Application Of Nucleus Pulposus Research Articles

Changes in ion-channels in the dorsal root ganglion after exposure to autologous nucleus pulposus and TNF. A rat experimental study

PurposeIt is known that contact of nucleus pulposus with the dorsal root ganglion may induce changes in nerve conduction and pain behavior. It has also been suggested that the behavioristic changes are caused by changes in voltage-gated ion channels, which in turn have been upregulated by TNF. Such upregulations have previously been shown for NaV 1.8 and NaV 1.9. In this investigation, we expanded the number of studied ion channels after the application of nucleus pulposus or TNF. MethodsFollowing removal of the left L4-5 fact joint, a disc puncture was performed and the dorsal root ganglion was exposed to nucleus pulposus (n = 5) and TNF (n = 5). Operated rats without disc puncture served as sham (n = 5) and 5 non-operated (naïve) rats were included. After 24 h, the DRGs were harvested and analyzed by quantitative PCR on validated pre-spotted primer plates displaying genes for 90 voltage-gated ion channels. ResultsIt was evident that the changes in operated animals were separate from the naïve rats. It was also apparent that gene expression changes in rats with nucleus pulposus or TNF application showed similar trends and were also separated from sham-operated animals. ConclusionThe application of nucleus pulposus and TNF onto the DRG in rats induces comparable changes in gene expression of several ion channels. Since the changes induced by TNF and NP are similar, one might also suspect that TNF mediates the NP-induced changes. However, such a mechanism needs further investigation.

Microglia-independent peripheral neuropathic pain in male and female mice.

The dominant view in the field of pain is that peripheral neuropathic pain is driven by microglia in the somatosensory processing region of the spinal dorsal horn. Here, to the contrary, we discovered a form of neuropathic pain that is independent of microglia. Mice in which the nucleus pulposus (NP) of the intervertebral disc was apposed to the sciatic nerve developed a constellation of neuropathic pain behaviours: hypersensitivity to mechanical, cold, and heat stimuli. However, NP application caused no activation of spinal microglia nor was pain hypersensitivity reversed by microglial inhibition. Rather, NP-induced pain hypersensitivity was dependent on cells within the NP which recruited macrophages to the adjacent nerve. Eliminating macrophages systemically or locally prevented NP-induced pain hypersensitivity. Pain hypersensitivity was also prevented by genetically disrupting the neurotrophin brain-derived neurotrophic factor selectively in macrophages. Moreover, the behavioural phenotypes as well as the molecular mechanisms of NP-induced pain hypersensitivity were not different between males and females. Our findings reveal a previously unappreciated mechanism for by which a discrete peripheral nerve lesion may produce pain hypersensitivity, which may help to explain the limited success of microglial inhibitors on neuropathic pain in human clinical trials.

Protectin DX Attenuates Lumbar Radicular Pain of Non-compressive Disc Herniation by Autophagy Flux Stimulation via Adenosine Monophosphate-Activated Protein Kinase Signaling.

Background: Neuroinflammation plays a crucial role in initiating and sustaining lumbar radicular pain (LRP). Protectin DX (PDX) has been experimentally verified to possess pro-resolving properties and anti-inflammatory effects. This study aimed to observe the analgesic effects of PDX and its potential mechanisms in LRP rats with non-compressive lumbar disc herniation (NCLDH).Method: Only male rats were selected to avoid gender-related interferences. Rat models of NCLDH were established, and rats were randomly divided into four groups: the sham group, the vehicle group, the PDX (10 ng PDX) group, and the PDX (100 ng PDX) group. Changes in the mechanical withdrawal threshold and thermal withdrawal latency were observed for 7 days. The mRNAs of pro-inflammatory and anti-inflammatory mediators were evaluated via real-time polymerase chain reaction, whereas western blot and immunohistochemistry were separately conducted to assess the expression levels of autophagy-related proteins and adenosine monophosphate-activated protein kinase (AMPK) signaling.Results: Intrathecal delivery of PDX reduced interleukin (IL)-6 and IL-1β mRNA levels and facilitated mRNA transcription of transforming growth factor-β1, with attenuation of mechanical and thermal hyperalgesia in LRP rat models. With the application of nucleus pulposus to the dorsal root ganglion, autophagy flux and AMPK signaling were severely disrupted in the spinal dorsal horns, and intrathecal treatment with PDX could dose-dependently restore the dysfunction of autophagy flux and AMPK signaling.Conclusion: These data suggest that PDX possesses pro-resolving properties and exerts potent analgesic effects in LRP by affecting autophagy flux via AMPK signaling.

