Expression In Dorsal Horn Research Articles

The transcription factor Tbx5-dependent epigenetic modification contributes to neuropathic allodynia by activating TRPV1 expression in dorsal horn.

Nerve injury can induce aberrant changes in the spine; these changes are due to, or at least partly governed by, transcription factors that contribute to the genesis of neuropathic allodynia. Here, we showed that spinal nerve ligation (SNL, a clinical neuropathic allodynia model) increased the expression of the transcription factor Tbx5 in the injured dorsal horn in male Sprague Dawley rats. In contrast, blocking this upregulation alleviated SNL-induced mechanical allodynia, and there was no apparent effect on locomotor function. Moreover, SNL-induced Tbx5 upregulation promoted the recruitment and interaction of GATA4 and Brd4 by enhancing its binding activity to H3K9Ac, which was enriched at the Trpv1 promotor, leading to an increase in TRPV1 transcription and the development of neuropathic allodynia. In addition, nerve injury-induced expression of Fbxo3, which abates Fbxl2-dependent Tbx5 ubiquitination, promoted the subsequent Tbx5-dependent epigenetic modification of TRPV1 expression during SNL-induced neuropathic allodynia. Collectively, our findings indicated that spinal Tbx5-dependent TRPV1 transcription signaling contributes to the development of neuropathic allodynia via Fbxo3-dependent Fbxl2 ubiquitination and degradation. Thus, we propose a potential medical treatment strategy for neuropathic allodynia by targeting Tbx5.Significance Statement Nerve injury-induced epigenetic changes in Tbx5 enhance TRPV1 expression in the dorsal horn, with Tbx5 recruiting GATA4 and Brd4 for epigenetic modification of TRPV1. The Fbxo3-mediated Fbxl2 ubiquitination pathway stabilizes Tbx5, increasing its transcriptional activity and enhancing TRPV1 expression, contributing to the initiation and maintenance of neuropathic pain via epigenetic reprogramming.

Sustained morphine exposure alters spinal NMDA receptor and astrocyte expression and exacerbates chronic pain behavior in female rats.

Sustained opioid use has long-term negative impacts on future pain experience, particularly in women. This study aimed to investigate the underlying spinal neurobiology of this clinical observation in an experimental model of joint pain. In this study, we tested the hypothesis that sustained opioid treatment exacerbates chronic pain responses and alters spinal cord dorsal horn astrogliosis and the expression of GluN2B-containing N-methyl-d-aspartate receptors in female rats. Subcutaneous morphine (3 mg/kg) or saline was administered twice daily for 1 week before inducing a model of joint knee pain (intra-articular injection of 2 mg of monosodium iodoacetate [MIA]) in adult female Sprague-Dawley rats, with pain-free controls receiving 50 µL of saline. Pain behavior (weight-bearing and mechanical paw withdrawal thresholds) was measured at baseline and at intervals thereafter. Twice-daily morphine/saline treatment was continued for up to 3 weeks after intra-articular injections, and spinal cord tissue was collected for Western blot analyses. Area under the curve analysis of weight-bearing asymmetry confirmed a significant exacerbation of pain behavior in the morphine/MIA group, compared with the saline/MIA group (F(3,18) = 46.3, P < 0.0001), despite comparable joint damage in both groups. Sustained morphine treatment was associated with significant elevations in dorsal horn expression of astrocytic glial fibrillary acidic protein (27 ± 5% increase) and neuronal GluN2B (80 ± 30% increase), but not microglial IBA1, irrespective of the model of joint pain. These data suggest that sustained morphine treatment in female rats drives spinal cord plasticity, including spinal astrogliosis and the expression of GluN2B-containing N-methyl-d-aspartate receptors, priming the dorsal horn to incoming sensory inputs and producing exacerbated pain responses.

Cannabinoid CB1 Receptor Expression and Localization in the Dorsal Horn of Male and Female Rat and Human Spinal Cord

ABSTRACT Background Preclinical and clinical evidence suggests that cannabis has potential analgesic properties. However, cannabinoid receptor expression and localization within spinal cord pain processing circuits remain to be characterized across sex and species. Aims We aimed to investigate the differential expression of the cannabinoid type 1 (CB1) receptor across dorsal horn laminae and cell populations in male and female adult rats and humans. Methods To investigate and quantify CB1 receptor expression in the spinal dorsal horn across species, we refined immunohistochemical procedures for successful rat and human fixed tissue staining and confocal imaging. Immunohistochemical results were complemented with analysis of CB1 gene (CNR1) expression within rodent and human dorsal horn using single-cell/nuclei RNA sequencing data sets. Results We found that CB1 was preferentially localized to the neuropil within the superficial dorsal horn of both rats and humans, with CB1 somatic staining across dorsal horn laminae. CB1 receptor immunoreactivity was significantly higher in the superficial dorsal horn compared to the deeper dorsal horn laminae for both rats and humans, which was conserved across sex. Interestingly, we found that CB1 immunoreactivity was not primarily localized to peptidergic afferents in rats and humans and that CNR1 (CB1) but not CNR2 (CB2) was robustly expressed in dorsal horn neuron subpopulations of both rodents and humans. Conclusions The conserved preferential expression of CB1 receptors in the superficial dorsal horn in male and female rodents and humans has significant implications for understanding the roles of this cannabinoid receptor in spinal mechanisms of nociception and analgesia.

