Photobiomodulation inhibits neuronal firing in the superficial but not deep layer of a rat spinal dorsal horn.

Photobiomodulation (PBM) has attracted attention as a treatment for chronic pain. Previous studies have reported that PBM of the sciatic nerve inhibits neuronal firing in the superficial layers (lamina I-II) of the spinal dorsal horn of rats, which is evoked by mechanical stimulation that corresponds to noxious stimuli. However, the effects of PBM on the deep layers (lamina III-IV) of the spinal dorsal horn, which receive inputs from innocuous stimuli, remain poorly understood. In this study, we examined the effect of PBM of the sciatic nerve on firing in the deep layers of the spinal dorsal horn evoked by mechanical stimulation. Before and after PBM, mechanical stimulation was administered to the cutaneous receptive field using 0.6-26.0 g von Frey filaments (vFFs), and vFF-evoked firing in the deep layers of the spinal dorsal horn was recorded. The vFF-evoked firing frequencies were not altered after the PBM for any of the vFFs. The inhibition rate for 26.0 g vFF-evoked firing was approximately 13 % in the deep layers and 70 % in the superficial layers. This suggests that PBM selectively inhibits the transmission of pain information without affecting the sense of touch. PBM has the potential to alleviate pain while preserving the sense of touch.

Neutrophil extracellular traps as a unique target in the treatment of inflammatory pain.

Pain is a widespread motivation for seeking healthcare and stands as a substantial global public health concern. Despite comprehensive investigations into the mechanisms of pain sensitization induced by inflammation, efficacious treatments options remain scarce. Neutrophil extracellular traps (NETs) have been associated with the progression and tissue damage of diverse inflammatory diseases. This study aims to explore the impact of NETs on the progression of inflammatory pain and explore potential therapeutic approaches. Initially, we observed neutrophil infiltration and the formation of NETs in the left hind paw of mice with inflammatory pain induced by complete Freund's adjuvant (CFA). Furthermore, we employed the peptidyl arginine deiminase 4 (PAD4) inhibitor Cl-amidine (diluted at 50 mg/kg in saline, administered via tail vein injection once daily for three days) to impede NETs formation and administered DNase1 (diluted at 10 mg/kg in saline, once daily for three days) to break down NETs. We investigated the pathological importance of peripheral NETs formation in inflammatory pain and its influence on the activation of spinal dorsal horn microglia. The findings indicate that neutrophils infiltrating locally generate NETs, leading to an increased release of inflammatory mediators that worsen peripheral inflammatory reactions. Consequently, this results in the transmission of more harmful peripheral stimuli to the spinal cord, triggering microglial activation and NF-κB phosphorylation, thereby escalating neuroinflammation and fostering pain sensitization. Suppression of peripheral NETs can mitigate peripheral inflammation in mice with inflammatory pain, reverse mechanical and thermal hypersensitivity by suppressing microglial activation in the spinal cord, ultimately diminishing inflammatory pain. In conclusion, these discoveries propose that obstructing or intervening with NETs introduces a novel therapeutic avenue for addressing inflammatory pain.

Spinal nerve transection-induced upregulation of SAP97 via promoting membrane trafficking of GluA1-containing AMPA receptors in the dorsal horn contributes to the pathogenesis of neuropathic pain.

Emerging evidence has implicated an important role of synapse-associated protein-97 (SAP97)-regulated GluA1-containing AMPARs membrane trafficking in cocaine restate and in contextual episodic memory of schizophrenia. Herein, we investigated the role of SAP97 in neuropathic pain following lumbar 5 spinal nerve transection (SNT) in rats. Our results showed that SNT led to upregulation of SAP97, enhanced the interaction between SAP97 and GluA1, and increased GluA1-containing AMPARs membrane trafficking in the dorsal horn. Microinjection of AAV-EGFP-SAP97 shRNA in lumbar 5 spinal dorsal horn inhibited SAP97 production, decreased SAP97-GluA1 interaction, reduced the membrane trafficking of GluA1-containing AMPARs, and partially attenuated neuropathic pain following SNT. Intrathecal injections of SAP97 siRNA or NASPM, an antagonist of GluA1-containing AMPARs, also partially reversed neuropathic pain on day 7, but not on day 14, after SNT. Spinal overexpression of SAP97 by AAV-EGFP-SAP97 enhanced SAP97-GluA1 interaction, increased the membrane insertion of GluA1-containing AMPARs, and induced abnormal pain in naïve rats. In addition, treatment with SAP97 siRNA or NASPM i.t. injection alleviated SNT-induced allodynia and hyperalgesia and exhibited a longer effect in female rats. Together, our results indicate that the SNT-induced upregulation of SAP97 via promoting GluA1-containing AMPARs membrane trafficking in the dorsal horn contributes to the pathogenesis of neuropathic pain. Targeting spinal SAP97 might be a promising therapeutic strategy to treatment of chronic pain.

