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

Disturbances of circadian rhythm and dysregulation of clock gene expression are involved in the induction of various neurological disorder states, including chronic pain. However, the relationship between the CNS circadian-clock gene system and nociception remains poorly defined. Significant circadian oscillations of Period (Per1, Per2), Bmal1 and Cryptochrome 1 (Cry1) mRNA expression have been observed in the lumbar spinal dorsal horn of naïve mice. The current study examined the expression of clock genes in the lumbar spinal dorsal horn of mice with neuropathic pain due to a partial sciatic nerve ligation (PSNL). Seven days after PSNL, the mice displayed a robust unilateral hind paw mechanical hypersensitivity. The normal circadian oscillations of Per1, Per2 and Cry1, but not Bmal1, mRNA expression were significantly suppressed in the ipsilateral lumbar spinal dorsal horn of PSNL mice 7 days 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.     

Role of the spinal cord NR2B-containing NMDA receptors in the development of neuropathic pain

Activation of N-methyl-d-aspartate (NMDA) receptors in the spinal dorsal horn has been shown to be essential for the initiation of central sensitization and the hyperexcitability of dorsal horn neurons in chronic pain. However, whether the spinal NR2B-containing NMDA (NMDA-2B) receptors are involved still remains largely unclear. Using behavioral test and in vivo extracellular electrophysiological recording in L5 spinal nerve-ligated (SNL) neuropathic rats, we investigate the roles of spinal cord NMDA-2B receptors in the development of neuropathic pain. Our study showed that intrathecal (i.t.) injection of Ro 25-6981, a selective NMDA-2B receptor antagonist, had a dose-dependent anti-allodynic effect without causing motor dysfunction. Furthermore, i.t. application of another NMDA-2B receptor antagonist ifenprodil prior to SNL also significantly inhibited the mechanical allodynia but not the thermal hyperalgesia. These data suggest that NMDA-2B receptors at the spinal cord level play an important role in the development of neuropathic pain, especially at the early stage following nerve injury. In addition, spinal administration of Ro 25-6981 not only had a dose-dependent inhibitory effect on the C-fiber responses of dorsal horn wide dynamic range (WDR) neurons in both normal and SNL rats, but also significantly inhibited the long-term potentiation (LTP) in the C-fiber responses of WDR neurons induced by high-frequency stimulation (HFS) applied to the sciatic nerve. These results indicate that activation of the dorsal horn NMDA-2B receptors may be crucial for the spinal nociceptive synaptic transmission and for the development of long-lasting spinal hyperexcitability following nerve injury. In conclusion, the spinal cord NMDA-2B receptors play a role in the development of central sensitization and neuropathic pain via the induction of LTP in dorsal horn nociceptive synaptic transmission. Therefore, the spinal cord NMDA-2B receptor is likely to be a target for clinical pain therapy.     

Comparison of the spinal neuropathic pain induced by intraspinal injection of N-methyl-d-aspartate and quisquate in rats

Objective

Excitatory amino acids play important roles in the development of secondary pathology following spinal cord injury (SCI). This study was designed to evaluate morphological changes in the dorsal horn of the spinal cord and assess profiles of pain behaviors following intraspinal injection of N-methyl-D-aspartate (NMDA) or quisqualate (QUIS) in rats.

Methods

Forty male Sprague-Dawley rats were randomized into three groups : a sham, and two experimental groups receiving injections of 125 mM NMDA or QUIS into their spinal dorsal horn. Following injection, hypersensitivity to cold and mechanical stimuli, and excessive grooming behaviors were assessed serially for four weeks. At the end of survival periods, morphological changes in the spinal cord were evaluated.

Results

Cold allodynia was developed in both the NMDA and QUIS groups, which was significantly higher in the QUIS group than in the NMDA group. The mechanical threshold for the ipsilateral hind paw in both QUIS and NMDA groups was significantly lower than that in the control group. The number of groomers was significantly higher in the NMDA group than in the QUIS group. The size of the neck region of the spinal dorsal horn, but not the superficial layer, was significantly smaller in the NMDA and QUIS groups than in the control group.