Motilin and its receptor are expressed in the dorsal horn in a rat model of acute incisional pain: Intrathecal motilin injection alleviates pain behaviors.

To observe the effects of intrathecal administration of motilin on pain behavior and expression of motilin (MTL)/motilin receptor (MTLR) in the spinal cord of a rat model of acute incisional pain. An incisional pain model was established in rats using a unilateral plantar incision. The rats were also injected intrathecally with 1, 5, or 25 μg of motilin. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were determined. MTL/MTLR expression in the spinal cord was detected by western blotting and immunofluorescence. The expression of MTL in the spinal cord, stomach, duodenum, and plasma was determined by enzyme-linked immunosorbent assay (ELISA). Motilin/motilin receptor were detected in the spinal cord. Spinal cord MTL/MTLR expression peaks at 2 h after modeling (P < 0.05) and start to decrease at 24 h (P < 0.05) to almost reach baseline levels at 72 h. The changes in gastric, duodenal, plasma, and spinal cord motilin levels correlated with MWT and TWL (all R 2 > 0.82). The intrathecal injection of 1, 5, or 25 μg of motilin could increase the pain threshold of rats with incisional pain within 72 h in a dose-dependent manner. This study showed for the first time that MTL/MTLR are expressed in rats' spinal dorsal horn. Acute pain increased MTL/MTLR expression in the spinal dorsal horn. Also, for the first time, this study showed that motilin intrathecal injection alleviates pain in rat models of acute incisional pain. These results suggest that MTL/MTLR could be a novel target for the management of acute pain.

Differential Activation of pERK1/2 and c-Fos Following Injury to Different Regions of Primary Sensory Neuron.

Nerve injury causes hyperexcitability of the dorsal root ganglion (DRG) and spinal dorsal horn (DH) neurons, which results in neuropathic pain. We have previously demonstrated that partial dorsal rhizotomy (PDR) produced less severe pain-like behavior than chronic constriction injury (CCI) or chronic compression of DRG (CCD) and did not enhance DRG neuronal excitability. However, the mechanisms underlying such discrepancy remain unclear. This study was designed to compare the activation of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) in DRG and DH, and c-Fos in DH following treatments of CCI, CCD, and PDR. We confirmed that thermal hyperalgesia produced by PDR was less severe than that produced by CCI or CCD. We showed that pERK1/2 in DRG and DH was greatly activated by CCI or CCD, whereas PDR produced only transient and mild pERK1/2 activation. CCI, CCD, and PDR induced robust c-Fos expression in DH; nevertheless, c-Fos+ neurons following PDR were much fewer than that following CCI or CCD. Blocking retrograde axonal transport by colchicine proximal to the CCI injury site diminished thermal hyperalgesia and inhibited pERK1/2 and c-Fos activation. These findings demonstrate that less severe pain-like behavior produced by PDR than CCI or CCD attributes to less activation of pERK1/2 and c-Fos. Such neurochemical activation partially relies on retrograde axonal transport of certain “injury signals” from the peripheral injured site to DRG somata.

126 Regulator of G Protein Signaling 4 (RGS4) Gene Expression is Upregulated in the Superficial Spinal Dorsal Horn and in Dorsal Root Ganglion Neurons in a Murine Model of Neuropathic Pain