Neural Circuitry Polarization in the Spinal Dorsal Horn (SDH): A Novel Form of Dysregulated Circuitry Plasticity during Pain Pathogenesis.

Pathological pain emerges from nociceptive system dysfunction, resulting in heightened pain circuit activity. Various forms of circuitry plasticity, such as central sensitization, synaptic plasticity, homeostatic plasticity, and excitation/inhibition balance, contribute to the malfunction of neural circuits during pain pathogenesis. Recently, a new form of plasticity in the spinal dorsal horn (SDH), named neural circuit polarization (NCP), was discovered in pain models induced by HIV-1 gp120 and chronic morphine administration. NCP manifests as an increase in excitatory postsynaptic currents (EPSCs) in excitatory neurons and a decrease in EPSCs in inhibitory neurons, presumably facilitating hyperactivation of pain circuits. The expression of NCP is associated with astrogliosis. Ablation of reactive astrocytes or suppression of astrogliosis blocks NCP and, concomitantly, the development of gp120- or morphine-induced pain. In this review, we aim to compare and integrate NCP with other forms of plasticity in pain circuits to improve the understanding of the pathogenic contribution of NCP and its cooperation with other forms of circuitry plasticity during the development of pathological pain.

Development and characterization of a Gucy2d-cre mouse to selectively manipulate a subset of inhibitory spinal dorsal horn interneurons.

Recent transcriptomic studies identified Gucy2d (encoding guanylate cyclase D) as a highly enriched gene within inhibitory dynorphin interneurons in the mouse spinal dorsal horn. To facilitate investigations into the role of the Gucy2d+ population in somatosensation, Gucy2d-cre transgenic mice were created to permit chemogenetic or optogenetic manipulation of this subset of spinal neurons. Gucy2d-cre mice created via CRISPR/Cas9 genomic knock-in were bred to mice expressing a cre-dependent reporter (either tdTomato or Sun1.GFP fusion protein), and the resulting offspring were characterized. Surprisingly, a much wider population of spinal neurons was labeled by cre-dependent reporter expression than previous mRNA-based studies would suggest. Although the cre-dependent reporter expression faithfully labeled ~75% of cells expressing Gucy2d mRNA in the adult dorsal horn, it also labeled a substantial number of additional inhibitory neurons in which no Gucy2d or Pdyn mRNA was detected. Moreover, cre-dependent reporter was also expressed in various regions of the brain, including the spinal trigeminal nucleus, cerebellum, thalamus, somatosensory cortex, and anterior cingulate cortex. Injection of AAV-CAG-FLEX-tdTomato viral vector into adult Gucy2d-cre mice produced a similar pattern of cre-dependent reporter expression in the spinal cord and brain, which excludes the possibility that the unexpected reporter-labeling of cells in the deep dorsal horn and brain was due to transient Gucy2d expression during early stages of development. Collectively, these results suggest that Gucy2d is expressed in a wider population of cells than previously thought, albeit at levels low enough to avoid detection with commonly used mRNA-based assays. Therefore, it is unlikely that these Gucy2d-cre mice will permit selective manipulation of inhibitory signaling mediated by spinal dynorphin interneurons, but this novel cre driver line may nevertheless be useful to target a broader population of inhibitory spinal dorsal horn neurons.

Dual effect of anandamide on spinal nociceptive transmission in control and inflammatory conditions.

