Disc Herniation Model Research Articles

Sequential Activation of AMPA Receptors and Glial Cells in a Pain Model of Lumbar Spine Disc Herniation.

ObjectiveTo investigate the glial cell and AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor activity after surgery for disc herniation pain model.MethodsIn total, 83 Sprague-Dawley rats were randomly assigned to the following groups: control (n=16), sham-operated (n=4), rats for pain behavior evaluation (n=3), nucleus pulposus-exposed groups for AMPA receptors (n=30), and glial cell (n=30). The rats were tested for mechanical allodynia; immunohistochemical staining for AMPA receptors (GluA1 and GluA2) and glial cells (OX-42 and glial fibrillary acid protein [GFAP]) in the spinal dorsal horn was performed on postoperative days 3, 7, and 14.ResultsMechanical withdrawal thresholds decreased after surgery, and this effect was maintained for up to 14 days. Immunohistochemical expression of GluA1 and GluA2 in the spinal dorsal horn had increased quantitatively on postoperative days 3 and 7 (p<0.05) to levels similar to that of the controls on postoperative day 14. Moreover, immunohistochemical expression of OX-42 and GFAP showed similar changes to AMPA receptors after surgery. Although the activity of AMPA receptors and glial cells achieved normalcy, the mechanical withdrawal threshold of the hind paw remained decreased 38 days after surgery.ConclusionThe rat model of lumbar disc herniation showed increased expression of AMPA receptor and glial cell activity in the spinal dorsal horn 3 and 7 days after surgery, which deceased to control levels at 14 days. The AMPA receptors and glial cell activations showed similar patterns after disc herniation surgery.

Lipoxin A4 Inhibits NLRP3 Inflammasome Activation in Rats With Non-compressive Disc Herniation Through the JNK1/Beclin-1/PI3KC3 Pathway.

BackgroundNon-compressive disc herniation is induced by an inflammatory response from the nucleus pulposus tissue and nerve roots. Lipoxins (LXs) are important endogenous anti-inflammatory mediators in the body, helping to inhibit neutrophil recruitment and stimulate autophagy in monocytes and macrophages. Here, we investigated the molecular mechanisms underlying the effects of exogenous lipoxin administration on rats with non-compressive disc herniation.MethodA non-compressive disc herniation model was established in rats. Fifty rats were randomly divided into: sham group, model group, PI3K inhibitor (LY294002) group, lipoxin A4 group (LXA4), and PI3K inhibitor and lipoxin A4 group (LY294002 + LXA4). Similar groupings were established for rat spinal neurons. Changes in the mechanical pain threshold and thermal pain threshold were monitored at different times. The expression of proinflammatory and anti-inflammatory mediators was assessed by ELISA, while immunohistochemistry was employed to measure the expression levels of NLRP3 and p-JNK1. The expression levels of autophagy-related proteins were measured by western blot.ResultsIn vivo, the pain threshold was markedly decreased in the model group at each time point examined compared with that in sham group. LY294002 treatment further reduced the pain threshold. After LXA4 injection, the pain threshold was significantly increased, and the effect of LY294002 was significantly weakened (p < 0.05). The levels of proinflammatory cytokines were increased in rats with non-compressive disc herniation, and these levels were further increased by LY294002 treatment (p < 0.05). However, treatment with LXA4 significantly reduced the levels of these proinflammatory cytokines in the model group (p < 0.05). The opposite effect was observed for anti-inflammatory mediators. The expression of NLRP3 was largely increased in the model group compared with that in the sham group (p < 0.05). Treatment with LY294002 also increased the NLRP3 expression level, while the administration of LXA4 elicited the opposite effect. Furthermore, western blot analysis showed that the expression of autophagy-related proteins was greatly decreased in the model group, whereas it was significantly increased in the LXA4 group (p < 0.05). The in vitro results were consistent with the outcomes observed in vivo.ConclusionsThese data suggested that LXA4 inhibited NLRP3 activation in rats with non-compressive disc herniation by regulating the JNK1/beclin-1/PI3KC3 pathway.

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.

SFKs/p38 Pathway is Involved in Radicular Pain by Promoting Spinal Expression of Pro-Inflammatory Cytokines in a Rat Model of Lumbar Disc Herniation.