INTRODUCTION: Regulator of G protein signaling 4 (RGS4), a potent negative regulator of G-protein coupled receptor signaling, is a key gene in the maintenance of mechanical and cold allodynia associated with chronic pain states in mice. RGS4 is abundantly expressed across the pain matrix, and is upregulated in response to peripheral inflammation and nerve injury. METHODS: To understand the dynamics of RGS4 gene expression after nerve injury, we perform the spared-nerve injury (SNI) model of neuropathic pain in C57BL/6. At 21 days (DRG) or 2.5 months (spinal cord) post-surgery (SNI vs sham), animals were sacrificed and specimens were harvested for tissue processing. RNAScope, an ultrasensitive RNA ISH method, was used to visualize and quantify single-molecule expression of RGS4, prodynorphin/PDYN (exclusively expressed by inhibitory dorsal horn interneurons), and somatostatin/SST (expressed by excitatory interneurons) within the cellular topography of the lumbar superficial dorsal horn. RESULTS: RGS4 expression increased significantly in the dorsal horn following spared-nerve injury (p = 0.0384). In sham-treated mice, there were no differences in cell-type-specific (PDYN vs. SST) RGS4 expression patterns in the dorsal horn (p = 0.4170). Cell-type-specific RGS4 expression in the dorsal horn was significantly different (F (2,27) = 8.353; p = 0.0015; one-way ANOVA) following SNI. SNI-induced RGS4 expression was significantly enriched in PDYN vs SST-expressing interneurons (p = 0.0300), and overall RGS4 expression was significantly downregulated in SST-expressing cells compared to all cells (p = 0.0012). In addition, neuronal-specific RGS4 expression was significantly increased in the DRG in SNI vs Sham-injured mice (p = 0.0050). CONCLUSION: Our data suggest that upregulation of RGS4 expression in the superficial dorsal horn and DRG may play a crucial role in maintenance of chronic pain following nerve injury.

Chronic pain‐mediated Regulator of G protein signaling 4 (RGS4) gene expression in superficial dorsal horn of spinal cord

Regulator of G protein signaling 4 (RGS4) is a potent negative regulator of G-protein coupled receptor signaling duration. Recent studies have identified RGS4 as a key gene in the maintenance of mechanical and cold allodynia associated with chronic pain states in mice. RGS4 is abundantly expressed across the pain matrix, and several studies have demonstrated upregulation of RGS4 mRNA in response to both peripheral inflammation and nerve injury. Here, we tested the hypothesis that peripheral nerve injury alters RGS4 neuronal expression broadly, and with specific expression-patterning among excitatory and inhibitory interneuron populations within the superficial dorsal horn of the mouse spinal cord. Chronic neuropathic pain was induced in C57BL/6 male mice via spared-nerve injury (SNI). At 2.5 months post-surgery (SNI vs sham), animals were sacrificed and lumbar spinal cords were harvested for tissue processing. RNAScope, an ultrasensitive RNA ISH method, was used to visualize and quantify single-molecule expression of RGS4, prodynorphin/PDYN (exclusively expressed by inhibitory dorsal horn interneurons), and somatostatin/SST (expressed by excitatory interneurons) within the cellular topography of the lumbar superficial dorsal horn. RGS4 expression increased significantly in the dorsal horn following spared-nerve injury (P =0.0384). RGS4 mRNA remained unchanged (SNI vs sham) among expression-positive dorsal horn neurons (p=0.6327). In sham-treated mice, there were no differences in cell-type specific (PDYN vs. SST) RGS4 expression patterns in the dorsal horn (p=0.4170). Cell-type specific RGS4 expression in the dorsal horn was significantly different (F (2,27) = 8.353; P =0.0015; one-way ANOVA) following spared-nerve injury. SNI-induced RGS4 expression was significantly enriched in PDYN vs SST-expressing interneurons (p=0.0300), and overall RGS4 expression was significantly downregulated in SST-expressing cells compared to all cells (p=0.0012). These data demonstrate that nerve injury induces upregulation of RGS4 in the superficial dorsal horn, and suggest that RGS4-positive inhibitory interneurons may be implicated in influencing persistent pain signaling within the dorsal horn of the spinal cord.

P2X3 receptor expression in dorsal horn of spinal cord and pain threshold after estrogen therapy for prevention therapy in neuropathic pain

IntroductionNeuropathic pain may arise from conditions that affecting the central or peripheral nervous system. This study was held to determine the difference P2X3 receptor expression in the dorsal horn of the spinal cord and pain threshold after estrogen therapy in neuropathic pain. MethodsThis study design was an experimental research laboratory. The 24 mice samples divided into negative control group, positive control, and treatment groups. The treatment groups were given subcutaneous injections of estrogen 0.4 ml and also examined for the onset of thermal hyperalgesia in every rat. On day 15, an autopsy was performed on rats, and the spine was taken. The spinal cord was stained by hematoxylin-eosin, and the expression of P2X3 receptors was investigated. P2X3 receptor expression was examined in the dorsal horn on each sample. ResultsFrom 24 subjects of the study revealed an increase in the onset of thermal hyperalgesia on the estrogen group compared with the placebo group, a higher start. This study also obtained a decrease in the expression of P2X3 on the therapy group compared to the positive control group with significant differences. Statistical test results revealed the appearance of the P2X3 estrogen group had a substantial difference with the placebo group (p = 0.000) and the mean of the negative control group (p = 0.030). The placebo group had a significant difference from the negative control group (p = 0.035). ConclusionEstrogen could decrease the expression of P2X3 receptors and prolonged the onset of thermal hyperalgesia. So, both of these explained that estrogen has a role in preventing the occurrence of neuropathic pain after peripheral nerve lesions.