Anandamide (AEA) is an important modulator of nociception in the spinal dorsal horn, acting presynaptically through Cannabinoid (CB1) and Transient receptor potential vanilloid (TRPV1) receptors. The role of AEA (1 µM, 10 µM, and 30 µM) application on the modulation of nociceptive synaptic transmission under control and inflammatory conditions was studied by recording miniature excitatory postsynaptic currents (mEPSCs) from neurons in spinal cord slices. Inhibition of the CB1 receptors by PF514273, TRPV1 by SB366791, and the fatty acid amide hydrolase (FAAH) by URB597 was used. Under naïve conditions, the AEA application did not affect the mEPSCs frequency (1.43±0.12 Hz) when all the recorded neurons were considered. The mEPSC frequency increased (180.0±39.2%) only when AEA (30 µM) was applied with PF514273 and URB597. Analysis showed that one sub-population of neurons had synaptic input inhibited (39.1% of neurons), the second excited (43.5%), whereas 8.7% showed a mixed effect and 8.7% did not respond to the AEA. With inflammation, the AEA effect was highly inhibitory (72.7%), while the excitation was negligible (9.1%), and 18.2% were not modulated. After inflammation, more neurons (45.0%) responded even to low AEA by mEPSC frequency increase with PF514273/URB597 present. AEA-induced dual (excitatory/inhibitory) effects at the 1st nociceptive synapse should be considered when developing analgesics targeting the endocannabinoid system. These findings contrast the clear inhibitory effects of the AEA precursor 20:4-NAPE application described previously and suggest that modulation of endogenous AEA production may be more favorable for analgesic treatments.

Alterations in the spinal cord, trigeminal nerve ganglion, and infraorbital nerve through inducing compression of the dorsal horn region at the upper cervical cord in trigeminal neuralgia.

BACKGROUND: Idiopathic trigeminal neuralgia (TN) cases encountered frequently in daily practice indicate significant gaps that still need to be illuminated in the etiopathogenesis. In this study, a novel TN animal model was developed by compressing the dorsal horn (DH) of the upper cervical spinal cord. METHODS: Eighteen rabbits were equally divided into three groups, namely control (CG), sham (SG), and spinal cord compression (SCC) groups. External pressure was applied to the left side at the C3 level in the SCC group. Dorsal hemilaminectomy was performed in the SG, and the operative side was closed without compression. No procedure was implemented in the control group. Samples from the SC, TG, and ION were taken after seven days. For the histochemical staining, damage and axons with myelin were scored using Hematoxylin and Eosin and Toluidine Blue, respectively. Immunohistochemistry, nuclei, apoptotic index, astrocyte activity, microglial labeling, and CD11b were evaluated. RESULTS: Mechanical allodynia was observed on the ipsilateral side in the SCC group. In addition, both the TG and ION were partially damaged from SC compression, which resulted in significant histopathological changes and increased the expression of all markers in both the SG and SCC groups compared to that in the CG. There was a notable increase in tissue damage, an increase in the number of apoptotic nuclei, an increase in the apoptotic index, an indication of astrocytic gliosis, and an upsurge in microglial cells. Significant increases were noted in the SG group, whereas more pronounced significant increases were observed in the SCC group. Transmission electron microscopy revealed myelin damage, mitochondrial disruption, and increased anchoring particles. Similar changes were observed to a lesser extent in the contralateral spinal cord. CONCLUSION: Ipsilateral trigeminal neuropathic pain was developed due to upper cervical SCC. The clinical finding is supported by immunohistochemical and ultrastructural changes. Thus, alterations in the DH due to compression of the upper cervical region should be considered as a potential cause of idiopathic TN.

Multimodal sensory control of motor performance by glycinergic interneurons of the mouse spinal cord deep dorsal horn.

Sensory feedback is integral for contextually appropriate motor output, yet the neural circuits responsible remain elusive. Here, we pinpoint the medial deep dorsal horn of the mouse spinal cord as a convergence point for proprioceptive and cutaneous input. Within this region, we identify a population of tonically active glycinergic inhibitory neurons expressing parvalbumin. Using anatomy and electrophysiology, we demonstrate that deep dorsal horn parvalbumin-expressing interneuron (dPV) activity is shaped by convergent proprioceptive, cutaneous, and descending input. Selectively targeting spinal dPVs, we reveal their widespread ipsilateral inhibition onto pre-motor and motor networks and demonstrate their role in gating sensory-evoked muscle activity using electromyography (EMG) recordings. dPV ablation altered limb kinematics and step-cycle timing during treadmill locomotion and reduced the transitions between sub-movements during spontaneous behavior. These findings reveal a circuit basis by which sensory convergence onto dorsal horn inhibitory neurons modulates motor output to facilitate smooth movement and context-appropriate transitions.