A controlled, randomized, animal study. The aim of this study was to investigate the role of src-family kinases/p38 pathway in a rat model of lumbar disc herniation (LDH). LDH always generates radicular pain, and the mechanism remains unclear. We have reported that spinal src-family kinases (SFKs) may be involved in the process, but the downstream mechanism needs further investigation. LDH was induced by implantation of autologous nucleus pulposus (NP), harvest from the tail, in lumbar 4/5 spinal nerve roots of rat. Von Frey filaments and radiant heat tests were performed to determine mechanical and thermal pain threshold respectively. Basso, Beattie, and Bresnahan (BBB) scale was assessed to test the locomotor function. The protein level of p-SFKs, t-SFKs, p-p38, t-p38 in spinal cord was examined by western blotting analysis. Cellular location of p-p38 was determined by immunochemistry staining. Spinal tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6 levels were detected by enzyme-linked immunosorbent assay (ELISA). Rats with NP implantation showed persistent ipsilateral mechanical allodynia and thermal hyperalgesia, which manifested as obvious decrease of paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). BBB scale indicated the locomotor function of hindpaws in rats with NP implantation kept intact. Western blotting and immunohistochemistry staining revealed that phosphorylated SFKs (p-SFKs) and phosphorylated p38 MAPK (p-p38) were sequentially upregulated in ipsilateral spinal dorsal horn, but not in contralateral side of rats with NP. Intrathecal delivery of SFKs inhibitor reduced spinal p-p38 expression. Both SFKs and p38 inhibitors alleviated pain behaviors in a dose-responsive manner without disturbing locomotor function and reduced spinal expression of TNF-α, IL-1β, and IL-6 in rats with NP. Spinal SFKs contribute to radicular pain by activation of p38 MAPK and increasing pro-inflammatory cytokines expression in rats with NP implantation. Targeting SFKs/p38 pathway may be helpful for alleviating radicular pain. N/A.

Comorbidity of Pain and Depression in a Lumbar Disc Herniation Model: Biochemical Alterations and the Effects of Fluoxetine.

Summary of Background Data: Depression is one of the most common comorbidities in patients with chronic low back pain. However, the mechanisms of depression in chronic low back pain patients and the effect of antidepressants on the comorbidity of pain and depression need to be further explored. The establishment of the appropriate animal models and of more effective therapies is critical for this comorbidity. Lumbar disc herniation (LDH) is the most common disease that causes low back pain. The current study examined whether an LDH model shows behavioral and biochemical alterations that are in accordance with the characteristics of the comorbidity of pain and depression and tested the effect of fluoxetine (FLX) on these measures.Objective: The current study examined whether an LDH model showed the behavioral and biochemical alterations that were in accordance with the characteristics of the comorbidity of pain and depression and tested the effect of FLX on these measures.Methods: The LDH animal model was generated by the implantation of the autologous nucleus pulposus on the left L5 nerve root just proximal to the dorsal root ganglion in Wistar rats. Pain intensity was evaluated by mechanical allodynia and thermal hyperalgesia, and changes in depressive behavior were examined by the taste preference and forced swim tests. Hippocampal serotonin (5-HT) levels were measured by liquid chromatography-mass spectrometry, and tumor necrosis factor-α (TNF-α) mRNA was quantified using real-time reverse transcriptase PCR.Results: LDH resulted in chronic pain, which further induced depressive behavior that persisted for 6 weeks after surgery. There were decreased 5-HT concentrations and upregulated TNF-α mRNA levels that were accompanied by behavioral changes. FLX treatment improved depressive behavior and moderately alleviated pain through increased 5-HT concentrations, and inhibited TNF-α mRNA expression.Conclusions: In summary, our studies provide initial evidence that the LDH chronic pain model might serve as a model of the comorbidity of low back pain and depression. The finding that FLX improved depressive behavior and pain through normalized 5-HT concentrations and TNF-α mRNA expression establishes the initial mechanism of the comorbidity of pain and depression.

AMP-Activated Protein Kinase Activation in Dorsal Root Ganglion Suppresses mTOR/p70S6K Signaling and Alleviates Painful Radiculopathies in Lumbar Disc Herniation Rat Model.