Conclusion

Intraspinal injection of NMDA or QUIS can be used as an excitotoxic model of SCI for further research on spinal neuropathic pain.     

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

Spinal cord astrocytes are critical in the maintenance of neuropathic pain. Connexin 43 (Cx43) expressed on spinal dorsal horn astrocytes modulates synaptic neurotransmission, but its role in nociceptive transduction has yet to be fully elaborated. In mice, Cx43 is mainly expressed in astrocytes, not neurons or microglia, in the spinal dorsal horn. Hind paw mechanical hypersensitivity was observed beginning 3 days after partial sciatic nerve ligation (PSNL), but a persistent downregulation of astrocytic Cx43 in ipsilateral lumbar spinal dorsal horn was not observed until 7 days post-PSNL, suggesting that Cx43 downregulation mediates the maintenance and not the initiation of nerve injury-induced hypersensitivity. Downregulation of Cx43 expression by intrathecal treatment with Cx43 siRNA also induced mechanical hypersensitivity. Conversely, restoring Cx43 by an adenovirus vector expressing Cx43 (Ad-Cx43) ameliorated PSNL-induced mechanical hypersensitivity. The sensitized state following PSNL is likely maintained by dysfunctional glutamatergic neurotransmission, as Cx43 siRNA-induced mechanical hypersensitivity was attenuated with intrathecal treatment of glutamate receptor antagonists MK801 and CNQX, but not neurokinin-1 receptor antagonist CP96345 or the Ca²⁺ channel subunit α₂δ₁ blocker gabapentin. The source of this dysfunctional glutamatergic neurotransmission is likely decreased clearance of glutamate from the synapse rather than increased glutamate release into the synapse. Astrocytic expression of glutamate transporter GLT-1, but not GLAST, and activity of glutamate transport were markedly decreased in mice intrathecally injected with Cx43-targeting siRNA but not non-targeting siRNA. Glutamate release from spinal synaptosomes prepared from mice treated with either Cx43-targeting siRNA or non-targeting siRNA was unchanged. Intrathecal injection of Ad-Cx43 in PSNL mice restored astrocytic GLT-1 expression. The cytokine tumor necrosis factor (TNF) has been implicated in the induction of central sensitization, particularly through its actions on astrocytes, in the spinal cord following peripheral injury. Intrathecal injection of TNF in naïve mice induced the downregulation of both Cx43 and GLT-1 in spinal dorsal horn, as well as hind paw mechanical hypersensitivity, as observed in PSNL mice. Conversely, intrathecal treatment of PSNL mice with the TNF inhibitor etanercept prevented not only mechanical hypersensitivity but also the downregulation of Cx43 and GLT-1 expression in astrocytes. The current findings indicate that spinal astrocytic Cx43 are essential for the maintenance of neuropathic pain following peripheral nerve injury and suggest modulation of Cx43 as a novel target for developing analgesics for neuropathic pain.     

Activation of the bile acid receptors TGR5 and FXR in the spinal dorsal horn alleviates neuropathic pain

Aims

Beyond digestion, bile acids have been recognized as signaling molecules with broad paracrine and endocrine functions by activating plasma membrane receptor (Takeda G protein-coupled receptor 5, TGR5) and the nuclear farnesoid X receptor (FXR). The present study investigated the role of bile acids in alleviating neuropathic pain by activating TGR5 and FXR.

Method

Neuropathic pain was induced by spared nerve injury (SNI) of the sciatic nerve. TGR5 or FXR agonist was injected intrathecally. Pain hypersensitivity was measured with Von Frey test. The amount of bile acids was detected using a bile acid assay kit. Western blotting and immunohistochemistry were used to assess molecular changes.