69. Ischemic spinal nerve root injury secondary to herniated lumbar intervertebral disc

BACKGROUND CONTEXT The painful clinical sequelae associated with sciatica secondary to intervertebral disc herniation have been traditionally attributed to mechanical compression of the spinal nerve root. More recent studies have also implicated inflammatory mediators and microvascular changes in the pathogenesis of acute sciatica. The application of nucleus pulposus (NP) to a spinal nerve root has been shown to induce thrombus formation in the associated nerve microvasculature and result in greater injury to the axons in the center than in the periphery, suggesting an ischemic mechanism of injury. The spinal nerve blood flow changes that are associated with nerve dysfunction seen in acute sciatica offer a potential target for therapeutic intervention. The VEGF angiogenic cytokines have been demonstrated to be potent stimulators of the differentiation and function of vascular endothelial cells after injury. PURPOSE To determine the relationship between the severity of spinal nerve root injury in patients with acute sciatica secondary to lumbar disc herniation (AS/LDH) undergoing surgical discectomy and the levels of VEGF-A and VEGF-B in serum, spinal nerve root lavage and disc tissue homogenate. STUDY DESIGN/SETTING ELISA cytokine immunoassay. PATIENT SAMPLE Twenty-four patients with AS/LDH of less than 16 weeks duration, undergoing laminectomy discectomy surgery. OUTCOME MEASURES VEGF-A and VEGF-Bconcentrations (pg/ml) in herniated nucleus pulposus homogenate (HNP-H), spinal nerve root lavagate (SN-L) and serum. METHODS At the time of surgery, the spinal nerve root was lavaged with 5 cc of saline, the extruded disc tissue removed surgically and serum samples were obtained. Disc and lavagate samples were stored in physiological buffer with protease inhibitors at -80° C. The concentrations of VEGF-A and VEGF-Bin disc tissue homogenate, serum and lavagate samples were measured in triplicate using ELISA immunoassay. Research ethics committee approval was obtained. RESULTS In AS/LDH patients, VAS pain and significant spinal nerve root deficit scores (3.0 - 4.0/5) correlated closely with serum and HNP-H tissue levels of VEGF-A and VEGF-B (Pearson r=0.78). In 11/24 patients, elevated VEGF-A and B concentrations correlated to a moderate degree with TNFα and IL-2, 6 and 8 inflammatory cytokine levels (r=0.61). Low concentrations of VEGF and inflammatory cytokine concentrations were seen in all SN-L test samples. CONCLUSIONS In patients with acute sciatica secondary to lumbar disc herniation, the severity of spinal nerve injury appears to be related to a combined mechanism, involving ischemic and inflammatory components. This research study was funded by grants from the National Institutes of Health, Bethesda, USA and from the University of British Columbia Orthopaedic Research Excellence Fund. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

Maresin 1 Attenuates Radicular Pain Through the Inhibition of NLRP3 Inflammasome-Induced Pyroptosis via NF-κB Signaling.

BackgroundThe exposure of the nucleus pulposus (NP) causes an immune and inflammatory response, which is intrinsically linked to the pathogenesis of radicular pain. As a newly discovered pro-resolving lipid mediator, maresin 1 (MaR1) could exert powerful inflammatory resolution, neuroprotection, and analgesic activities. In the present research, the analgesic effect of MaR1 was observed. Then, the potential mechanism by which MaR1 attenuated radicular pain was also analyzed in a rat model.MethodsIntrathecal administration of MaR1 (10 or 100 ng) was successively performed in a rat with non-compressive lumbar disk herniation for three postoperative days. Mechanical and thermal thresholds were determined to assess pain-related behavior from days 1 to 7 (n = 8/group). On day 7, the tissues of spinal dorsal horns from different groups were gathered to evaluate expression levels of inflammatory cytokines (IL-1β, IL-18, and TNF-α), the NLRP3 inflammasome and pyroptosis indicators (GSDMD, ASC, NLRP3, and Caspase-1), together with NF-κB/p65 activation (n = 6/group). TUNEL and PI staining were performed to further examine the process of pyroptosis.ResultsAfter intrathecal administration in the rat model, MaR1 exhibited potent analgesic effect dose-dependently. MaR1 significantly prompted the resolution of the increased inflammatory cytokine levels, reversed the up-regulated expression of the inflammasome and pyroptosis indicators, and reduced the cell death and the positive activation of NF-κB/p65 resulting from the NP application on the L5 dorsal root ganglion.ConclusionThis study indicated that the activation of NLRP3 inflammasome and pyroptosis played a significant role in the inflammatory reaction of radicular pain. Also, MaR1 could effectively down-regulate the inflammatory response and attenuate pain by inhibiting NLRP3 inflammasome-induced pyroptosis via NF-κB signaling.