Animal experiment: a rat model of lumbar disc herniation (LDH) induced painful radiculopathies. To investigate the role and mechanism of AMP-activated protein kinase (AMPK) in dorsal root ganglia (DRG) neurons in LDH-induced painful radiculopathies. Overactivation of multiple pain signals in DRG neurons triggered by LDH is crucial to the development of radicular pain. AMPK is recognized as a cellular energy sensor, as well as a pain sensation modulator, but its function in LDH-induced pain hypersensitivity remains largely unknown. The LDH rat model was established by autologous nucleus pulposus transplantation into the right lumbar 5 (L5) nerve root. At different time points after AMPK agonist metformin (250 mg/kg/d) or mammalian target of rapamycin (mTOR) inhibitor rapamycin (5 mg/kg) intraperitoneal administration, thermal and mechanical sensitivity were evaluated by measuring paw withdrawal latency (PWL) and 50% paw withdrawal thresholds (PWT). The levels of AMPK, mTOR, and p70S6K phosphorylation were determined by Western blot. We also investigated the proportion of p-AMPK positive neurons in the right L5 DRG neurons using immunofluorescence. LDH evoked persistent thermal hyperalgesia and mechanical allodynia on the ipsilateral paw, as indicated by the decreased PWL and 50% PWT. These pain hypersensitive behaviors were accompanied with significant inhibition of AMPK and activation of mTOR in the associated DRG neurons. Pharmacological activation of AMPK in the DRG neurons not only suppressed mTOR/p70S6K signaling, but also alleviated LDH-induced pain hypersensitive behaviors. We provide a molecular mechanism for the activation of pain signals based on AMPK-mTOR axis, as well as an intervention strategy by targeting AMPK-mTOR axis in LDH-induced painful radiculopathies. N/A.

Composite biomaterial repair strategy to restore biomechanical function and reduce herniation risk in an ex vivo large animal model of intervertebral disc herniation with varying injury severity.

Back pain commonly arises from intervertebral disc (IVD) damage including annulus fibrosus (AF) defects and nucleus pulposus (NP) loss. Poor IVD healing motivates developing tissue engineering repair strategies. This study evaluated a composite injectable IVD biomaterial repair strategy using carboxymethylcellulose-methylcellulose (CMC-MC) and genipin-crosslinked fibrin (FibGen) that mimic NP and AF properties, respectively. Bovine ex vivo caudal IVDs were evaluated in cyclic compression-tension, torsion, and compression-to-failure tests to determine IVD biomechanical properties, height loss, and herniation risk following experimentally-induced severe herniation injury and discectomy (4 mm biopsy defect with 20% NP removed). FibGen with and without CMC-MC had failure strength similar to discectomy injury suggesting no increased risk compared to surgical procedures, yet no biomaterials improved axial or torsional biomechanical properties suggesting they were incapable of adequately restoring AF tension. FibGen had the largest failure strength and was further evaluated in additional discectomy injury models with varying AF defect types (2 mm biopsy, 4 mm cruciate, 4 mm biopsy) and NP removal volume (0%, 20%). All simulated discectomy defects significantly compromised failure strength and biomechanical properties. The 0% NP removal group had mean values of axial biomechanical properties closer to intact levels than defects with 20% NP removed but they were not statistically different and 0% NP removal also decreased failure strength. FibGen with and without CMC-MC failed at super-physiological stress levels above simulated discectomy suggesting repair with these tissue engineered biomaterials may perform better than discectomy alone, although restored biomechanical function may require additional healing with the potential application of these biomaterials as sealants and cell/drug delivery carriers.

Temporal and Spatial Changes of μ-Opioid Receptors in the Brain, Spinal Cord and Dorsal Root Ganglion in a Rat Lumbar Disc Herniation Model

Controlled, interventional, animal study. To investigate the spatial and temporal changes of μ-opioid receptor (MOR) expression in a rat lumbar disc herniation (LDH) model. MORs widely express in the peripheral and central nervous systems, and opioid drugs produce an analgesic effect through their activation. However, the efficacy of opioid drugs is sometimes inadequate in several pathological conditions of pain. MORs in the brain as well as the spinal cord (SC) and dorsal root ganglion (DRG) are thought to be associated with pain-related behavior, but the underlying mechanisms are not completely understood. In all, 91 adult female Sprague-Dawley rats were used. Autologous nucleus pulposus (NP) was applied onto the left L5 DRG in the NP group rats. Rats were divided into two surgical groups, the NP and the sham group. The von Frey test of left hind paw was performed before surgery, and 2, 7, 14, 21 and 28 days after surgery. Immunohistochemistry and immunoblotting in the DRG, SC, Caudate putamen, nucleus accumbens (NAc) and periaqueductal grey matter were performed before surgery, and 2, 7, 14, 21 and 28 days after surgery. The thresholds in the NP group were significantly lower than those in the sham group from day 2 onwards. At days 7 and 14, MOR expression in the injured-side SC and DRG were significantly lower than those in the sham group. At day 21, MOR in the NAc was significantly decreased compared to that in the sham group. Changes of MOR expression in the NAc, SC and DRG were associated with pain-related behavior. This result might show the underling pathogenesis of the resistance to MOR agonists in the patient with LDH. N/A.