Results

We found that bile acids were downregulated, whereas the expression of cytochrome P450 cholesterol 7ahydroxylase (CYP7A1), a rate-limiting enzyme for bile acid synthesis, was upregulated exclusively in microglia in the spinal dorsal horn after SNI. Furthermore, the expression of the bile acid receptors TGR5 and FXR was increased in glial cells and GABAergic neurons in the spinal dorsal horn on day 7 after SNI. Intrathecal injection of either TGR5 or FXR agonist on day 7 after SNI alleviated the established mechanical allodynia in mice, and the effects were blocked by TGR5 or FXR antagonist. Bile acid receptor agonists inhibited the activation of glial cells and ERK pathway in the spinal dorsal horn. All of the above effects of TGR5 or FXR agonists on mechanical allodynia, on the activation of glial cells, and on ERK pathway were abolished by intrathecal injection of the GABA_A receptor antagonist bicuculline.

Conclusion

These results suggest that activation of TGR5 or FXR counteracts mechanical allodynia. The effect was mediated by potentiating function of GABA_A receptors, which then inhibited the activation of glial cells and neuronal sensitization in the spinal dorsal horn.     

High-frequency spinal cord stimulation produces long-lasting analgesic effects by restoring lysosomal function and autophagic flux in the spinal dorsal horn

High-frequency spinal cord stimulation(HF-SCS) has been established as an effective therapy for neuropathic pain. However, the analgesic mechanisms involved in HF-SCS remain to be clarified. In our study, adult rat neuropathic pain was induced by spinal nerve ligation. Two days after modeling, the rats were subjected to 4 hours of HF-SCS(motor threshold 50%, frequency 10,000 Hz, and pulse width 0.024 ms) in the dorsal horn of the spinal cord. The results revealed that the tactile allodynia of spinal nerve-injured rats was markedly alleviated by HFSCS, and the effects were sustained for 3 hours after the stimulation had ceased. HF-SCS restored lysosomal function, increased the levels of lysosome-associated membrane protein 2(LAMP2) and the mature form of cathepsin D(matu-CTSD), and alleviated the abnormally elevated levels of microtubule-associated protein 1 A/B-light chain 3(LC3)-II and sequestosome 1(P62) in spinal nerve-injured rats. HF-SCS also mostly restored the immunoreactivity of LAMP2, which was localized in neurons in the superficial layers of the spinal dorsal horn in spinal nerve-injured rats. In addition, intraperitoneal administration of 15 mg/kg chloroquine for 60 minutes reversed the expression of the aforementioned proteins and shortened the timing of the analgesic effects of HF-SCS. These findings suggest that HF-SCS may exhibit longlasting analgesic effects on neuropathic pain in rats through improving lysosomal dysfunction and alleviating autophagic flux. This study was approved by the Laboratory Animal Ethics Committee of China Medical University, Shenyang, China(approval No. 2017 PS196 K) on March 1, 2017.     

Contribution of the spinal cord BDNF to the development of neuropathic pain by activation of the NR2B-containing NMDA receptors in rats with spinal nerve ligation