A Novel Mechanism of Specialized Proresolving Lipid Mediators Mitigating Radicular Pain: The Negative Interaction with NLRP3 Inflammasome.

Inhibition of immune and inflammatory reaction induced by the expose of nucleus pulposus (NP) could effectively ameliorate neuropathic pain in the lumbar disc herniation. Maresin1 (MaR1), as a macrophage-derived mediator of inflammation resolution, displayed potent anti-inflammatory action. In the present study, we attempted to elucidate the impact of MaR1 on radicular pain and the interaction with NLRP3 inflammasome. We established a rat model of non-compressive lumbar disc herniation and different administration (MaR1 or Caspase-1 inhibitor) was given to them. The paw withdrawal latency (PWL) and paw withdrawal thresholds (PWTs) were observed to assess pain behaviors. The spinal cord horns were collected and the levels of IL-1β and IL-18 were measured by ELISA. The mRNA and protein expression levels of NLRP3 inflammasome components were tested by RT-PCR, western blot and immunohistochemistry. The endogenous MaR1 levels of the spinal cord were analyzed using LC-MS/MS. The application of NP in the models lead to mechanical and thermal hypersensitivity, increased IL-1β and IL-18 levels and expressions of NLRP3 inflammasome components, which were reversed markedly by administration of MaR1. Caspase-1 inhibition also improved mechanical hypersensitivity, decreased the expressions of inflammatory cytokines and restrained the activation of inflammasome. Meanwhile, Caspase-1 inhibitor promoted the endogenous MaR1 synthesis, which was hindered in the pain models. Altogether, our study indicated that the negative interaction between MaR1 and NLRP3 inflammasome mediated the inflammatory response in spinal dorsal horn, which involved in the pathogenesis of radicular pain.

Acetaminophen combined with tramadol is more effective than acetaminophen or tramadol to reduce neuropathic root pain: an experimental study with application of nucleus pulposus in a rat model.

Various drugs are used to treat patients with neuropathic pain; however, optimal treatment using acetaminophen (A) and/or tramadol (T) remains unclear. The evidence supporting the drug choice and the timing of administration is insufficient. Therefore, the objective of the present study was to investigate the effect of T and/or A on pain-related behavior in a nucleus pulposus (NP) rat model. Sprague-Dawley rats (n = 180) were divided into NP-A (52mg/kg), NP-T (6mg/kg), NP-AT (combined A and T), NP-S (saline), and sham groups (n = 36 per group). The rats received 0.2mL of treatment solution orally once daily for 7days after application of NP on the left L5 dorsal root ganglion (DRG). Behavioral testing and immunohistochemistry analysis for some markers' expressions in DRGs and the spinal cord were performed. Pain thresholds in the NP-AT group did not significantly differ from the sham at all time points, while those were significantly lower in the NP-A and in the NP-T groups at D7 and/or D14 (p < 0.05). Tumor necrosis factor-α in the NP-S group was significantly higher at D2 and D7 (p < 0.05). Among the three treatment groups, activating transcriptional factor 3 and growth-associated protein 43 showed a tendency toward an increase at D7-D21. Combined administration of acetaminophen and tramadol maintained in the pain threshold in the rat NP model. These findings suggest that the combination of acetaminophen and tramadol might be a potential therapeutic modality for patients with lumbar disc herniation. These slides can be retrieved under Electronic Supplementary Material.

Resolvin D2 Relieving Radicular Pain is Associated with Regulation of Inflammatory Mediators, Akt/GSK-3β Signal Pathway and GPR18.

