is a key component that mediates pain modulation. Although spinal cord glial cells appear to play an important role in chronic pain development, the precise mechanisms involving descending facilitation pathways from the PAG following nerve injury are poorly understood. This study shows that cellular events that occur during glial activation in the VL-PAG may promote descending facilitation from the PAG during neuropathic pain. Chronic constriction nerve injury (CCI) was induced by ligature construction of the sciatic nerve in male Sprague-Dawley rats. Behavioral responses to noxious mechanical (paw withdrawal threshold; PWT) and thermal (paw withdrawal latency; PWL) stimuli were evaluated. After CCI, immunohistochemical and Western blot analysis of microglia and astrocytes in the VL-PAG showed morphological and quantitative changes indicative of activation in microglia and astrocytes. Intra-VL-PAG injection of microglial or astrocytic inhibitors attenuated PWT and PWL at days 7 and 14, respectively, following CCI. We also evaluated the effects of intra-VL-PAG administration of the phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) inhibitor SB 203580 at day 7 after CCI. This treatment abolished microglial activation and produced a significant time-dependent attenuation of PWT and PWL. Western blot analysis showed localized expression of p-p38 in the VL-PAG after CCI. P-p38 was expressed in labeled microglia of the VL-PAG but was not present in astrocytes and neurons on day 7 after CCI. These results demonstrate that CCI-induced neuropathic pain is associated with glial activation in the VL-PAG, which likely participates in descending pain facilitation through the p38 MAPK signaling pathway.

Tachykinin receptor 3 in the lateral habenula alleviates pain and anxiety comorbidity in mice.

The coexistence of chronic pain and anxiety is a common clinical phenomenon. Here, the role of tachykinin receptor 3 (NK3R) in the lateral habenula (LHb) in trigeminal neuralgia and in pain-associated anxiety was systematically investigated. First, electrophysiological recording showed that bilateral LHb neurons are hyperactive in a mouse model of trigeminal neuralgia made by partial transection of the infraorbital nerve (pT-ION). Chemicogenetic activation of bilateral LHb glutamatergic neurons in naive mice induced orofacial allodynia and anxiety-like behaviors, and pharmacological activation of NK3R in the LHb attenuated allodynia and anxiety-like behaviors induced by pT-ION. Electrophysiological recording showed that pharmacological activation of NK3R suppressed the abnormal excitation of LHb neurons. In parallel, pharmacological inhibition of NK3R induced orofacial allodynia and anxiety-like behavior in naive mice. The electrophysiological recording showed that pharmacological inhibition of NK3R activates LHb neurons. Neurokinin B (NKB) is an endogenous high-affinity ligand of NK3R, which binds NK3R and activates it to perform physiological functions, and further neuron projection tracing showed that the front section of the periaqueductal gray (fPAG) projects NKB-positive nerve fibers to the LHb. Optogenetics combined with electrophysiology recordings characterize the functional connections in this fPAG NKB → LHb pathway. In addition, electrophysiological recording showed that NKB-positive neurons in the fPAG were more active than NKB-negative neurons in pT-ION mice. Finally, inhibition of NKB release from the fPAG reversed the analgesic and anxiolytic effects of LHb Tacr3 overexpression in pT-ION mice, indicating that fPAG NKB → LHb regulates orofacial allodynia and pain-induced anxious behaviors. These findings for NK3R suggest the cellular mechanism behind pT-ION in the LHb and suggest that the fPAG NKB → LHb circuit is involved in pain and anxiety comorbidity. This previously unrecognized pathway might provide a potential approach for relieving the pain and anxiety associated with trigeminal neuralgia by targeting NK3R.

Antipruritic effects of geraniol on acute and chronic itch via modulating spinal GABA/GRPR signaling.