Examining the role of transient receptor potential canonical 5 (TRPC5) in osteoarthritis

IntroductionOsteo-arthritis (OA) involves joint degradation and usually pain; with mechanisms poorly understood and few treatment options. There is evidence that the transient receptor potential canonical 5 (TRPC5) mRNA expression is reduced in OA patients’ synovia. Here we examine the profile of TRPC5 in DRG and involvement in murine models of OA. DesignTRPC5 KO mice were subjected to partial meniscectomy (PMNX) or injected with monoiodoacetate (MIA) and pain-related behaviours were determined. Knee joint pathological scores were analysed and gene expression changes in ipsilateral synovium and dorsal root ganglia (DRG) determined. c-Fos protein expression in the ipsilateral dorsal horn of the spinal cord was quantified. ResultsTRPC5 KO mice developed a discrete enhanced pain-related phenotype. In the MIA model, the pain-related phenotype correlated with c-Fos expression in the dorsal horn and increased expression of nerve injury markers ATF3, CSF1 and galanin in the ipsilateral DRG. There were negligible differences in the joint pathology between WT and TRPC5 KO mice, however detailed gene expression analysis determined increased expression of the mast cell marker CD117 as well as extracellular matrix remodelling proteinases MMP2, MMP13 and ADAMTS4 in MIA-treated TRPC5 KO mice. TRPC5 expression was defined to sensory subpopulations in DRG. ConclusionsDeletion of TRPC5 receptor signalling is associated with exacerbation of pain-like behaviour in OA which correlates with increased expression of enzymes involved in extracellular remodelling, inflammatory cells in the synovium and increased neuronal activation and injury in DRG. Together, these results identify a modulating role for TRPC5 in OA-induced pain-like behaviours.

NFATc2-dependent epigenetic upregulation of CXCL14 is involved in the development of neuropathic pain induced by paclitaxel

BackgroundThe major dose-limiting toxicity of paclitaxel, one of the most commonly used drugs to treat solid tumor, is painful neuropathy. However, the molecular mechanisms underlying paclitaxel-induced painful neuropathy are largely unclarified.MethodsPaw withdrawal threshold was measured in the rats following intraperitoneal injection of paclitaxel. The qPCR, western blotting, protein or chromatin immunoprecipitation, ChIP-seq identification of NFATc2 binding sites, and microarray analysis were performed to explore the molecular mechanism.ResultsWe found that paclitaxel treatment increased the nuclear expression of NFATc2 in the spinal dorsal horn, and knockdown of NFATc2 with NFATc2 siRNA significantly attenuated the mechanical allodynia induced by paclitaxel. Further binding site analysis utilizing ChIP-seq assay combining with gene expression profile revealed a shift of NFATc2 binding site closer to TTS of target genes in dorsal horn after paclitaxel treatment.We further found that NFATc2 occupancy may directly upregulate the chemokine CXCL14 expression in dorsal horn, which was mediated by enhanced interaction between NFATc2 and p300 and consequently increased acetylation of histone H4 in CXCL14 promoter region. Also, knockdown of CXCL14 in dorsal horn significantly attenuated mechanical allodynia induced by paclitaxel.ConclusionThese results suggested that enhanced interaction between p300 and NFATc2 mediated the epigenetic upregulation of CXCL14 in the spinal dorsal horn, which contributed to the chemotherapeutic paclitaxel-induced chronic pain.

Down-Regulation of NOX4 Expression in Dorsal Horn of Spinal Cord Could Alleviate Cancer-Induced Bone Pain in Rats by Reducing Oxidative Stress Response.

IntroductionCancer-induced bone pain (CIBP) is very common in patients with advanced cancer. Recent studies have shown that reactive oxygen species (ROS) can sense and regulate pain response process through spinal cord mechanism, and play a role in CIBP. NADPH oxidase (NOX) is a group of protease complexes that produce ROS. In the current study, we investigated the expression of NOX4 in the spinal dorsal horn of rats with CIBP and its related role and molecular mechanism.Materials and MethodsA rat CIBP model was established by injecting Walker-256 cells into the tibia medullary cavity, and the expression of NOX4 in spinal dorsal horn was down-regulated by injecting lentivirus into spinal cord. RT-PCR, Western blot and immunofluorescence staining were used to detect the expression of NOX4 in CIBP rats, cell localization and its effect on CIBP rats, and the effect of down-regulating the expression of NOX4 on the expression of H2O2, nitric oxide synthase nNO, antioxidant enzyme SOD, and the activity of neuro-receptor in spinal dorsal horn of rats.ResultsIn rats with CIBP, the expression of NOX4 was significantly increased, and immunofluorescence showed that NOX4 was mainly expressed in microglia in the dorsal horn of spinal cord. Down-regulating the expression of NOX4 in rats can reduce the level of H2O2 and nNO in dorsal horn tissue, and increase the expression of SOD to reduce the oxidative stress response. At the same time, down-regulating NOX4 can reduce the sensitivity of spinal cord and relieve the pain of bone cancer by inhibiting the expression of NMDAR and GABAA-γ2 in dorsal horn tissue.ConclusionNOX4 is a promising therapeutic target in CIBP, and down-regulation of NOX4 expression can alleviate CIBP in rats by reducing oxidative stress and weakening spinal cord sensitization.