Chemogenetics Modulation of Electroacupuncture Analgesia in Mice Spared Nerve Injury-Induced Neuropathic Pain through TRPV1 Signaling Pathway.

Neuropathic pain, which is initiated by a malfunction of the somatosensory cortex system, elicits inflammation and simultaneously activates glial cells that initiate neuroinflammation. Electroacupuncture (EA) has been shown to have therapeutic effects for neuropathic pain, although with uncertain mechanisms. We suggest that EA can reliably cure neuropathic disease through anti-inflammation and transient receptor potential V1 (TRPV1) signaling pathways from the peripheral to the central nervous system. To explore this, we used EA to treat the mice spared nerve injury (SNI) model and explore the underlying molecular mechanisms through novel chemogenetics techniques. Both mechanical and thermal pain were found in SNI mice at four weeks (mechanical: 3.23 ± 0.29 g; thermal: 4.9 ± 0.14 s). Mechanical hyperalgesia was partially attenuated by 2 Hz EA (mechanical: 4.05 ± 0.19 g), and thermal hyperalgesia was fully reduced (thermal: 6.22 ± 0.26 s) but not with sham EA (mechanical: 3.13 ± 0.23 g; thermal: 4.58 ± 0.37 s), suggesting EA's specificity. In addition, animals with Trpv1 deletion showed partial mechanical hyperalgesia and no significant induction of thermal hyperalgesia in neuropathic pain mice (mechanical: 4.43 ± 0.26 g; thermal: 6.24 ± 0.09 s). Moreover, we found increased levels of inflammatory factors such as interleukin-1 beta (IL1-ß), IL-3, IL-6, IL-12, IL-17, tumor necrosis factor alpha, and interferon gamma after SNI modeling, which decreased in the EA and Trpv1-/- groups rather than the sham group. Western blot and immunofluorescence analysis showed similar tendencies in the dorsal root ganglion, spinal cord dorsal horn, somatosensory cortex (SSC), and anterior cingulate cortex (ACC). In addition, a novel chemogenetics method was used to precisely inhibit SSC to ACC activity, which showed an analgesic effect through the TRPV1 pathway. In summary, our findings indicate a novel mechanism underlying neuropathic pain as a beneficial target for neuropathic pain.

[Tacrolimus Induces Pain by Upregulating the Synaptic Expression of AMPA Receptors in the Spinal Cord Dorsal Horn]. / ä»–å ‹èŽ«å¸é€šè¿‡ä¸Šè°ƒè„Šé«“èƒŒè§’AMPA受体的突触表达诱发疼痛.

Objective: To investigate the effects of long-term administration of tacrolimus (also known as FK506) on the pain-related behaviors in mice and to study the underlying mechanism of pain induced by FK506 via measuring the effect of FK506 on the synaptic expression and phosphorylation of alpha-amino-3-hyroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the spinal cord dorsal horn of mice. Methods: 1) A total of 24 mice were evenly and randomly assigned to two groups, a FK506 group and a Saline group. The FK506 group was given daily intraperitoneal injection of FK506 and the Saline group received normal saline. Both groups received injection once a day for 7 days in a row. Some of the mice ( n=6 in each group) were monitored for the changes in the paw withdrawal threshold (PWT), the paw withdrawal latency (PWL), and the spontaneous pain behaviors to establish the pain model. The other mice ( n=6 in each group) of each group underwent isolation of the dorsal horn when obvious pain symptoms were induced on day 7 of injection. Then, immunoblotting was performed to determine the synaptic expression and phosphorylation levels of GluA1 and GluA2 subunits of AMPA receptors. 2) The mice were randomly divided into two groups, FK506+calcineurin (CaN) group and FK506+Saline group ( n=6 in each group). After the pain model was constructed, the mice were given intrathecal injection of recombinant CaN (also know as 33 U) or normal saline. Then, 60 minutes later, the PWT and the PWL of the mice were measured to investigate the role of CaN in FK506-induced pain. 3) Another18 mice were selected. The mice were randomly and evenly assigned to three groups, a control group (receiving intraperitoneal injection of normal saline followed by intrathecal injection of normal saline), FK506+Saline group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of normal saline) and FK506+CaN group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of CaN). Then, 60 minutes later, the spinal cords were isolated and subjected to immunoblotting assay to determine the role of CaN in FK506-induced AMPA receptor modification. Results: 1) After 7 consecutive days of intraperitoneal injection of FK506, the PWT and PWL of mice dropped significantly, reaching on day 7 as low as 22.3%±0.05% and 66.6%±0.05% of the control group, respectively ( P<0.01). The FK506-treated mice displayed evident spontaneous pain behavior, presenting significantly increased licking activities ( P<0.01). These results indicated that FK506-induced pain model was successfully established. Immunoblotting assay showed that the total expressions of GluA1 and GluA2 subunits in the spinal dorsal horn of the FK506 group remained unchanged in comparison with those of the Saline group. However, FK506 specifically induced an increase in the synaptic expression of GluA1. In addition, the phosphorylation levels of GluA1 at Ser845 and Ser831 in FK506-treated mice were significantly increased in comparison with those of the control group ( P<0.05). 2) Compared with those of the mice in the FK506+Saline group, the PWT and the PWL of mice in the FK506+CaN group were significantly increased ( P<0.05). 3) Compared with those of the FK506+Saline group, the synaptic expression of GluA1 were decreased in FK506+CaN group ( P<0.01) and the phosphorylation levels of GluA1 at Ser845 and Ser831 were significantly downregulated ( P<0.001). Conclusion: The hyper-expression and hyperphosphorylation of GluA1 subunit in the spinal cord dorsal horn resulting from CaN inhibition contributes to the FK506-induced pain syndrome. FK506 induces the synaptic hyper-expression and hyperphosphorylation of GluA1 in the dorsal horn of the spinal cord through CaN inhibition, thereby inducing pain.