Neuroinflammation induced by protruded nucleus pulposus (NP) has been shown to play a significant role in facilitation of radicular pain. Resolvin D2 (RvD2), a novel member of resolvin family, exhibits potent anti-inflammatory, pro-resolving and antinociceptive effects. But the effect of RvD2 in radicular pain remains unknown. The radicular pain rat models were induced by application of NP to L5 dorsal root ganglion. Each animal received intrathecal injections of vehicle or RvD2 (10ng µl-1 or 100ng µl-1). Mechanical thresholds were determined by measuring the paw withdrawal threshold for 7 days. The expressions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and transforming growth factor-β1 (TGF-β1) in ipsilateral lumbar segment of rat spinal dorsal horns were measured by using ELISA and real time-PCR. Western blot was used to measure the expressions of phosphorylated Akt (p-Akt) and phosphorylated glycogen synthase kinase 3 beta (p-GSK-3β). The expressions and distributions of RvD2 receptor, G-protein-coupled receptor 18 (GPR18), were also explored in the spinal cord of rats by using double-label immunofluorescence. RvD2 treatment caused significant reductions in the intensity of mechanical hypersensitivity and spinal expressions of TNF-α and IL-6. Meanwhile, RvD2 increased the expressions of TGF-β1 and regulated Akt/GSK-3β signaling. Furthermore, immunofluorescence showed that GPR18 colocalized with neurons and astrocytes in spinal cord. The results suggested that RvD2 might attenuate mechanical allodynia via regulating the expressions of inflammatory mediators and activation of Akt/GSK-3β signal pathway. RvD2 might offer a hopeful method for radicular pain therapy.

Gua Sha attenuates thermal hyperalgesia and decreases proinflammatory cytokine expression in serum in rats with lumbar disc herniation induced by autologous nucleus pulposus

OBJECTIVETo investigate the analgesic effect of Gua Sha and its underlying mechanism in rats with noncompressive lumbar disk herniation induced by autologous nucleus pulposus. METHODSA rat model of noncompressive lumbar disk herniation was established and rats were randomly divided into model group, sham group, and Gua Sha group (24 in each group). Gua Sha was performed from the 5th day after the surgery, once every other day, 3 times for a course of treatment, and totally 3 courses. The thermal withdrawal latency was evaluated using the intelligent hot plate one day before the surgery, and on days 4 (the day before the treatment), 10 (the end of the first course), 16 (the end of the second course) and 22 (the end of the third course). On days 4, 10, 16 and 22, six rats in each group were picked randomly and their blood samples were drawn to assess the expression of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). RESULTSCompared to rats in the sham group, the application of nucleus pulposus to right L5 dorsal root ganglion induced prolonged thermal hyperalgesia, and up-regulated the expression of IL-1β, IL-6 and TNF-α in serum (P < 0.01). The therapy of Gua Sha attenuated thermal hyperalgesia potently, inhibited the expression of IL-1β, IL-6 and TNF-α in a time-dependent manner (P < 0.01). There were no significant differences in the thermal withdrawal latency and the expression of inflammatory cytokines between the sham and Gua Sha groups at the end of the treatment (P > 0.01). CONCLUSIONThe current study showed that Gua Sha might alleviate thermal hyperalgesia in rats with lumbar disc herniation induced by autologous nucleus pulposus via inhibiting the expression of proinflammatory cytokins.

Bone mesenchymal stem cells attenuate radicular pain by inhibiting microglial activation in a rat noncompressive disk herniation model.

Spinal disk herniation can induce radicular pain through chemical irritation caused by proinflammatory and immune responses. Bone marrow mesenchymal stem cells (BMSCs) are a unique type of adult stem cell with the functions of suppressing inflammation and modulating immune responses. This study was undertaken to observe the effect of intrathecal BMSCs on the treatment of mechanical allodynia and the suppression of microglial activation in a rat noncompressive disk herniation model. The model was induced by the application of nucleus pulposus (NP) to the L5 dorsal root ganglion (DRG). The study found that the use of NP in the DRG can induce abnormal mechanical pain, increase the contents of the proinflammatory factors TNF-α and IL-1β, decrease the content of the anti-inflammatory cytokine TGF-β1 and activate microglia in the spinal dorsal horns (L5) (P < 0.05). BMSC administration could increase the mechanical withdrawal thresholds dramatically, decrease the contents of IL-1β and TNF-α, increase the content of TGF-β1 significantly (P < 0.05) and inhibit microglial activation in the bilateral spinal dorsal horn. Our results indicate that BMSC administration can reduce mechanical allodynia and downregulate the expression of proinflammatory cytokines by inhibiting microglial activation in the spinal dorsal horn in a rat noncompressive disk herniation model.