Suppression of WNK1-SPAK/OSR1 Attenuates Bone Cancer Pain by Regulating NKCC1 and KCC2

Our preliminary experiment indicated the activation of with-nolysine kinases 1 (WNK1) in bone cancer pain (BCP) rats. This study aimed to investigate the underlying mechanisms via which WNK1 contributed to BCP. A rat model of BCP was induced by Walker-256 tumor cell implantation. WNK1 expression and distribution in the lumbar spinal cord dorsal horn and dorsal root ganglion were examined. SPS1-related proline/alanine-rich kinase (SPAK), oxidative stress-responsive kinase 1 (OSR1), sodium-potassium-chloride cotransporter 1 (NKCC1), and potassium-chloride cotransporter 2 (KCC2) expression were assessed. Pain behaviors including mechanical allodynia and movement-evoked pain were measured. BCP rats exhibited significant mechanical allodynia, with increased WNK1 expression in the dorsal horn and dorsal root ganglion neurons, elevated SPAK/OSR1 and NKCC1 expression in the dorsal root ganglion, and decreased KCC2 expression in the dorsal horn. WNK1 knock-down by small interfering alleviated mechanical allodynia and movement-evoked pain, inhibited WNK1-SPAK/OSR1-NKCC1 activities, and restored KCC2 expression. In addition, closantel (a WNK1-SPAK/OSR1 inhibitor) improved pain behaviors, downregulated SPAK/OSR1 and NKCC1 expression, and upregulated KCC2 expression in BCP rats. Activation of WNK1-SPAK/OSR1 signaling contributed to BCP in rats by modulating NKCC1 and KCC2 expression. Therefore, suppression of WNK1-SPAK/OSR1 may serve as a potential target for BCP therapy. PerspectiveOur findings demonstrated that the WNK1-SPAK/OSR1 signaling contributed to BCP in rats via regulating NKCC1 and KCC2. Suppressing this pathway reduced pain behaviors. Based on these findings, the WNK1-SPAK/OSR1 signaling may be a potential target for BCP therapy.

An NPY Y1 receptor antagonist unmasks latent sensitization and reveals the contribution of protein kinase A and Epac to chronic inflammatory pain.

Peripheral inflammation produces a long-lasting latent sensitization of spinal nociceptive neurons, that is, masked by tonic inhibitory controls. We explored mechanisms of latent sensitization with an established four-step approach: (1) induction of inflammation; (2) allow pain hypersensitivity to resolve; (3) interrogate latent sensitization with a channel blocker, mutant mouse, or receptor antagonist; and (4) disrupt compensatory inhibition with a receptor antagonist so as to reinstate pain hypersensitivity. We found that the neuropeptide Y Y1 receptor antagonist BIBO3304 reinstated pain hypersensitivity, indicative of an unmasking of latent sensitization. BIBO3304-evoked reinstatement was not observed in AC1 knockout mice and was prevented with intrathecal co-administration of a pharmacological blocker to the N-methyl-D-aspartate receptor (NMDAR), adenylyl cyclase type 1 (AC1), protein kinase A (PKA), transient receptor potential cation channel A1 (TRPA1), channel V1 (TRPV1), or exchange protein activated by cAMP (Epac1 or Epac2). A PKA activator evoked both pain reinstatement and touch-evoked pERK expression in dorsal horn; the former was prevented with intrathecal co-administration of a TRPA1 or TRPV1 blocker. An Epac activator also evoked pain reinstatement and pERK expression. We conclude that PKA and Epac are sufficient to maintain long-lasting latent sensitization of dorsal horn neurons that is kept in remission by the NPY-Y1 receptor system. Furthermore, we have identified and characterized 2 novel molecular signaling pathways in the dorsal horn that drive latent sensitization in the setting of chronic inflammatory pain: NMDAR→AC1→PKA→TRPA1/V1 and NMDAR→AC1→Epac1/2. New treatments for chronic inflammatory pain might either increase endogenous NPY analgesia or inhibit AC1, PKA, or Epac.