BACKGROUND AND PURPOSE: Itch (pruritus) is a common unpleasant feeling, often accompanied by the urge of scratching the skin. It is the main symptom of many systemic and skin diseases, which can seriously affect the patient's quality of life. Geraniol (GE; trans-3,7-dimethyl-2,6-octadien-1-ol) is a natural monoterpene with diverse effects, including anti-inflammatory, antioxidant, neuroprotective, anti-nociceptive, and anticancer properties. The study aims to examine the effects of GE on acute and chronic itch, and explore the underlying mechanisms. METHODS: Acute itch was investigated by using Chloroquine and compound 48/80 induced model, followed by manifestation of diphenylcyclopropenone (DCP)-induced allergic contact dermatitis and the acetone-ether-water (AEW)-induced dry skin model in mice. The scratching behavior, skin thickness, c-Fos expression, and GRPR protein expression in the spinal cord were subsequently monitored and evaluated by behavioral tests as well as pharmacological and pharmacogenetic technologies. RESULTS: Dose-dependent intraperitoneal injection of GE alleviated the acute itch, induced by chloroquine and compound 48/80, as well as increased the spinal c-Fos expression. Intrathecal administration of GE suppressed the GABAA receptor inhibitor bicuculline-induced itch, GRP-induced itch, and the GABAergic neuron inhibition-induced itch. Furthermore, the subeffective dose of bicuculline blocked the anti-pruritic effect of GE on the chloroquine and compound 48/80 induced acute itch. GE also attenuated DCP and AEW-induced chronic itch, as well as the increase of spinal GRPR expression in DCP mice. CONCLUSION AND IMPLICATIONS: GE alleviates both acute and chronic itch via modulating the spinal GABA/GRPR signaling in mice. Findings of this study reveal that GE may provide promising therapeutic options for itch management. Also, considering the pivotal role of essential oils in aromatherapy, GE has great application potential in aromatherapy for treating skin diseases, and especially the skin with severe pruritus.

Foxg1 Modulation of the Prkcd Gene in the Lateral Habenula Mediates Trigeminal Neuralgia-Associated Anxiety-Like Behaviors in Mice.

Trigeminal Neuralgia (TN) is a debilitating disorder frequently accompanied by mood complications such as depression and anxiety. The current study sought to elucidate the molecular underpinnings that contribute to the pathogenesis of TN and its associated anxiety. Employing a partial transection of the infraorbital nerve (pT-ION) in a murine model, we successfully induced sustained primary and secondary orofacial allodynia alongside anxiety-like behavioral manifestations. Transcriptome-wide gene microarray analyses revealed a marked upregulation of Foxg1 subsequent to pT-ION. Targeted knockdown of Foxg1, achieved through bilateral microinjection of adeno-associated virus harboring Foxg1-specific shRNA into the lateral habenula (LHb), resulted in a significant attenuation of both orofacial pain and anxiety-like behaviors. Subsequent RNA sequencing implicated Prkcd as a downstream effector gene modulated by Foxg1. Pharmacological inhibition of protein kinase C delta, encoded by Prkcd, within the LHb markedly ameliorated pT-ION-induced symptomatology. The dual luciferase assay revealed that Foxg1 substantially enhances the transcriptional activity of the Prkcd gene. Collectively, these findings indicate that trigeminal nerve injury leads to Foxg1 upregulation in the LHb, which in turn elevates the expression of Prkcd, culminating in the manifestation of orofacial pain and anxiety-like behaviors. This work offers promising therapeutic targets and a conceptual framework for the clinical management of TN and its psychological comorbidities.

New tricks for an old slug: descending serotonergic system in pain.

A large body of research including animal and human studies has confirmed the crucial role of the serotonin (5-HT) system in the regulation of nociception and chronic pain-related behaviors. In recent years, the functional status of the 5-HT system in descending inhibition and facilitation of spinal nociceptive processing has been reevaluated by novel genetic manipulation techniques and selective agents for 5-HT receptor subtypes. Although these studies shed more light on several aspects of descending 5-HT and spinal 5-HT receptors functioning in descending modulation in pain perception, the current knowledge about the specific role of descending 5-HT system in the induction and maintenance of persistent pain remains fragmentary. In this paper, we review the available data from recent studies of the inhibitory or facilitatory influence from descending 5-HT-spinal 5-HT receptor system in acute and persistent pain, attempt to dissect the involvement of this signaling pathway in neural circuits of maintenance of persistent pain and discuss some issues that need to be considered for further pain research.

Neuronal toll like receptor 9 contributes to complete Freund's adjuvant-induced inflammatory pain in mice.

Toll like receptor 9 (TLR9) is a critical sensor for danger-associated molecular patterns (DAMPs) and a crucial marker of non-sterile/sterile inflammation among all TLRs. However, the significance of TLR9 in inflammatory pain remains unclear. Here, we subcutaneously injected Complete Freund's adjuvant (CFA) into the plantar surface of the hind paw, to established a mouse model of inflammatory pain, and we examined expression and distribution of TLR9 in this model. There was a significant increase of TLR9 mRNA and reduction of mechanical paw withdrawal threshold in mice intraplantar injected with CFA. By contrast, mechanical paw withdrawal threshold significantly increased in mice treated with TLR9 antagonist ODN2088. Furthermore, TLR9 is found predominantly distributed in the neurons by immunofluorescence experiment. Accordingly, neuronal TLR9 downregulation in the spinal cord prevented CFA-induced persistent hyperalgesia. Overall, these findings indicate that neuronal TLR9 in the spinal cord is closely related to CFA-induced inflammatory pain. It provides a potential treatment option for CFA-induced inflammatory pain by applying TLR9 antagonist.