IL-33/ST2 signaling contributes to radicular pain by modulating MAPK and NF-\u03baB activation and inflammatory mediator expression in the spinal cord in rat models of noncompressive lumber disk herniation

BackgroundImmune and inflammatory responses occurring in the spinal cord play a pivotal role in the progression of radicular pain caused by intervertebral disk herniation. Interleukin-33 (IL-33) orchestrates inflammatory responses in a wide range of inflammatory and autoimmune disorders of the nervous system. Thus, the purpose of this study is to investigate the expression of IL-33 and its receptor ST2 in the dorsal spinal cord and to elucidate whether the inhibition of spinal IL-33 expression significantly attenuates pain-related behaviors in rat models of noncompressive lumbar disc herniation.MethodsLentiviral vectors encoding short hairpin RNAs that target IL-33 (LV-shIL-33) were constructed for gene silencing. Rat models of noncompressive lumber disk herniation were established, and the spines of rats were injected with LV-shIL-33 (5 or 10 μl) on the first day after the operation. Mechanical thresholds were evaluated during an observation period of 21 days. Moreover, the expression levels of spinal tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and cyclooxygenase 2 (COX-2) and the activation of the mitogen-activated protein kinases (MAPK) and nuclear factor-κB (NF-κB) pathways were evaluated to gain insight into the mechanisms related to the contribution of IL-33/ST2 signaling to radicular pain.ResultsThe application of nucleus pulposus (NP) to the dorsal root ganglion (DRG) induced an increase in IL-33 and ST2 expression in the spinal cord, mainly in the dorsal horn neurons, astrocytes, and oligodendrocytes. Spinally delivered LV-shIL-33 knocked down the expression of IL-33 and markedly attenuated mechanical allodynia. In addition, spinal administration of LV-shIL-33 reduced the overexpression of spinal IL-1β, TNF-α, and COX-2 and attenuated the activation of C-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and NF-κB/p65 but not p38.ConclusionsThis study indicates that spinal IL-33/ST2 signaling plays an important role in the development and progression of radicular pain in rat models of noncompressive lumber disk herniation. Thus, the inhibition of spinal IL-33 expression may provide a potential treatment to manage radicular pain caused by intervertebral disk herniation.

Investigation of the Effect of Diabetes on Radiculopathy Induced by Nucleus Pulposus Application to the DRG in a Spontaneously Diabetic Rat Model.

A controlled, interventional animal study. The aim of this study was to evaluate the effect of diabetes mellitus (DM) on radiculopathy due to lumbar disc herniation (LDH), by investigating pain-related behavior and the expression of tumor necrosis factor-alpha (TNF-α) and growth-associated protein 43 (GAP43) in type 2 diabetic rats following application of nucleus pulposus (NP) to the dorsal root ganglion (DRG). Previous clinical studies suggested negative effects of DM on radiculopathy due to LDH, and that inflammation and nerve regeneration could interact with DM and radiculopathy. We applied autologous NP to the left L5 DRG of adult male Wistar rats and Goto-Kakizaki rats. Behavioral testing measured the mechanical withdrawal threshold of rats. We immunohistochemically evaluated the localization of ionized calcium-binding adapter molecule-1 (Iba-1), receptor of advanced glycation end products (RAGE), and TNF-α in DRGs. TNF-α and GAP43 expression levels in DRG were determined by quantitative real-time PCR and western blotting. The mechanical withdrawal threshold significantly declined in the non-DM NP group compared with the non-DM sham group for 28 days, whereas the decline in threshold extended to 35 days in the DM NP group compared with the DM sham group. RAGE and TNF-α expression in DRGs was colocalized in Iba-1 positive cells. The non-DM NP rats had higher TNF-α protein expression levels versus the non-DM sham rats on day 7, and the DM NP group had higher levels versus the DM sham group on days 7 and 14. The non-DM NP group had higher GAP43 mRNA expression than the non-DM sham group for 28 days, while the DM NP group had a higher level than the DM sham group for 35 days. DM prolongs the pain-related behavior caused by NP. The prolonged inflammation and nerve regeneration could elucidate the pathogenesis of continuous pain of radiculopathy initiated by LDH. N /A.