Puerarin alleviate radicular pain from lumbar disc herniation by inhibiting ERK-dependent spinal microglia activation

Lumbar disc herniation is a common cause of radicular pain, but the mechanism remains ambiguous and the treatment stays unsatisfied. Many studies revealed a traditional Chinese medicine puerarin may moderate chronic pain from diabetes and nerve injury. Thus far, the role and mechanism of puerarin in radicular pain is still unknown. In this study, by using a rat model of lumbar disc herniation, which was induced by autologous nucleus pulposus (NP) implantation, the analgesic effect of puerarin on radicular pain was tested. Puerarin was delivered intraperitoneally form 1 h before surgery, and once daily for 7 days. The results demonstrated that NP implantation induced long-lasting pain, characterized by decrease of paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) in ipsilateral hindpaws, as long as day 20 after surgery. Spinal phosphorylated extracellular signal-regulated kinase (p-ERK) was up-regulated from day 5 to day 20 after surgery in ipsilateral but not contralateral side, and p-ERK was mainly co-localized with microglia. Puerarin decreased p-ERK expression from day 7 to day 20 after surgery. Puerarin or ERK inhibitor PD98059 alleviated pain behaviors, decreased expression of microglia marker ionized calcium-binding adaptor molecule 1 (Iba-1) in rats with NP implantation. The results suggested puerarin may alleviate radicular pain by inhibiting ERK-dependent or accompanied spinal microglia activation.

Effect of exogenous resolvin D2 on radicular pain in a rat model of non-compressive lumbar intervertebral disc herniation

Objective To evaluate the effect of exogenous resolvin D2 on radicular pain in a rat model of non-compressive lumbar intervertebral disc herniation. Methods Thirty-six clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 230-270 g, were divided into 3 groups (n=12 each) using a random number table method: sham operation group (group S), radicular pain induced by non-compressive lumbar intervertebral disc herniation group (group P) and exogenous resolvin D2 group (group R). The right L5 dorsal root ganglions were covered by autologous nucleus pulposus tissues to establish the model of non-compressive lumbar disc herniation in P and R groups.The corresponding surgical site was only exposed in group S. The corresponding drugs were intrathecally injected within 3 days after establishing the model, phosphate buffer solution 10 μl was injected in group P, 1 ng/μl resolvin D2 solution 10 μl was injected in group R, and normal saline 10 μl was given for pipe washing after administration in the three groups.The mechanical paw withdrawal threshold (MWT) was measured on 1 day before establishing the model and 1-7 days after establishing the model.The spinal dorsal horns of lumbar enlargement segments were removed on day 7 after establishing the model for determination of the expression of glial fibrillary acidic protein (GFAP) (by Western blot) and co-expression of G-protein-coupled receptor 18 with GFAP (by double-label immunofluorescence). Results Compared with group S, the MWT was significantly decreased at 1-7 days after establishing the model, and the expression of GFAP was up-regulated in group P (P<0.05). Compared with group P, the MWT was significantly increased at 3-7 days after establishing the model, and the expression of GFAP was down-regulated in group R (P<0.05). G-protein-coupled receptor 18 was co-expressed with GFAP. Conclusion Exogenous resolvin D2 can reduce radicular pain in a rat model of non-compressive lumbar intervertebral disc herniation, and the mechanism is related to inhibiting activation of astrocytes in the spinal dorsal horns. Key words: Docosahexaenoic acids; Neuralgia; Intervertebral disc displacement; Glial fibrillary acidic protein

CDMP1 overexpression mediates inflammatory cytokine‑induced apoptosis via inhibiting the Wnt/β‑Catenin pathway in rat dorsal root ganglia neurons.

Cartilage-derived morphogenetic protein-1 (CDMP1) is a polypeptide growth factor with specific cartilage inducibility, which is predominantly expressed in the developmental long bone cartilage core and in the pre-cartilage matrix in the embryonic stage. The aim of the present study was to investigate the roles and the mechanisms of CDMP1 overexpression on the apoptosis of rat dorsal root ganglia (DRG) neurons that were induced by inflammatory cytokines. Cell counting Kit-8 assay, flow cytometry and TdT-mediated dUTP nick-end labeling assay were performed to examine cell viability and apoptosis. ELISA, hematoxylin and eosin staining and immunohistochemistry assays were performed to examine the levels of several factors in DRG tissues. Western blot analysis and reverse transcription-quantitative polymerase chain reaction assays were used to determine the mRNA and protein expression levels, respectively. The results demonstrated that CDMP1 expression was downregulated, while inflammatory cytokine expression was upregulated in DRG tissues derived from lumbar disc herniation (LDH) model rats. In addition, DRG cells from LDH rats exhibited increased apoptosis compared with control rats. CDMP1 overexpression enhanced the cell viability of inflammatory cytokine-induced DRG cells, and suppressed the apoptosis of inflammatory cytokine-induced DRG cells via regulating the expression levels of Caspase-3/8/9, BCL2 apoptosis regulator, and BCL2 associated X. Furthermore, CDMP1 overexpression was demonstrated to affect the Wnt/β-Catenin pathway in the inflammatory cytokine-induced DRG cells. In conclusion, the present findings suggested that CDMP1 overexpression mediated inflammatory cytokine-induced apoptosis via Wnt/β-Catenin signaling in rat DRG cells.