The NMDA receptor and the brain-derived neurotrophic factor (BDNF) are involved in central sensitization and synaptic plasticity in the spinal cord. To determine whether the spinal cord BDNF contributes to the development and maintenance of neuropathic pain by activation of the dorsal horn NR2B-containing NMDA (NMDA-2B) receptors, this study was designed to investigate if alterations in BDNF and its TrkB receptor in the spinal dorsal horn would parallel the timeline of the development of neuropathic pain in lumbar 5 (L5) spinal nerve ligated (SNL) rats. The enzyme-linked immunosorbent assay (ELISA) showed that the BDNF concentration significantly increased during 24 h post-surgery, and the maximal enhancement lasted for 48 h. It declined as time progressed and returned to the level of pre-operation at 28 days after SNL. In parallel with the alteration of BDNF concentration in the spinal dorsal horn, the 50% paw withdrawal threshold (PWT) of the ipsilateral hind paw in SNL rats also showed a significant decrease during 24–48 h after SNL as compared with those in sham-operated rats. The correlation analysis revealed that the BDNF concentration had a negative correlation with 50% PWT in early stage (0–48 h) (r = -0.974, p = 0.001), but not late stage (3–28 days) (r = 0.3395, p = 0.6605), after SNL. Similarly, the immunohistochemical staining revealed that a significant up-regulation of BDNF expression in the spinal dorsal horn appeared as early as 12 h post-operation in SNL rats, peaked at 24–48 h, declined at 3 days and disappeared at 14 days after SNL. In contrast, an increase in NMDA-2B receptors expression in the spinal dorsal horn was delayed to 48 h after SNL. The increase reached peak at 3 days, lasted for 14 days, and returned to the control level of pre-operation at 28 days after SNL. The maximal enhancement of BDNF expression occurred in early stage (24–48 h) after nerve injury, while the peak of NMDA-2B receptors expression appeared in late stage (3–14 days) post-nerve ligation. As compared with the dynamic changes of 50% PWT in the timeline after nerve injury, the maximal enhancement of BDNF expression closely paralleled the maximal decline in the slope of 50% PWT, while the peak of NMDA-2B receptors expression corresponded with the plateau of the decreased 50% PWT. Therefore, the increased BDNF in the spinal dorsal horn was likely to be associated with the initiation of neuropathic pain in early stage (0–48 h), while the activation of NMDA-2B receptors was involved in the maintenance of persistent pain states in late stage (2–14 days) after nerve injury. Moreover, the present study also demonstrated that the BDNF/TrkB-mediated signaling pathway within the spinal cord might be involved in the induction of neuropathic pain in early stage after nerve injury, and the selective NMDA-2B receptors antagonist (Ro 25-6981) almost completely blocked the BDNF-induced mechanical allodynia in all of the tested rats. These data suggested that the BDNF/TrkB-mediated signaling pathway in the spinal cord was involved in the development of nerve injury-induced neuropathic pain through the activation of dorsal horn NMDA-2B receptors.     

Cytokine and growth factor immunohistochemical spinal profiles in two animal models of mononeuropathy

Nerve injury leads to central neuroimmunologic responses that may be integral to the development and maintenance of chronic neuropathic pain in humans. Recent data have demonstrated that cytokines and growth factors may be strongly implicated in the generation of pain states at both peripheral and central nervous system sites. We utilized immunohistochemical methods to investigate this phenomenon in rat models of neuropathic pain. Specifically, we employed well-characterized models of neuropathy that result in behaviors suggestive of neuropathic pain in humans; a freeze lesion of the sciatic nerve, termed sciatic cryoneurolysis, and a chronic constriction sciatic nerve injury. We used immunohistochemistry to examine spinal localization of the cytokines, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and the growth factors, basic fibroblast growth factor (bFGF), and transforming growth factor-β1 (TGF-β) at 3, 14, and 35 days following sciatic cryoneurolysis or 6 days following chronic constriction injury as compared with normal, unoperated rats. There was minimal, diffuse cytokine/growth factor staining in lumbar spinal tissue from the normal group. However, cell profile quantification demonstrated increases in lumbar spinal IL-1β-, TNF-α- and TGF-β-like immunoreactivity (LI) in both mononeuropathy models studied. At 3 days following sciatic cryoneurolysis, intense bFGF LI was present in the ipsilateral dorsal and ventral horn. By 14 days bFGF LI was also observed in contralateral dorsal and ventral horns. In contrast, we found no obvious staining differences in lumbar spinal cord following the chronic constriction injury. This study demonstrated increased specific cytokine and growth factor-like expression in the spinal cord following peripheral nerve injuries. It also showed a differential expression of bFGF in two distinct mononeuropathy models. These results provide further evidence that central cytokine production via a neuroimmune cascade may be involved in the development and maintenance of behaviors that mimic neuropathic pain following nerve injury.     

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

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

Spinal cord stimulation in a patient with multiple sclerosis and failed back surgery syndrome

In the present article, we described a case of treating intractable pain from failed back surgery syndrome (FBSS) and multiple sclerosis (MS) after implantation of spinal cord stimulation (SCS) in a patient. We are reporting a case where SCS has been used for treating a patient with both FBSS and MS.