Upregulation of nuclear factor‑κB and acid sensing ion channel 3 in dorsal root ganglion following application of nucleus pulposus onto the nerve root in rats.

The nucleus pulposus (NP) is an avascular, hydrated tissue that permits the intervertebral disc to resist compressive loads to the spine. To determine the mechanisms by which intervertebral disc degeneration is caused by the nucleus pulposus, the expression and regulation of nuclear factor (NF)‑κB and acid sensing ion channel 3 (ASIC3) were examined. For the intervertebral disc degeneration model, NP was harvested from the tail of rats and applied to the L5 dorsal root ganglion (DRG). The mechanical pain withdrawal threshold (PWT) in NP model rats was assessed. Reverse transcription‑quantitative polymerase chain reaction and western blotting were used to examine NF‑κB and ASIC3 expression levels in DRG. Finally, the effect of the NF‑κB inhibitor pyrrolidine dithiocarbamate (PDTC) and the ASIC3 signaling pathway blocker amiloride were examined. Rats exposed to NP exhibited decreased PWT for 12days, and NF‑κB and ASIC3 was upregulated in DRG induced by NP 14days after surgery. After administration of amiloride and PDTC to DRG affected by NP, the levels of nitric oxide (NO), tumor necrosis factor‑α (TNF‑α), interleukin‑6 (IL‑6), NF‑κB and ASIC3 were downregulated, and the levels of aquaporin (AQP) 1 and AQP3 were significantly increased for 14days. In conclusion, these results suggested that NF‑κB and ASIC3 may serve an important role in intervertebral disc degeneration caused by NP.

Local up-regulation of interferon-γ (IFN-γ) following disc herniation is involved in the inflammatory response underlying acute lumbar radicular pain

Lumbar radicular pain after disc herniation may be associated with release of pro-inflammatory cytokines from nucleus pulposus (NP) tissue. In the present study we examined the role of interferon-γ (IFN-γ) and cluster of differentiation 68 (CD68) in the acute phase of this process.First, in an animal model mimicking the clinical situation after disc herniation, the role of IFN-γ close to the dorsal nerve roots was studied. Next, in patients with lumbar radicular pain due to disc herniation, we examined how two single nucleotide polymorphisms (SNPs; rs2069705 and rs2069718) are important for the IFN-γ expression influenced the pain behavior.The animal data demonstrated a significant increase in the nociceptive activity at the spinal level after local application of NP and IFN-γ onto the dorsal nerve roots. A positive correlation between IFN-γ and CD68 in the NP tissue was also demonstrated. In the patients, a significant increase in Oswestry Disability Index (ODI) score was observed in carriers of the IFN-γ SNPs; rs2069705 A and rs2069718 G alleles.The present data suggest that IFN-γ close to the dorsal nerve roots may contribute to the pathogenesis, the nociceptive activity and the pain behavior following lumbar disc herniation.

Effect of an Acid-sensing Ion Channels Inhibitor on Pain-related Behavior by Nucleus Pulposus Applied on the Nerve Root in Rats.

Controlled, interventional animal study. To examine the effect of an inhibitor of acid-sensing ion channel 3 (ASIC3) on pain-related behavior induced by application of the nucleus pulposus (NP) onto the dorsal root ganglion (DRG) in rats. ASIC3 is associated with acidosis pain in inflamed or ischemic tissues and is expressed in sensory neurons and NP cells. The ASIC3 inhibitor, APETx2, increases the mechanical threshold of pain in models of knee osteoarthritis or postoperative pain. However, the efficacy of APETx2 for pain relief in the NP application model remains unknown. Autologous NP was applied to the left L5 nerve root of 183 adult female Sprague-Dawley rats. The DRGs were treated with NP plus one of the following four treatments: saline solution (SM), low (0.01 μg: LD), medium (0.1 μg: MD), or high dose (1.0 μg: HD) of APETx2. Behavioral testing was performed to investigate the mechanical withdrawal threshold using von Frey hairs. Expression of nerve growth factor, hypoxia-inducible factor-1α (HIF1α), activating transcription factor-3, and ionized calcium-binding adaptor molecule-1 was evaluated using immunohistochemistry. Statistical differences among multiple groups were assessed using the Steel test, the Tukey-Kramer test, and the Dunnett test. P < 0.05 were considered significant. The thresholds in the HD group were higher than those in the SM group at Days 14 and 21 (P < 0.05). In the MD group, the threshold was higher than in the SM group at Day 14 (P < 0.05). High doses of APETx2 reduced the expression of HIF1α after Day 14 compared with the SM group (P < 0.05). APETx2 significantly improved pain-related behavior in a dose-dependent manner. APETx2 may inhibit ASIC3 and partly inhibit Nav1.8 channels. This ASIC3 channel inhibitor may be a potential therapeutic agent in early-stage lumbar disc herniation. N/A.