Measurement of mechanical withdrawal thresholds and gait analysis using the CatWalk method in a nucleus pulposus-applied rodent model

BackgroundThere are some previous reports of gait analysis using a rodent pain model. Applying the CatWalk method, objective measurements of pain-related behavior could be evaluated, but this method has not been investigated using the nucleus pulposus (NP) applied model, which was developed as a model of lumber disc herniation. We aimed to measure mechanical withdrawal thresholds and analyze gait patterns using the CatWalk method for the evaluation of the pain-related behavior caused by NP application.MethodsTwenty-four nine-week-old female Sprague-Dawley rats were randomly divided into two experimental groups, the NP group (n = 12), in which autologous NP from the tail was applied to the left L5 dorsal root ganglion, and the sham-operated group (n = 12). Measurements of mechanical withdrawal thresholds were performed using von Frey filaments touching the left footpads, and gait analysis was performed using the CatWalk method. These experiments were conducted 1 day before surgery and 7, 14, 21, and 28 days after surgery. Data were statistically analyzed using the Wilcoxon rank-sum test.ResultsThe NP group showed significantly lower withdrawal thresholds than the sham group at days 14 and 21. Stand (duration of contact of a paw with the glass plate) was significantly higher in the NP group at days 7 and 14, whereas step cycle (duration between two consecutive initial contacts of the same paw) and duty cycle (stand as a percentage of step cycle) were the same at day 7. Long initial dual stance (duration of ground contact for both hind paws simultaneously, but the first one in a step cycle of a target hind paw) of the right hind paw was measured at days 7 and 14. The left hind paw per right hind paw ratio of the stand index (speed at which the paw loses contact with the glass plate) and mean intensity (mean intensity of the complete paw) changed at day 7 or 14. Phase dispersion (parameter describing the temporal relationship between placement of two paws) of the hind paws decreased at day 7.ConclusionsRats with applied NP showed a decreased withdrawal threshold and abnormal gait. The differences in gait parameters between the NP and sham groups were observed at an earlier time point than the withdrawal thresholds. Gait analysis could be an effective method for understanding pain caused by applied NP.

A more realistic disc herniation model incorporating compression, flexion and facet-constrained shear: a mechanical and microstructural analysis. Part II: high rate or 'surprise' loading.

Part I of this study explored mechanisms of disc failure in a complex posture incorporating physiological amounts of flexion and shear at a loading rate considerably lower than likely to occur in a typical in vivo manual handling situation. Given the strain-rate-dependent mechanical properties of the heavily hydrated disc, loading rate will likely influence the mechanisms of disc failure. Part II investigates the mechanisms of failure in healthy discs subjected to surprise-rate compression while held in the same complex posture. 37 motion segments from 13 healthy mature ovine lumbar spines were compressed in a complex posture intended to simulate the situation arising when bending and twisting while lifting a heavy object at a displacement rate of 400mm/min. Seven of the 37 samples reached the predetermined displacement prior to a reduction in load and were classified as early stage failures, providing insight to initial areas of disc disruption. Both groups of damaged discs were then analysed microstructurally using light microscopy. The average failure load under high rate complex loading was 6.96kN (STD 1.48kN), significantly lower statistically than for low rate complex loading [8.42kN (STD 1.22kN)]. Also, unlike simple flexion or low rate complex loading, direct radial ruptures and non-continuous mid-wall tearing in the posterior and posterolateral regions were commonly accompanied by disruption extending to the lateral and anterior disc. This study has again shown that multiple modes of damage are common when compressing a segment in a complex posture, and the load bearing ability, already less than in a neutral or flexed posture, is further compromised with high rate complex loading.

A more realistic disc herniation model incorporating compression, flexion and facet-constrained shear: a mechanical and microstructural analysis. Part I: Low rate loading.