Priming of Adult Incision Response by Early-Life Injury: Neonatal Microglial Inhibition Has Persistent But Sexually Dimorphic Effects in Adult Rats.

Neonatal hindpaw incision primes developing spinal nociceptive circuitry, resulting in enhanced hyperalgesia following reinjury in adulthood. Spinal microglia contribute to this persistent effect, and microglial inhibition at the time of adult reincision blocks the enhanced hyperalgesia. Here, we pharmacologically inhibited microglial function with systemic minocycline or intrathecal SB203580 at the time of neonatal incision and evaluated sex-dependent differences following adult reincision. Incision in adult male and female rats induced equivalent hyperalgesia and spinal dorsal horn expression of genes associated with microglial proliferation (Emr1) and transformation to a reactive phenotype (Irf8). In control adults with prior neonatal incision, the enhanced degree and duration of incision-induced hyperalgesia and spinal microglial responses to reincision were equivalent in males and females. However, microglial inhibition at the time of the neonatal incision revealed sex-dependent effects: the persistent mechanical and thermal hyperalgesia following reincision in adulthood was prevented in males but unaffected in females. Similarly, reincision induced Emr1 and Irf8 gene expression was downregulated in males, but not in females, following neonatal incision with minocycline. To evaluate the distribution of reincision hyperalgesia, prior neonatal incision was performed at different body sites. Hyperalgesia was maximal when the same paw was reincised, and was increased following prior incision at ipsilateral, but not contralateral, sites, supporting a segmentally restricted spinal mechanism. These data highlight the contribution of spinal microglial mechanisms to persistent effects of early-life injury in males, and sex-dependent differences in the ability of microglial inhibition to prevent the transition to a persistent pain state span developmental stages.SIGNIFICANCE STATEMENT Following the same surgery, some patients develop persistent pain. Contributory mechanisms are not fully understood, but early-life experience and sex/gender may influence the transition to chronic pain. Surgery and painful procedural interventions in vulnerable preterm neonates are associated with long-term alterations in somatosensory function and pain that differ in males and females. Surgical injury in neonatal rodents primes the developing nociceptive system and enhances reinjury response in adulthood. Neuroimmune interactions are critical mediators of persistent pain, but sex-dependent differences in spinal neuroglial signaling influence the efficacy of microglial inhibitors following adult injury. Neonatal microglial inhibition has beneficial long-term effects on reinjury response in adult males only, emphasizing the importance of evaluating sex-dependent differences at all ages in preclinical studies.

Ablation of α2δ-1 inhibits cell-surface trafficking of endogenous N-type calcium channels in the pain pathway in vivo

The auxiliary α2δ calcium channel subunits play key roles in voltage-gated calcium channel function. Independent of this, α2δ-1 has also been suggested to be important for synaptogenesis. Using an epitope-tagged knockin mouse strategy, we examined the effect of α2δ-1 on CaV2.2 localization in the pain pathway in vivo, where CaV2.2 is important for nociceptive transmission and α2δ-1 plays a critical role in neuropathic pain. We find CaV2.2 is preferentially expressed on the plasma membrane of calcitonin gene-related peptide-positive small nociceptors. This is paralleled by strong presynaptic expression of CaV2.2 in the superficial spinal cord dorsal horn. EM-immunogold localization shows CaV2.2 predominantly in active zones of glomerular primary afferent terminals. Genetic ablation of α2δ-1 abolishes CaV2.2 cell-surface expression in dorsal root ganglion neurons and dramatically reduces dorsal horn expression. There was no effect of α2δ-1 knockout on other dorsal horn pre- and postsynaptic markers, indicating the primary afferent pathways are not otherwise affected by α2δ-1 ablation.

Ulinastatin attenuates neuropathic pain via the ATP/P2Y2 receptor pathway in rat models

Ulinastatin, a serine protease inhibitor, which has anti-inflammatory properties and neuroprotective effects, is used to treat acute inflammatory disorders. Recent evidence indicates that administration of ulinastatin alleviates pain in rat model of neuropathic pain (NPP). However, its effect on NPP and the underlying mechanism requires further study. In this study, we evaluated the role of intrathecal administration of ulinastatin in rats with sciatic nerve ligation and observed the effect of ulinastatin on the ATP/P2Y2 receptor pathway. We performed mechanical and thermal sensitivity measurements, immunohistochemistry and double-label immunofluoresence studies to evaluate P2Y2 receptor and adenosine 5′-monophosphate-activated protein kinase (AMPK) expression in the dorsal horn of the lumbar enlargement region of the spinal cord, and a luciferase assay for the detection of ATP levels in the cerebrospinal fluid. The results showed that ulinastatin prevented the development of mechanical allodynia and thermal hypersensitivity in the rat sciatic nerve ligation model. Ulinastatin reduced the level of extracellular ATP, down-regulated P2Y2 receptor and AMPK expression in the spinal dorsal horn of the chronic constrictive injury model. We found that increased expression of P2Y2 receptor in microglia was likely involved in the activation of microglia after nerve injury, and ulinastatin inhibited the abnormal microglia activation in the dorsal horn after nerve injury. These findings demonstrated that ulinastatin might be a potential and effective drug for the treatment of NPP via the suppression of the ATP/P2Y2 receptor pathway.