Esketamine inhibits the c-Jun N-terminal kinase pathway in the spinal dorsal horn to relieve bone cancer pain in rats.

Cancer-induced bone pain (CIBP) is one of the most common and feared symptoms in patients with advanced tumors. The X-C motif chemokine ligand 12 (CXCL12) and the CXCR4 receptor have been associated with glial cell activation in bone cancer pain. Moreover, mitogen-activated protein kinases (MAPKs), as downstream CXCL12/CXCR4 signals, and c-Jun, as activator protein AP-1 components, contribute to the development of various types of pain. However, the specific CIBP mechanisms remain unknown. Esketamine is a non-selective N-methyl-d-aspartic acid receptor (NMDA) inhibitor commonly used as an analgesic in the clinic, but its analgesic mechanism in bone cancer pain remains unclear. We used a tumor cell implantation (TCI) model and explored that CXCL12/CXCR4, p-MAPKs, and p-c-Jun were stably up-regulated in the spinal cord. Immunofluorescence images showed activated microglia in the spinal cord on day 14 after TCI and co-expression of CXCL12/CXCR4, p-MAPKs (p-JNK, p-ERK, p-p38 MAPK), and p-c-Jun in microglia. Intrathecal injection of the CXCR4 inhibitor AMD3100 reduced JNK and c-Jun phosphorylations, and intrathecal injection of the JNK inhibitor SP600125 and esketamine also alleviated TCI-induced pain and reduced the expression of p-JNK and p-c-Jun in microglia. Overall, our data suggest that the CXCL12/CXCR4-JNK-c-Jun signaling pathway of microglia in the spinal cord mediates neuronal sensitization and pain hypersensitivity in cancer-induced bone pain and that esketamine exerts its analgesic effect by inhibiting the JNK-c-Jun pathway.

BDNF in Neuropathic Pain; the Culprit that Cannot be Apprehended.

In males but not in females, brain derived neurotrophic factor (BDNF) plays an obligatory role in the onset and maintenance of neuropathic pain. Afferent terminals of injured peripheral nerves release colony stimulating factor (CSF-1) and other mediators into the dorsal horn. These transform the phenotype of dorsal horn microglia such that they express P2X4 purinoceptors. Activation of these receptors by neuron-derived ATP promotes BDNF release. This microglial-derived BDNF increases synaptic activation of excitatory dorsal horn neurons and decreases that of inhibitory neurons. It also alters the neuronal chloride gradient such the normal inhibitory effect of GABA is converted to excitation. By as yet undefined processes, this attenuated inhibition increases NMDA receptor function. BDNF also promotes the release of pro-inflammatory cytokines from astrocytes. All of these actions culminate in the increase dorsal horn excitability that underlies many forms of neuropathic pain. Peripheral nerve injury also alters excitability of structures in the thalamus, cortex and mesolimbic system that are responsible for pain perception and for the generation of co-morbidities such as anxiety and depression. The weight of evidence from male rodents suggests that this preferential modulation of excitably of supra-spinal pain processing structures also involves the action of microglial-derived BDNF. Possible mechanisms promoting the preferential release of BDNF in pain signaling structures are discussed. In females, invading T-lymphocytes increase dorsal horn excitability but it remains to be determined whether similar processes operate in supra-spinal structures. Despite its ubiquitous role in pain aetiology neither BDNF nor TrkB receptors represent potential therapeutic targets.