Osthole, a Coumadin Analog from Cnidium monnieri (L.) Cusson, Ameliorates Nucleus Pulposus-Induced Radicular Inflammatory Pain by Inhibiting the Activation of Extracellular Signal-Regulated Kinase in Rats

Aim: This study was aimed at assessing the role of extracellular signal regulated kinase (ERK) in mechanical allodynia resulting from lumbar disc herniation (LDH) and exploring the osthole's anti-nociceptive effect on ERK activation. Methods: Radicular pain was generated by applying nucleus pulposus (NP) to the L₅ dorsal root ganglion (DRG). Allodynia was measured using Von Frey filaments to calculate the mechanical pain threshold. Phosphorylated ERK and total ERK protein in the lumbar spinal dorsal horn was detected by using the Western blot technique. Cyclooxygenase 2 (COX-2) mRNA was assessed by real-time reverse-transcription polymerase chain reaction. Results: The application of NP to L₅ DRG induced mechanical hypersensitivity which lasted for at least 28 days, and a significant increase of ERK phosphorylation in the ipsilateral spinal dorsal horn from postoperative day (POD) 1 to POD 21. ERK inhibitor attenuated NP-induced hyperalgesia compared to the dimethyl sulfoxide-(vehicle control) administered group (p < 0.05). Epidural treatment with osthole could ameliorate NP-evoked hyperalgesia by suppressing the activation of ERK rather than decreasing the expression of ERK protein. Osthole could also inhibit the increased expression of COX-2 mRNA in spinal dorsal horn, which was a known downstream effect of ERK signaling pathway. Conclusions: Our results suggest that ERK activation in the spinal dorsal horn plays a vital role in NP-evoked hyperalgesia. Osthole exerts analgesic effect on radicular inflammatory pain in LDH rat model, by down-regulating the mRNA expression of the target gene of COX-2 via inhibiting ERK activation in the spinal dorsal horn.

Activation of epidermal growth factor receptors (EGFRs) following disc herniation induces hyperexcitability in the pain pathways

Abstract Aims Low back pain and sciatica after disc herniation may be caused by mechanical compression of the nerve roots, but also by the release of pro-inflammatory agents including growth factors from the nucleus pulposus (NP). Methods Here, in an animal model mimicking the clinical situation following disc herniation, CLIA protein analyses, extracellular single-cell recordings in the spinal dorsal horn and qPCR were performed to examine the nociceptive signaling due to disc herniation. Results The present data demonstrated that EREG may be released from NP - and that administration of EREG onto the spinal dorsal nerve roots increased spontaneous activity in nociceptive neurons. An up-regulation of EGFR and HER4 in the dorsal horn as well as an up-regulation of HER3 in the DRG were demonstrated after application of NP onto the dorsal nerve roots. Conclusion Our findings suggest that EREG and signaling through its receptors may be involved in pain hypersensitivity and other sensory abnormalities after disc herniation.

Resolvin D1 Inhibits Mechanical Hypersensitivity in Sciatica by Modulating the Expression of Nuclear Factor-κB, Phospho-extracellular Signal-regulated Kinase, and Pro- and Antiinflammatory Cytokines in the Spinal Cord and Dorsal Root Ganglion.