Multiple Sclerosis (MS) is a chronic demyelinating autoimmune disease and the most frequently occurring type of neuronal demyelination. Multiple Sclerosis may show various neurological manifestations, including neuropathic pain, dysesthesia and paraesthesia in approximately 75% of the patients, based on the severity of nerve damage.1 The associated neuropathic pain accounts for 30% of all MS symptomatic medication. However, medical treatment is generally unresponsive or inadequate for the management of this pain.1Failed back surgery syndrome is the most common indication for spinal cord stimulation (SCS). It is often characterised by disabled back or radicular limb pain following spinal surgery, with neuropathic and nociceptive components that are often insufficiently responsive to conventional therapies such as reoperation or medical treatment.2Neuropathic pain may develop secondary to a primary lesion in the nervous system or due to nervous system dysfunction. Depending on the location of the lesion in the nervous system, it is classified as either central or peripheral neuropathic pain. Due to insufficient epidemiological research and follow-up, the exact prevalence of neuropathic pain remains unknown. However, it is estimated that approximately 1-1.5% of the population is affected by it.Spinal cord laminae I, II and V receive information from nociceptive afferent fibres of the dorsal root ganglia.3 Although nociceptive information is transmitted to higher centres for processing, it can be modified at primary afferent nerve terminals and at the presynaptic junction of projection neurones in the dorsal horn of the spinal cord.Spinal cord stimulation-induced analgesia involves neurophysiological and neurochemical mechanisms. Spinal cord stimulation activates the dorsal column and inhibitory interneurons that attenuate pain.3Neurostimulation is a known effective treatment for neuropathic pain.1 Therefore, we aimed to determine whether SCS is successful in treating intractable pain in a patient with MS and FBSS.     

Expression of acetylated histone 3 in the spinal cord and the effect of morphine on inflammatory pain in rats

In this study, a rat model of inflammatory pain was produced by injecting complete Freund’s adjuvant into the hind paw, and the expression of acetylated histone 3 in the spinal cord dorsal horn was examined using immunohistochemical staining. One day following injection, there was a dramatic decrease in acetylated histone 3 expression in spinal cord dorsal horn neurons. However, on day 7, expression recovered in adjuvant-injected rats. While acetylated histone 3 labeling was present in dorsal horn neurons, it was more abundant in astrocytes and microglial cells. The recovery of acetylated histone 3 expression was associated with a shift in expression of the protein from neurons to glial cells. Morphine injection significantly upregulated the expression of acetylated histone 3 in spinal cord dorsal horn neurons and glial cells 1 day after injection, especially in astrocytes, preventing the transient downregulation. Our results indicate that inflammatory pain induces a transient downregulation of acetylated histone 3 in the spinal cord dorsal horn at an early stage following adjuvant injection, and that this effect can be reversed by morphine. Thus, the downregulation of acetylated histone 3 may be involved in the development of inflammatory pain.     

Amitriptyline upregulates EAAT1 and EAAT2 in neuropathic pain rats

Excitatory amino acid transporters (EAATs) appear to participate in the pathogenesis of neuropathic pain. The present study was performed to evaluate the effects of the tricyclic antidepressant amitriptyline on the expressions of EAATs in neuropathic pain rats. Using spared nerve injured (SNI) male Sprague Dawley rats, we found that SNI induced an initial EAATs upregulation on postoperative day 1 within the ipsilateral spinal cord dorsal horn, followed by a downregulation on postoperative days 3 and 5. Intraperitoneal administration of amitriptyline reversed the downregulation of EAATs in SNI rats on postoperative days 3–5 and attenuated the mechanical allodynia. We further demonstrated that administration of amitriptyline alone induced an upregulation of EAATs in sham-operated rat but do not produce an antinociceptive effect. These results indicate that amitriptyline could increase the expression of EAATs which may be one of its mechanisms in the treatment of neuropathic pain.     