High mobility group box-1: A therapeutic target for analgesia and associated symptoms in chronic pain.

The number of patients with chronic pain continues to increase against the background of an ageing society and a high incidence of various epidemics and disasters. One factor contributing to this situation is the absence of truly effective analgesics. Chronic pain is a persistent stress for the organism and can trigger a variety of neuropsychiatric symptoms. Hence, the search for useful analgesic targets is currently being intensified worldwide, and it is anticipated that the key to success may be molecules involved in emotional as well as sensory systems. High mobility group box-1 (HMGB1) has attracted attention as a therapeutic target for a variety of diseases. It is a very unique molecule having a dual role as a nuclear protein while also functioning as an inflammatory agent outside the cell. In recent years, numerous studies have shown that HMGB1 acts as a pain inducer in primary sensory nerves and the spinal dorsal horn. In addition, HMGB1 can function in the brain, and is involved in the symptoms of depression, anxiety and cognitive dysfunction that accompany chronic pain. In this review, we will summarize recent research and discuss the potential of HMGB1 as a useful drug target for chronic pain.

Methylglyoxal activates transient receptor potential A1/V1 via reactive oxygen species in the spinal dorsal horn.

Methylglyoxal (MGO), a highly reactive dicarbonyl metabolite of glucose primarily formed during the glycolytic pathway, is a precursor of advanced glycation end-products (AGEs). Recently, numerous studies have shown that MGO accumulation can cause pain and hyperalgesia. However, the mechanism through which MGO induces pain in the spinal dorsal horn remains unclear. The present study investigated the effect of MGO on spontaneous excitatory postsynaptic currents (sEPSC) in rat spinal dorsal horn neurons using blind whole-cell patch-clamp recording. Perfusion of MGO increased the frequency and amplitude of sEPSC in spinal horn neurons in a concentration-dependent manner. Additionally, MGO administration increased the number of miniature EPSC (mEPSC) in the presence of tetrodotoxin, a sodium channel blocker. However, 6-cyano-7-nitroqiunocaline-2,3-dione (CNQX), an AMPA/kainate receptor antagonist, blocked the enhancement of sEPSC by MGO. HC-030031, a TRP ankyrin-1 (TRPA1) antagonist, and capsazepine, a TRP vanilloid-1 (TRPV1) antagonist, inhibited the action of MGO. Notably, the effects of MGO were completely inhibited by HC-030031 and capsazepine. MGO generates reactive oxygen species (ROS) via AGEs. ROS also potentially induce pain via TRPA1 and TRPV1 in the spinal dorsal horn. Furthermore, we examined the effect of MGO in the presence of N-tert-butyl-α-phenylnitrone (PBN), a non-selective ROS scavenger, and found that the effect of MGO was completely inhibited. These results suggest that MGO increases spontaneous glutamate release from the presynaptic terminal to spinal dorsal horn neurons through TRPA1, TRPV1, and ROS and could enhance excitatory synaptic transmission.

Suppression of HCN channels in the spinal dorsal horn restores KCC2 expression and attenuates diabetic neuropathic pain.