The Different Dynamic Changes of Nerve Growth Factor in the Dorsal Horn and Dorsal Root Ganglion Leads to Hyperalgesia and Allodynia in Diabetic Neuropathic Pain

Background: Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes and more than half of the patients with DPN have self-reported symptoms referring to painful diabetic neuropathy (PDN). Nerve growth factor (NGF) is a key factor for the nervous system, but the role of it in the neuropathic pain of diabetic patients is unclear. Objective: This study aimed to investigate the relationship between the dynamic expression of NGF in dorsal horn and dorsal root ganglion (DRG) of diabetic rats and hyperalgesia and allodynia in diabetic neuropathic pain. It also aimed to explore the effects of exogenous mouse NGF (mNGF) on NGF expression in dorsal horn, DRG, and mechanical pain threshold. Study Design: Animal research study. Setting: Experimental research laboratory. Methods: The model of diabetes was established by a single intraperitoneal injection of streptozocin (STZ 55 mg/kg). Firstly, the rats were randomly divided into 2 groups: control group (n = 10) and diabetes group (n = 40). The diabetes group contained 4 subgroups: diabetes week 1 group (DM1, n = 10), diabetes week 2 group (DM2, n = 10), diabetes week 4 group (DM4, n = 10), and diabetes week 8 group (DM8, n = 10). Then, the other rats were randomly divided into 2 groups: control group (n = 10) and treatment group (n = 30). The treatment group contained 3 subgroups: saline group (n = 10), low dose mNGF group (mNGF1, n = 10), and high dose mNGF group (mNGF2, n = 10). Mechanical pain threshold was assessed using Von Frey hairs, before the establishment of the diabetes model and 1, 2, 4, and 8 weeks after the establishment. The NGF expression in dorsal horn and DRG was measured by western blot. Results: The mechanical pain threshold decreased one week after the establishment of the diabetes model, which continued for 8 weeks. The NGF expression in the dorsal horn was reduced 2 weeks after diabetes induction and the decreased NGF expression continued for 4 weeks. However, the NGF expression in DRG was reduced one week after diabetes induction and remained at a low level for 8 weeks. Hyperalgesia occurred when the NGF expression in the DRG decreased and further reduction in the NGF expression in the dorsal horn caused concomitant allodynia. The mechanical pain threshold was significantly elevated 2 weeks after mNGF treatment. Limitations: The course of diabetes should be much longer and there is not a precise analysis of the quantitative relation between the NGF expression in the dorsal horn/DRG and hyperalgesia/ allodynia. Conclusion: In diabetic neuropathic pain, the dynamic changes of the NGF expression in dorsal horn and DRG is involved in the development of hyperalgesia and allodynia respectively. Exogenous mNGF may relieve diabetic neuropathic pain by increasing the NGF expression in dorsal horn and DRG. Key words: Nerve growth factor, diabetic peripheral neuropathy, pain, hyperalgesia, allodynia, dorsal horn, dorsal root ganglion Pain Physician 2017; 20:E551

A central role for spinal dorsal horn neurons that express neurokinin-1 receptors in chronic itch.

We investigated roles for spinal neurons expressing the neurokinin-1 receptor (NK1R) and/or gastrin-releasing peptide receptor (GRPR) in a mouse model of ovalbumin (OVA)-induced chronic atopic dermatitis. Mice receiving repeated topical application of OVA exhibited atopic-like skin lesions and behavioral signs of chronic itch including spontaneous scratching, touch-evoked scratching (alloknesis), and enhancement of chloroquine-evoked scratching (hyperknesis). Substance P-saporin (SP-SAP) and bombesin-saporin (BB-SAP) were intrathecally injected into OVA-sensitized mice to neurotoxically ablate NK1R- or GRPR-expressing spinal neurons, respectively. SP-SAP diminished the expression of NK1R in the superficial spinal dorsal horn and significantly attenuated all behavioral signs of chronic itch. BB-SAP reduced the spinal dorsal horn expression of GRPR and significantly attenuated hyperknesis, with no effect on spontaneous scratching or alloknesis. To investigate whether NK1R-expressing spinal neurons project in ascending somatosensory pathways, we performed a double-label study. The retrograde tracer, Fluorogold (FG), was injected into either the somatosensory thalamus or lateral parabrachial nucleus. In the upper cervical (C1-2) spinal cord, most neurons retrogradely labeled with FG were located in the dorsomedial aspect of the superficial dorsal horn. Of FG-labeled spinal neurons, 89% to 94% were double labeled for NK1R. These results indicate that NK1R-expressing spinal neurons play a major role in the expression of symptoms of chronic itch and give rise to ascending somatosensory projections. Gastrin-releasing peptide receptor-expressing spinal neurons contribute to hyperknesis but not to alloknesis or ongoing itch. NK1R-expressing spinal neurons represent a potential target to treat chronic itch.