LRP1 deficiency in microglia blocks neuro-inflammation in the spinal dorsal horn and neuropathic pain processing

Following injury to the peripheral nervous system (PNS), microglia in the spinal dorsal horn (SDH) become activated and contribute to the development of local neuro-inflammation, which may regulate neuropathic pain processing. The molecular mechanisms that control microglial activation and its effects on neuropathic pain remain incompletely understood. We deleted the gene encoding the plasma membrane receptor, LDL Receptor-related Protein-1 (LRP1), conditionally in microglia using two distinct promoter-Cre recombinase systems in mice. LRP1 deletion in microglia blocked development of tactile allodynia, a neuropathic pain-related behavior, after partial sciatic nerve ligation (PNL). LRP1 deletion also substantially attenuated microglial activation and pro-inflammatory cytokine expression in the SDH following PNL. Because LRP1 shedding from microglial plasma membranes generates a highly pro-inflammatory soluble product, we demonstrated that factors which activate spinal cord microglia, including lipopolysaccharide (LPS) and colony-stimulating factor-1, promote LRP1 shedding. Proteinases known to mediate LRP1 shedding, including ADAM10 and ADAM17, were expressed at increased levels in the SDH after PNL. Furthermore, LRP1-deficient microglia in cell culture expressed significantly decreased levels of interleukin-1β and interleukin-6 when treated with LPS. We conclude that in the SDH, microglial LRP1 plays an important role in establishing and/or amplifying local neuro-inflammation and neuropathic pain following PNS injury. The responsible mechanism most likely involves proteolytic release of LRP1 from the plasma membrane to generate a soluble product that functions similarly to pro-inflammatory cytokines in mediating crosstalk between cells in the SDH and in regulating neuropathic pain.     

Activation of extracellular signal-regulated protein kinases 5 in the spinal cord contributes to the neuropathic pain behaviors induced by CCI in rats

Abstract

Objective: To investigate whether activation and translocation of extracellular signal-regulated kinase 5 (ERK5) is involved in the induction and maintenance of neuropathic pain and observe the effects of activation and translocation of ERK5 on the expression of phosphorylated cAMP response element binding (pCREB) in the chronic neuropathic pain.

Methods: Lumbar intrathecal catheters were chronically implanted in male Sprague–Dawley rats. The left sciatic nerve was loosely ligated proximal to the sciatica's trifurcation at ～ 1.0 mm intervals with 4-0 silk sutures. The phosphorothioate-modified antisense oligonucleotides (AS-ODNs) were intrathecally administered every 12 hours, 1 day pre-chronic constriction injury (CCI) and 3 day post-CCI. Thermal and mechanical nociceptive thresholds were assessed with the paw withdrawal latency to a radiant heat and von Frey filaments. Expressions of phosphorylated ERK5 (pERK5), pCREB, were assessed by both Western blotting and immunohistochemical analysis.

Results: Intrathecal injection of ERK5 AS-ODN significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia. CCI significantly increased the expression of pERK5 neurons in the ipsilateral spinal dorsal horn to injury, not in the contralateral spinal dorsal horn. The time courses of pERK5 expression showed that the levels of both cytosol and nuclear pERK5 were increased at all points after CCI and reached a peak level on post-operative day 5. CCI significantly increased the expression of pERK5 neurons in the laminae I and II of ipsilateral spinal dorsal horn to injury, not in the contralateral spinal dorsal horn. Phospho-CREB-positive neurons were distributed in all laminae of the bilateral spinal cord. Intrathecal injection AS-ODN markedly suppressed the increase of CCI-induced pERK5, pCREB expression in the spinal cord.

Conclusion: The activation of ERK5 pathways contributes to neuropathic pain in CCI rats, and the function of pERK5 may partly be accomplished via the CREB protein-dependent gene expression.     

Paeonol alleviates neuropathic pain by modulating microglial M1 and M2 polarization via the RhoA/p38MAPK signaling pathway

Background

This study aimed to investigate the potential mechanism of paeonol in the treatment of neuropathic pain.