Antiallodynic effects of propentofylline Elicited by interrupting spinal glial function in a rat model of bone cancer pain.

The activation of microglia and astrocytes in the spinal cord is involved in the progress of cancer pain. Propentofylline (PPF), a glial modulating agent, alleviates pain hypersensitivity in neuropathic pain models. The present study investigated the potential roles of PPF in a preclinical rat model of bone caner pain established by inoculating Walker 256 cells into the left tibia. At day 9 postinoculation, single administration of PPF (10 µg/10 µl, i.t.) significantly but transiently suppressed mechanical allodynia induced by bone cancer. Repeated application of PPF (10 µg/10 µl, i.t., once daily from days 9 to 12) persistently relieved mechanical allodynia on the side ipsilateral to surgery. Immunohistochemistry and ELISA showed that microglia and astrocytes in the spinal cord were activated, and the production of glia-derived proinflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) markedly increased at day 12 postinoculation in the cancer group. Intrathecal injection of PPF (10 µg/10 µl) significantly inhibited the activation of spinal glial cells and the expression of proinflammatory cytokines. These results suggest that the glial modulating agent PPF has antiallodynic effects on bone cancer pain and has potential utility for clinical treatment of cancer pain.

Glial IL-33 signaling through an ST2-to-CXCL12 pathway in the spinal cord contributes to morphine-induced hyperalgesia and tolerance.

Morphine and other opiates are highly effective for treating moderate to severe pain. However, morphine-induced hyperalgesia and analgesic tolerance prevent durable efficacy in patients. Here, we investigated the underlying molecular mechanisms of this phenomenon. We found that repeated subcutaneous injections of morphine in mice increased the abundance of the cytokine interleukin-33 (IL-33) primarily in oligodendrocytes and astrocytes and that of its receptor ST2 mainly in astrocytes. Pharmacological inhibition or knockdown of IL-33 or ST2 in the spinal cord attenuated morphine-induced hyperalgesia and analgesic tolerance in mice, as did global knockout of either Il33 or St2, which also reduced morphine-enhanced astroglial activation and excitatory synaptic transmission. Furthermore, a pathway mediated by tumor necrosis factor receptor­associated factor 6 (TRAF6) and the kinase JNK in astrocytes was required for IL-33­mediated hyperalgesia and tolerance through promoting the production of the chemokine CXCL12 in the spinal cord. The findings suggest that targeting IL-33­ST2 signaling could enable opioids to produce sustained analgesic effects in chronic pain management.

Involvement of microglial P2X7 receptors and downstream signaling pathways in long-term potentiation of spinal nociceptive responses.

Tetanic stimulation of the sciatic nerve (TSS) produces long-term potentiation (LTP) of C-fiber-evoked field potentials in the spinal cord. This potentiation is considered to be a substrate for long-lasting sensitization in the spinal pain pathway. Because microglia have previously been shown to regulate the induction of spinal LTP, we hypothesize that P2X7 receptors (P2X7R), which are predominantly expressed in microglia and participate in the communication between microglia and neurons, may play a role in this induction. This study investigated the potential roles of P2X7Rs in spinal LTP and persistent pain induced by TSS in rats. OxATP or BBG, a P2X7R antagonist, prevented the induction of spinal LTP both in vivo and in spinal cord slices in vitro and alleviated mechanical allodynia. Down-regulation of P2X7Rs with P2X7-siRNA blocked the induction of spinal LTP and inhibited mechanical allodynia. Double immunofluorescence showed colocalization of P2X7Rs with the microglial marker OX-42, but not with the astrocytic marker GFAP or the neuronal marker NeuN. Intrathecal injection of BBG suppressed the up-regulation of microglial P2X7Rs and increased expression of Fos in the spinal superficial dorsal horn. Further, pre-administration of BBG inhibited increased expression of the microglial marker Iba-1, phosphorylated p38 (p-p38), interleukin 1ß (IL-1ß) and GluR1 following TSS. Pre-administration of the IL-1 receptor antagonist (IL-1ra) blocked both the induction of spinal LTP and the up-regulation of GluR1. These results suggest that microglial P2X7Rs and its downstream signaling pathways play a pivotal role in the induction of spinal LTP and persistent pain induced by TSS.