To date, the mechanisms of disc failure have been explored at a microstructural level in relatively simple postures. However, in vivo the disc is known to be subjected to complex loading in compression, bending and shear, and the influence of these factors on the mechanisms of disc failure is yet to be described at a microstructural level. The purpose of this study was to provide a microstructural analysis of the mechanisms of failure in healthy discs subjected to compression while held in a complex posture incorporating physiological amounts of flexion and facet-constrained shear. 30 motion segments from 10 healthy mature ovine lumbar spines were compressed in a complex posture intended to simulate the situation arising when bending and twisting while lifting a heavy object, and at a displacement rate of 40mm/min. Nine of the 30 samples reached the predetermined displacement prior to a reduction in load and were classified as early-stage failures, providing insight into initial areas of disc disruption. Both groups of damaged discs were then analysed microstructurally using light microscopy. Complex postures significantly reduced the load required to cause disc failure than earlier described for flexed postures [8.42kN (STD 1.22kN) compared to 9.69kN (STD 2.56kN)] and resulted in a very different failure morphology to that observed in either simple flexion or direct compression, involving infiltration of nucleus material in a circuitous path to the annular periphery. The complex posture as used in this study significantly reduced the load required to cause disc failure, providing further evidence that asymmetric postures while lifting should be avoided if possible.

Bay11-7082 attenuates neuropathic pain via inhibition of nuclear factor-kappa B and nucleotide-binding domain-like receptor protein 3 inflammasome activation in dorsal root ganglions in a rat model of lumbar disc herniation.

Lumbar disc herniation (LDH) is an important cause of radiculopathy, but the underlying mechanisms are incompletely understood. Many studies suggested that local inflammation, rather than mechanical compression, results in radiculopathy induced by LDH. On the molecular and cellular level, nuclear factor-kappa B (NF-κB) and nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome have been implicated in the regulation of neuroinflammation formation and progression. In this study, the autologous nucleus pulposus (NP) was implanted in the left L5 dorsal root ganglion (DRG) to mimic LDH in rats. We investigated the expression of NF-κB and the components of NLRP3 inflammasome in the DRG neurons in rats. Western blotting and immunofluorescence for the related molecules, including NLRP3, apoptosis-associated speck-like protein containing caspase-1 activator domain (ASC), caspase-1, interleukin (IL)-1β, IL-18, IκBα, p-IκBα, p65, p-p65, and calcitonin gene-related peptide (CGRP) were examined. In the NP-treated group, the activations of NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 in DRG neurons in rats were elevated at 1 day after surgery, and the peak occurred at 7 days. Treatment with Bay11-7082, an inhibitor of the actions of IKK-β, was able to inhibit expression and activation of the molecules (NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65) and relieve the pain in rats. Our study shows that NF-κB and NLRP3 inflammasome are involved in the maintenance of NP-induced pain, and that Bay11-7082 could alleviate mechanical allodynia and thermal hyperalgesia by inhibiting NF-κB and NLRP3 inflammasome activation.

Changes in Neuroglial Activity in Multiple Spinal Segments after Caudal Epidural Pulsed Radiofrequency in a Rat Model of Lumbar Disc Herniation