Methods

Relevant mechanisms were explored through microglial pseudotime analysis and the use of specific inhibitors in cell experiments. In animal experiments, 32 SD rats were randomly divided into the sham operation group, the chronic constrictive injury (CCI) group, the ibuprofen group, and the paeonol group. We performed behavioral testing, ELISA, PCR, Western blotting, immunohistochemistry, and immunofluorescence analysis.

Results

The pseudotime analysis of microglia found that RhoA, Rock1, and p38MAPK were highly expressed in activated microglia, and the expression patterns of these genes were consistent with the expression trends of the M1 markers CD32 and CD86. Paeonol decreased the levels of M1 markers (IL1β, iNOS, CD32, IL6) and increased the levels of M2 markers (IL10, CD206, ARG-1) in LPS-induced microglia. The expression of iNOS, IL1β, RhoA, and Rock1 was significantly increased in LPS-treated microglia, while paeonol decreased the expression of these proteins. Thermal hyperalgesia occurred after CCI surgery, and paeonol provided relief. In addition, paeonol decreased the levels of IL1β and IL8 and increased the levels of IL4 and TGF-β in the serum of CCI rats. Paeonol decreased expression levels of M1 markers and increased expression levels of M2 markers in the spinal cord. Paeonol decreased IBA-1, IL1β, RhoA, RhoA-GTP, COX2, Rock1, and p-p38MAPK levels in the spinal dorsal horn.

Conclusion

Paeonol relieves neuropathic pain by modulating microglial M1 and M2 phenotypes through the RhoA/p38 MAPK pathway.     

KDM6B epigenetically regulated-interleukin-6 expression in the dorsal root ganglia and spinal dorsal horn contributes to the development and maintenance of neuropathic pain following peripheral nerve injury in male rats

The lysine specific demethylase 6B (KDM6B) has been implicated as a coregulator in the expression of proinflammatory mediators, and in the pathogenesis of inflammatory and arthritic pain. However, the role of KDM6B in neuropathic pain has yet to be studied. In the current study, the neuropathic pain was determined by assessing the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) following lumbar 5 spinal nerve ligation (SNL) in male rats. Immunohistochemistry, Western blotting, qRT-PCR, and chromatin immunoprecipitation (ChIP)-PCR assays were performed to investigate the underlying mechanisms. Our results showed that SNL led to a significant increase in KDM6B mRNA and protein in the ipsilateral L4/5 dorsal root ganglia (DRG) and spinal dorsal horn; and this increase correlated a markedly reduction in the level of H3K27me3 methylation in the same tissue. Double immunofluorescence staining revealed that the KDM6B expressed in myelinated A- and unmyelinated C-fibers in the DRG; and located in neuronal cells, astrocytes, and microglia in the dorsal horn. Behavioral data showed that SNL-induced mechanical allodynia and thermal hyperalgesia were impaired by the treatment of prior to i.t. injection of GSK-J4, a specific inhibitor of KDM6B, or KDM6B siRNA. Both microinjection of AAV2-EGFP-KDM6B shRNA in the lumbar 5 dorsal horn and sciatic nerve, separately, alleviated the neuropathic pain following SNL. The established neuropathic pain was also partially attenuated by repeat i.t. injections of GSK-J4 or KDM6B siRNA, started on day 7 after SNL. SNL also resulted in a remarkable increased expression of interleukin-6 (IL-6) in the DRG and dorsal horn. But this increase was dramatically inhibited by i.t. injection of GSK-J4 and KDM6B siRNA; and suppressed by prior to microinjection of AAV2-EGFP-KDM6B shRNA in the dorsal horn and sciatic nerve. Results of ChIP-PCR assay showed that SNL-induced enhanced binding of STAT3 with IL-6 promoter was inhibited by prior to i.t. injection of GSK-J4. Meanwhile, the level of H3K27me3 methylation was also decreased by the treatment. Together, our results indicate that SNL-induced upregulation of KDM6B via demethylating H3K27me3 facilitates the binding of STAT3 with IL-6 promoter, and subsequently mediated-increase in the expression of IL-6 in the DRG and dorsal horn contributes to the development and maintenance of neuropathic pain. Targeting KDM6B might a promising therapeutic strategy to treatment of chronic pain.     