Accumulating evidence indicates that spinal inflammatory and immune responses play an important role in the process of radicular pain caused by intervertebral disk herniation. Resolvin D1 (RvD1) has been shown to have potent antiinflammatory and antinociceptive effects. The current study was undertaken to investigate the analgesic effect of RvD1 and its underlying mechanism in rat models of noncompressive lumbar disk herniation. Rat models of noncompressive lumber disk herniation were established, and mechanical thresholds were evaluated using the von Frey test during an observation period of 21 days (n = 8/group). Intrathecal injection of vehicle or RvD1 (10 or 100 ng) was performed for three successive postoperative days. On day 7, the ipsilateral spinal dorsal horns and L5 dorsal root ganglions (DRGs) were removed to assess the expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-10, and transforming growth factor-β1 (TGF-β1) and the activation of nuclear factor-κB (NF-κB)/p65 and phospho-extracellular signal-regulated kinase (p-ERK) signaling (n = 30/group). The application of nucleus pulposus to L5 DRG induced prolonged mechanical allodynia, inhibited the production of IL-10 and TGF-β1, and up-regulated the expression of TNF-α, IL-1β, NF-κB/p65, and p-ERK in the spinal dorsal horns and DRGs. Intrathecal injection of RvD1 showed a potent analgesic effect, inhibited the up-regulation of TNF-α and IL-1β, increased the release of IL-10 and TGF-β1, and attenuated the expression of NF-κB/p65 and p-ERK in a dose-dependent manner. The current study showed that RvD1 might alleviate neuropathic pain via regulating inflammatory mediators and NF-κB/p65 and p-ERK pathways. Its antiinflammatory and proresolution properties may offer novel therapeutic approaches for the management of neuropathic pain.

Hyperexcitability in Spinal WDR Neurons following Experimental Disc Herniation Is Associated with Upregulation of Fractalkine and Its Receptor in Nucleus Pulposus and the Dorsal Root Ganglion.

Introduction. Lumbar radicular pain following intervertebral disc herniation may be associated with a local inflammatory response induced by nucleus pulposus (NP) cells. Methods. In anaesthetized Lewis rats, extracellular single unit recordings of wide dynamic range (WDR) neurons in the dorsal horn and qPCR were used to explore the effect of NP application onto the dorsal nerve roots (L3–L5). Results. A clear increase in C-fiber response was observed following NP conditioning. In the NP tissue, the expression of interleukin-1β (IL-1β), colony stimulating factor 1 (Csf1), fractalkine (CX3CL1), and the fractalkine receptor CX3CR1 was increased. Minocycline, an inhibitor of microglial activation, inhibited the increase in neuronal activity and attenuated the increase in IL-1β, Csf1, CX3L1, and CX3CR1 expression in NP tissue. In addition, the results demonstrated an increase in the expression of TNF, CX3CL1, and CX3CR1 in the dorsal root ganglions (DRGs). Conclusion. Hyperexcitability in the pain pathways and the local inflammation after disc herniation may involve upregulation of CX3CL1 signaling in both the NP and the DRG.

Production of CCL20 on nucleus pulposus cells recruits IL-17-producing cells to degenerated IVD tissues in rat models.

IL-17-producing cells play an important role in various autoimmune diseases. A higher level of IL-17-producing cells is observed in patients with intervertebral disc (IVD) degeneration. However, the mechanism of the accumulation of IL-17-producing cells remains to be elucidated. Our aim is to demonstrate whether the interaction of CC chemokine ligand (CCL) 20 and its specific receptor CCR6 is involved in the recruitment of IL-17-producing cells to degenerated disc tissues in rat models. The well-accepted needle puncture model and the autologous nucleus pulposus application model of rats were established. All of the animals were randomly divided into four groups: the sham surgery group, the needle puncture group (NP group), the sham surgery + autologous nucleus pulposus application group (sham-ANPA group) and the needle puncture + autologous nucleus pulposus application group (NP-ANPA group). Immunohistochemical staining, western blot and real-time PCR were used to evaluate the expression levels of CCL20, IL-17 and CCR6 in the IVD samples. Moreover, the IL-17 concentrations in the serum were detected by ELISA. Pearson correlations were performed to analyse the correlation among the expressions of CCL20, CCR6 and IL-17. The expressions of CCL20, IL17 and CCR6 were dramatically elevated in comparison with the control groups. The circulating IL-17 in the serum of the NP-ANPA group was elevated compared to the sham surgery group. In addition, there was a positive correlation among the expression levels of CCL20, CCR6 and IL-17. The results suggest a potential mechanism for the recruitment of IL-17-producing cells to degenerated intervertebral discs via a CCL20/CCR6 system in vivo.