Background: Herniated lumbar discs can induce sciatica by mechanical compression and/ or chemical irritation. It was recently reported that neuroglial cellular activity after pulsed radiofrequency (PRF) application to a single dorsal root ganglion (DRG) attenuated neuroglial activity at the corresponding spinal dorsal horn. Recently, caudal epidural PRF has been used to manage neuropathic pain, but evidence of molecular changes after the administration of caudal epidural PRF to attenuate neuropathic pain is lacking, and it has not been determined whether caudal epidural PRF affects neuroglial activity at different spinal levels. Objectives: Using immunohistochemical methods in a rat model of lumbar disc herniation, the authors investigated the effects of caudal epidural PRF administration on pain-related behavior, on the activations of microglia and astrocytes in spinal cord, and on the expressions of calcitonin gene-related peptide (CGRP) and Transient receptor potential vanilloid 1(TRPV1) in the DRG at the L3, L4, L5, L6, and S1 levels. Study Design: Controlled animal trial. Setting: University hospital laboratory. Methods: Forty-five Sprague-Dawley rats were randomly assigned to a sham-operated group (n = 10) or a nucleus pulposus (NP)-exposed group (n = 35). Rats in the NP-exposed group were further subdivided into a NP-exposed with sham stimulation group (the NP-nonPRF group; n = 13) or a NP exposed with caudal epidural PRF stimulation group (the NP-PRF group; n = 22). Pulsed radiofrequency was administered on postoperative day 10 (POD 10) by placing an electrode in the caudal epidural space through the sacral hiatus and administering 5 Hz of PRF current for 600 seconds (maximum tip temperature 42°C). Rats were tested for mechanical allodynia on POD 10 and on days 7 and 14 after caudal epidural PRF administration (post-PRF). At 14 days post-PRF, sections of the spinal cord from L3, L4, L5, L6, and S1 were immunostained for ionized calciumbinding adapter molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP), and DRGs from the same levels were immunostained for CGRP and TRPV1. Results: Mechanical withdrawal thresholds increased at 7 days post-PRF (P = 0.04), and the immunohistochemical expression of Iba1 in the L5 spinal dorsal horn and of CGRP in the L5 DRG were quantitatively reduced (P &lt; 0.001) at 14 days post-PRF. Furthermore, the upregulations of Iba1 at L3, L4, L6, and S1 dorsal horns and CGRP at L6 DRG were also attenuated by caudal epidural PRF (P &lt; 0.001). Limitation: We examined molecular changes only in ipsilateral lumbar regions and at 14 days post-PRF. Conclusion: Caudal epidural PRF reduced mechanical allodynia and downregulated microglia activity and CGRP expression at the lumbar disc herniated level and in adjacent lumbar spinal levels in a rat model of lumbar disc herniation. Key words: Caudal, pulsed radiofrequency, multisegmental, lumbar disc herniation, microglia, calcitonin gene-related peptide

Protective effect of cannabidiol on hydrogen peroxide‑induced apoptosis, inflammation and oxidative stress in nucleus pulposus cells.

Cannabidiol, a major component of marijuana, protects nerves, and exerts antispasmodic, anti-inflammatory and anti‑anxiety effects. In the current study, the protective effect of cannabidiol was observed to prevent hydrogen peroxide (H2O2)‑induced apoptosis, inflammation and oxidative stress in nucleus pulposus cells. Nucleus pulposus cells were isolated from rats and cultured invitro, and H2O2 was used to construct the nucleus pulposus cell model. Cell viability of the nucleus pulposus cells was assessed using a 3‑(4,5-dimethylthiazol-2-yl)-2,5‑diphenyltetrazolium bromide assay. The ratio of apoptotic cells, and caspase‑3 or cyclooxygenase‑2 (COX‑2) mRNA expression was analyzed by annexinV‑fluorescein isothiocyanate/propidium‑iodide staining and reverse transcription‑quantitative polymerase chain reaction, respectively. The quantities of interleukin (IL)‑1β and interleukin‑6 were measured using a series of assay kits. B-cell lymphoma2 (Bcl‑2) and inducible nitric oxide synthase (iNOS) protein expression levels were analyzed using western blotting. The present study identified that cannabidiol enhanced cell viability and reduced apoptosis in H2O2‑treated nucleus pulposus cells invitro using a lumbar disc herniation (LDH) model. In addition, cannabidiol reduced caspase‑3 gene expression and augmented the Bcl‑2 protein expression levels in the nucleus pulposus cells following H2O2 exposure. Pre‑treatment with cannabidiol suppressed the promotion of COX‑2, iNOS, IL‑1β and IL‑6 expression in the nucleus pulposus cells following H2O2 exposure. Taken together, these results suggest that cannabidiol potentially exerts its protective effect on LDH via the suppression of anti‑apoptosis, anti‑inflammation and anti‑oxidative activities in nucleus pulposus cells.

The Effect of GCSB-5 a New Herbal Medicine on Changes in Pain Behavior and Neuroglial Activation in a Rat Model of Lumbar Disc Herniation.

ObjectiveLumbar disc herniation can induce sciatica by mechanical compression and/or chemical irritation. The aim of this study was to compare the effects of GCSB-5 (Shinbaro®) and NSAIDs on pain-related behavior and on the expressions of microglia, astrocytes, CGRP, TRPV1, IL-6, and CX3CL1 in a rat model of lumbar disc herniation.Methods112 male Sprague-Dawley rats underwent implantation of nucleus pulposus to a dorsal root ganglion (DRG). Rats were divided into five groups as follows; a saline group (the vehicle control group) (n=27), a 10 mg/kg aceclofenac group (the aceclofenac group) (n=22), and 100, 300 or 600 mg/kg GCSB-5 groups (the GCSB-5 100, 300, or 600 groups) (n=21 for each group). Rats were tested for mechanical allodynia at 3 days after surgery and at 1 day, 3 days, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days after treatment commencement. Immunohistochemical staining of microglia (Iba1), astrocytes (GFAP), CGRP, and TRPV1, and PCR for IL-6 and CX3CL1 were performed on spinal dorsal horns and DRGs at 56 days after medication commencement.ResultsAfter 56 days of GCSB-5 300 administration, mechanical withdrawal thresholds were significantly increased (p<0.05), and immunohisto-chemical expressions of Iba1, GFAP, CGRP, and TRPV1 were reduced than other groups, but this difference was not statistically significant.ConclusionThese results indicate GCSB-5 reduces mechanical allodynia and downregulates neuroglial activity and the expressions of CGRP and TRPV1 in the spinal segments of a rat model of lumbar disc herniation.