Previous studies have shown that the hyperpolarized cyclic nucleotide gated (HCN) ion channels in the spinal dorsal horn (SDH) might be involved in the development of diabetic neuropathic pain (DNP). Additionally, other studies have shown that the decreased potassium-chloride cotransporter 2 (KCC2) expression in the SDH promotes pain hypersensitivity. Both HCN channels and KCC2 were highly expressed in spinal substantia gelatinosa neurons. However, whether the K+ efflux induced by the activation of HCN channels in DNP modulate KCC2 function and subsequently affect the role of γ-aminobutyric acid (GABA)/GABA-A receptors of neurons in the SDH remains to be clarified. The purpose of this work was to investigate the underlying mechanisms of KCC2 participating in HCN channels to promote DNP. Here, we found that the analgesic role of HCN channels blocker ZD7288 was associated with the up-regulated KCC2 expression and could be prevented by DIOA, a KCC2 blocker. Furthermore, the level of GABA in DNP rats significantly increased, which was decreased by ZD72288. Moreover, DIOA pretreatment could partly block the inhibitory effect of ZD7288 on the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signaling activation of DNP rats. Finally, inhibition of cAMP-PKA signaling alleviated allodynia and elevated KCC2 expression in DNP rats. Altogether, this study reveals that the role of cAMP-PKA signaling-regulated HCN channels in DNP associated with decreased KCC2 expression in the spinal cord and altered GABA nature.

Nerve injury disrupts temporal processing in the spinal cord dorsal horn through alterations in PV+ interneurons.

How mechanical allodynia following nerve injury is encoded in patterns of neural activity in the spinal cord dorsal horn (DH) remains incompletely understood. We address this in mice using the spared nerve injury model of neuropathic pain and in vivo electrophysiological recordings. Surprisingly, despite dramatic behavioral over-reactivity to mechanical stimuli following nerve injury, an overall increase in sensitivity or reactivity of DH neurons is not observed. We do, however, observe a marked decrease in correlated neural firing patterns, including the synchrony of mechanical stimulus-evoked firing, across the DH. Alterations in DH temporal firing patterns are recapitulated by silencing DH parvalbumin+ (PV+) interneurons, previously implicated in mechanical allodynia, as are allodynic pain-like behaviors. These findings reveal decorrelated DH network activity, driven by alterations in PV+ interneurons, as a prominent feature of neuropathic pain and suggest restoration of proper temporal activity as a potential therapeutic strategy to treat chronic neuropathic pain.

12-(S)-Hydroxyeicosatetraenoic Acid and GPR31 Signaling in Spinal Cord in Neuropathic Pain.

Neuropathic pain is a pressing unmet medical need requiring novel nonopioid-based therapeutic approaches. Using unbiased transcriptomic analysis, we found that the expression of Gpr31, a G protein-coupled receptor, increased in the dorsal horn of the spinal cord in rats with traumatic nerve injury-induced neuropathic pain. Daily intrathecal injections of siGpr31 reversed behavioral hypersensitivities in a time-dependent manner. GPR31, a Gα i protein-coupled receptor, has recently been cloned and is a receptor for 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE]. The lack of commercially available GPR31 antagonists has hampered the understanding of this receptor in pathophysiological states, including pain. To investigate this, our first approach was to identify novel GPR31 antagonists. Using a multidisciplinary approach, including in silico modeling, we identified the first highly potent and selective small-molecule GPR31 antagonist, SAH2. Here, we characterize the pharmacological activity in well-described models of neuropathic pain in rodents and provide evidence that 12-(S)-HETE/GPR31-dependent behavioral hypersensitivities are mediated through mitogen-activated protein kinase (MAPK) activation in the spinal cord. Our studies provide the pharmacological rationale for investigating contributions of GPR31 along the pain neuroaxis and the development of nonopioid GPR31-targeted strategies. SIGNIFICANCE STATEMENT: We have identified the first highly selective GPR31 antagonist. Using this antagonist, we have demonstrated that GPR31 signaling in the spinal cord is pronociceptive and MAPK pathways provided signaling mechanisms downstream of GPR31 activation in these processes.

Inhibition of aquaporin-4 and its subcellular localization attenuates below-level central neuropathic pain by regulating astrocyte activation in a rat spinal cord injury model.