Involvement of CCL1/CCR8 in Spinal Cord Dorsal Horn in Remifentanil-Induced Hyperalgesia in Rats

Background: Numerous researches manifested that remifentanil infusion might be responsible for opioid-induced hyperalgesia (OIH), but most studies exploring OIH were hardly designed to clarify underlying mechanisms. Chemokine (C-C motif) ligand 1 (CCL1) was found to be implicated in neuropathic pain, and our previous study has also shown that proinflammatory cytokines have been associated with the induction and maintenance of OIH. However, whether CCL1 could contribute to hyperalgesia induced by remifentanil in rats remains unclear.Methods: To explore effect of CCL1 on OIH, a neutralizing antibody against CCL1 (anti-CCL1) was administrated intrathecally after remifentanil infusion in rats. Western blotting and immunohistochemistry (IHC) were applied to analyze time course of CCL1 and CCR8 (specific CCL1 receptor) expression in dorsal horn after remifentanil administration. Expression of inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 was evaluated by real-time quantitative polymerase chain reaction (RT-qPCR). Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured and recorded for 48 post-infusion hours to evaluate mechanical and thermal hyperalgesia.Results: We discovered that CCL1 and CCR8 expressions in dorsal horn were increased and maintained at a high level from 2 hours to 48 hours, the last examination time, after remifentanil infusion. We found that intrathecal delivery of anti-CCL1 could ameliorate remifentanil related thermal and mechanical hyperalgesia without affecting baseline nociceptive threshold. It was also shown that enhancement of inflammatory mediators (TNF-α, IL-1β, and IL-6) expression in dorsal horn after remifentanil infusion was reversed by anti-CCL1 administration.Conclusions: Our current results indicated that CCL1 and CCR8 might be implicated in the development of remifentanil-induced hyperalgesia via regulation of cytokines (TNF-α, IL-1β, and IL-6) in dorsal horn in rats. Citation: Lin- Lin Zhang, Rui- Chen Shu, Nan Li, Zhi-Fen Wang, Chun-Yan Wang, Hai- Yun Wang, et al. Involvement of CCL1/CCR8 in spinal cord dorsal horn in remifentanil- induced hyperalgesia in rats. J Anesth Perioper Med 2015; 2: 53-60. doi: 10.24015/JAPM.2015.0009This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Development and Characterization of a Novel, Anatomically Relevant Rat Model of Acute Postoperative Pain

Acute postoperative pain remains a significant health care issue. Development of anatomically relevant animal models of postoperative pain, with improved predictive validity, would advance understanding of postoperative pain mechanisms and improve treatment outcomes. This study aimed to develop, characterize, and validate a rat model of acute postoperative pain associated with inguinal hernia repair based on the Lichtenstein inguinal hernia repair procedure (without hernia induction). We hypothesized that the surgery would result in reduced spontaneous locomotor activity, which would represent a pain-related phenotype. Postsurgical characterization involved extensive monitoring of home cage and open field locomotor activity, as well as mechanical hypersensitivity and assessment of c-Fos expression in the dorsal horn of the spinal cord. In pharmacologic validation studies, rats received morphine, carprofen, or paracetamol 1 hour before, and/or immediately after, surgery. Rats that underwent hernia repair surgery exhibited significantly lower horizontal and vertical activities in the home cage and open field in the early postsurgical period, compared with sham rats or rats that underwent skin incision only. Morphine, carprofen, and paracetamol attenuated the surgery-induced reductions in locomotor activity, to varying degrees. Surgery was associated with significantly increased c-Fos expression in the ipsilateral dorsal horn of the spinal cord, an effect attenuated by carprofen treatment. These results support the development and characterization of a novel, anatomically relevant animal model of acute postoperative pain that may facilitate development of improved treatment regimens. PerspectiveAcute pain following inguinal hernia repair can be difficult to treat. Here we report, for the first time, the development of a novel, anatomically relevant rat model to facilitate improved understanding and treatment of acute postoperative pain following inguinal hernia repair.