Hyperexcitability and sensitization of sodium channels of dorsal root ganglion neurons in a rat model of lumber disc herniation.

Low back pain and sciatica are the most common symptoms of patients with lumbar disc herniation (LDH). The pathophysiology of lumbocrural pain and sciatica is not fully understood. The aim of the present study was to define the membrane properties and activities of voltage-gated sodium channels of dorsal root ganglion (DRG) neurons in a rat model of LDH. LDH was established by transplantation of autologous nucleus pulposus (NP) to lumbar 5 and 6 spinal nerves (L5-L6 DRG) of adult male rats. Mechanical paw withdrawal threshold (PWT) and thermal paw withdrawal latency (PWL) were measured 1 day before and through 35 days after transplantation of NP. Changes in expression of VGSCs were determined by western blotting. L5-L6 DRGs neurons innervating the hindpaw were labeled with DiI and acutely dissociated for measuring excitability and sodium channel currents under whole-cell patch clamp configurations. NP transplantation significantly reduced the PWT and PWL in association with a significant reduction in rheobase and an increase in numbers of action potentials evoked by 2X and 3X rheobase current stimulation. Voltage-gated sodium current density was significantly enhanced in L5-L6 DRG neurons from LDH rats. The inactivation curve showed a leftward shift in LDH rats while activation curve did not significantly alter. However, NP transplantation remarkably enhanced expression of NaV1.7 and NaV1.8 in L5-L6 DRGs but not in T10-12 DRGs. These data suggest that NP application produces pain-related behavior and potentiates sodium current density of DRG neurons, which is most likely mediated by enhanced expression of NaV1.7 and NaV1.8.

Lipoxin A4 attenuates radicular pain possibly by inhibiting spinal ERK, JNK and NF-κB/p65 and cytokine signals, but not p38, in a rat model of non-compressive lumbar disc herniation

Inflammatory response induced by protrused nucleus pulposus (NP) has been shown to play a crucial role in the process of radicular pain. Lipoxins represent a unique class of lipid mediators that have anti-inflammatory and pro-resolving action. The present study was undertaken to investigate if intrathecal lipoxin A4 (LXA4) could alleviate mechanical allodynia in the rat models of application of NP to the L5 dorsal root ganglion (DRG). Non-compressive models of application of NP to L5 DRG were established and intrathecal catheterization for drug administration was performed in rats. Daily intrathecal injection of vehicle or LXA4 (10ng or 100ng) was performed for three successive days post-operation. Mechanical thresholds were tested and the ipsilateral lumbar (L4–L6) segment of spinal dorsal horns were removed for the determination of tumor necrosis factor-α (TNF-α), IL-1β, transforming growth factor-β1 (TGF-β1) and IL-10 expression and NF-κB/p65, extracellular signal-regulated kinase (ERK), C-Jun N-terminal kinase (JNK) and P38 expression. Application of NP to DRG in rats induced mechanical allodynia, increased the expression of pro-inflammatory factors (TNF-α and IL-1β), NF-κB/p65, the phosphorylated-ERK (p-ERK), -JNK (p-JNK) and -P38 (p-p38) and decreased the expression of anti-inflammatory cytokines (TGF-β1 and IL-10) in the ipsilateral lumbar (L4–L6) segment of spinal dorsal horns. Intrathecal injection of LXA4 alleviated the development of neuropathic pain, inhibited the upregulation of pro-inflammatory cytokines (TNF-α and IL-1β), upregulated the expression of anti-inflammatory cytokines (TGF-β1 and IL-10) and attenuated the activation of NF-κB/p65, p-ERK, p-JNK, but not p-p38, in a dose-dependent manner.In this study, we have demonstrated that LXA4 potently alleviate radicular pain in a rat model of non-compressive lumbar disc herniation. The anti-inflammatory and pro-resolution properties of LXA4 have shown a great promise for the management of radicular pain caused by intervertebral disc herniation.