The mechanisms of central neuropathic pain (CNP) caused by spinal cord injury have not been sufficiently studied. We have found that the upregulation of astrocytic aquaporin-4 (AQP4) aggravated peripheral neuropathic pain after spinal nerve ligation in rats. Using a T13 spinal cord hemisection model, we showed that spinal AQP4 was markedly upregulated after SCI and mainly expressed in astrocytes in the spinal dorsal horn (SDH). Inhibition of AQP4 with TGN020 suppressed astrocyte activation, attenuated the development and maintenance of below-level CNP and promoted motor function recovery in vivo. In primary astrocyte cultures, TGN020 also changed cell morphology, diminished cell proliferation and suppressed astrocyte activation. Moreover, T13 spinal cord hemisection induced cell-surface abundance of the AQP4 channel and perivascular localization in the SDH. Targeted inhibition of AQP4 subcellular localization with trifluoperazine effectively diminished astrocyte activation in vitro and further ablated astrocyte activation, attenuated the development and maintenance of below-level CNP, and accelerated functional recovery in vivo. Together, these results provide mechanistic insights into the roles of AQP4 in the development and maintenance of below-level CNP. Intervening with AQP4, including targeting AQP4 subcellular localization, might emerge as a promising agent to prevent chronic CNP after SCI.

Chronic Adaptations in the Dorsal Horn Following a Cervical Spinal Cord Injury in Primates.

Spinal cord injury (SCI) is devastating, with limited treatment options and variable outcomes. Most in vivo SCI research has focused on the acute and early post-injury periods, and the promotion of axonal growth, so little is understood about the clinically stable chronic state, axonal growth over time, and what plasticity endures. Here, we followed animals into the chronic phase following SCI, to address this gap. Male macaques received targeted deafferentation, affecting three digits of one hand, and were divided into short (4-6 months) or long-term (11-12 months) groups, based on post-injury survival times. Monkeys were assessed behaviorally, where possible, and all exhibited an initial post-injury deficit in manual dexterity, with gradual functional recovery over 2 months. We previously reported extensive sprouting of somatosensory corticospinal (S1 CST) fibers in the dorsal horn in the first five post-injury months. Here, we show that by 1 year, the S1 CST sprouting is pruned, with the terminal territory resembling control animals. This was reflected in the number of putatively "functional" synapses observed, which increased over the first 4-5 months, and then returned to baseline by 1 year. Microglia density also increased in the affected dorsal horn at 4-6 months and then decreased, but did not return to baseline by 1 year, suggesting refinement continues beyond this time. Overall, there is a long period of reorganization and consolidation of adaptive circuitry in the dorsal horn, extending well beyond the initial behavioral recovery. This provides a potential window to target therapeutic opportunities during the chronic phase.

Protectin D1 ameliorates non-compressive lumbar disc herniation through SIRT1-mediated CGRP signaling.

Background. Neuro-inflammatory response promotes the initiation and sustenance of lumbar disc herniation (LDH). Protectin D1 (PD1), as a new type of specialized pro-resolving mediator (SPM), can improve the prognosis of various inflammatory diseases. Recent studies have shown that over representation of calcitonin gene-related peptides (CGRP) may activate nociceptive signaling following nerve injury. Silent information regulator 1 (SIRT1) is ubiquitously expressed in the dorsal horn of the spinal cord and plays a role in the pathogenesis of LDH. In this study, we investigated the analgesic effects of PD1 and elucidated the impact of neurogenic inflammation in the pathogenesis of neuropathic pain induced by non-compressive lumbar disc herniation (NCLDH) in a rat model. Methods. NCLDH models were established by applying protruding autologous nucleus pulposus to the L5 Dorsal root ganglion (DRG). PD1, SIRT1 antagonist or agonist, CGRP or antagonist were administered as daily intrathecal injections for three consecutive days postoperatively. Behavioral tests were conducted to assess mechanical and thermal hyperalgesia. The ipsilateral lumbar (L4-6) segment of the spinal dorsal horn was isolated for further analysis. Alterations in the release of SIRT1 and CGRP were explored using western blot and immunofluorescence. Results. Application of protruded nucleus (NP) materials to the DRG induced mechanical and thermal allodynia symptoms, and deregulated the expression of pro-inflammatory and anti-inflammatory cytokines in rats. Intrathecal delivery of PD1 significantly reversed the NCLDH-induced imbalance in neuro-inflammatory response and alleviated the symptoms of mechanical and thermal hyperalgesia. In addition, NP application to the DGRs resulted the spinal upregulation of CGRP and SIRT1 expression, which was almost restored by intrathecal injection of PD1 in a dose-dependent manner. SIRT1 antagonist or agonist and CGRP or antagonist treatment further confirmed the result. Conclusion. Our findings indicate PD1 has a potent analgesic effect, and can modulate neuro-inflammation by regulating SIRT1-mediated CGRP signaling in NCLDH.