Brain-derived neurotrophic factor contributes to spinal long-term potentiation and mechanical hypersensitivity by activation of spinal microglia in rat

It has been shown that following peripheral nerve injury brain-derived neurotrophic factor (BDNF) released by activated microglia contributes to neuropathic pain, but whether BDNF affects the function of microglia is still unknown. In the present work we found that spinal application of BDNF, which induced long-term potentiation (LTP) of C-fiber evoked field potentials, activated spinal microglia in naïve animals, while pretreatment with microglia inhibitor minocycline blocked BDNF-induced LTP. In addition, following LTP induction by BDNF, both phosphorylated Src-family kinases (p-SFKs) and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) were up-regulated only in spinal microglia but not in neurons and astrocytes, whilst spinal application of SFKs inhibitor (PP2 or SU6656) or p38 MAPK inhibitor (SB203580) blocked BDNF-induced LTP and suppressed microglial activation. As spinal LTP at C-fiber synapses is considered to underlie neuropathic pain, we subsequently examined whether BDNF may contribute to mechanical hypersensitivity by activation of spinal microglia using spared nerve injury (SNI) model. Following SNI BDNF and TrkB receptor were up-regulated mainly in dorsal horn neurons and in activated microglia, and p-SFKs and p-p38 MAPK were increased exclusively in microglia. Intrathecal injection of BDNF scavenger TrkB-Fc starting before SNI, which prevented the behavioral sign of neuropathic pain, suppressed both microglial activation and the up-regulation of p-SFKs and p-p38 MAPK produced by SNI. Thus, the increased BDNF/TrkB signaling in spinal dorsal horn may contribute to neuropathic pain by activation of microglia following peripheral nerve injury and inhibition of SFKs or p38 MAPK may selectively inhibit microglia in spinal dorsal horn.     

Chronic constriction injury of sciatic nerve induces the up-regulation of descending inhibitory noradrenergic innervation to the lumbar dorsal horn of mice

Peripheral nerve injury in rodents results in hypersensitivity to mechanical and thermal stimuli accompanied by reduced antinociceptive efficacy of opioids and, in some models, sensitivity to sympathetic blockade. 2-Adrenergic receptor agonists increase in potency and efficacy after nerve injury in rodents and effectively relieve neuropathic pain in humans who do not get pain relief from opioids. However, the underlying mechanisms are unclear. It has been well known that the major noradrenergic innervation of the spinal dorsal horn originates from the locus coeruleus nucleus (LC) in the brainstem. Therefore, the aim of this study is to examine whether peripheral nerve injury that causes neuropathic pain modulates the noradrenergic innervation to the lumbar dorsal horn, in order to determine the possible anatomical substrates underlying increased potency and efficacy of noradrenergic receptor agonists in alleviating neuropathic pain. At 2 weeks after chronic constriction injury (CCI) of the sciatic nerve, a remarkable increase in tyrosine-hydroxylase (TH) and dopamine β-hydroxylase (DβH) immunoreactive (IR) axonal terminals was observed in the ipsilateral L4–L6 dorsal horn. Consistently, greater numbers of both TH- and DβH-IR neurons were detected in the ipsilateral LC. Interestingly, in the lower lumbar and upper sacral spinal dorsal horn, numerous TH-IR neurons were observed in the superficial dorsal horn (primarily lamina I). CCI of the sciatic nerve did not change the number of these TH-IR cells. These findings suggest that augmented descending inhibitory noradrenergic innervation to the dorsal horn could be one of the mechanisms underlying the increased effectiveness in the anti-allodynic effect elicited by 2-adrenergic receptor agonists.