Epitranscriptomic analysis of m6A methylome in rats after lumbosacral nerve root avulsion.

Background: N6-methyladenosine (m6A) is the most prevalent modification in mRNAs but its role in lumbosacral nerve root avulsion (LSRA) remains elusive. Materials & methods:Mettl3 expression and global m6A level were detected by qPCR, western blot and immunostaining. Altered m6A-tagged transcript profiles were revealed by methylated RNA immunoprecipitation and RNA sequencing. Results:Mettl3 and global m6A level were upregulated in spinal cord tissues of LSRA rats. In all, 1087 m6A peaks were differentially modified by m6A, of which 654 were upregulated and 433 downregulated. Biological functions of these transcripts and the hypermethylated or hypomethylated transcripts were also identified. Conclusion: Our findings revealed a profound function of m6A modification in LSRA, which provides new insights into its pathogenesis.

[Effect of wheat-grain moxibustion on the expression of Beclin-1/GRP78 in spinal dorsal horn in rats with cervical spondylotic radiculopathy].

OBJECTIVE: To observe the effect of wheat-grain moxibustion at "Dazhui" (GV 14) on the expressions of Beclin-1 and GRP78 in spinal dorsal horn in rats with cervical spondylotic radiculopathy (CSR), and to explore the possible analgesic mechanism of wheat-grain moxibustion for CSR. METHODS: A total of 48 SD rats were randomly divided into a sham operation group, a model group, a wheat-grain moxibustion group and a wheat-grain moxibustion+3-MA group, 12 rats in each group. The CSR model was prepared by spinal cord insertion method. Three days after modeling, the rats in the model group were intraperitoneally injected with 1 mL of 0.9% sodium chloride solution; the rats in the wheat-grain moxibustion group were treated with wheat-grain moxibustion at "Dazhui" (GV 14, 6 cones per time) on the basis of the model group; the rats in the wheat-grain moxibustion+3-MA group were intraperitoneally injected with 3-MA solution and wheat-grain moxibustion at "Dazhui" (GV 14, 6 cones per time). The three groups were intervened for 7 days, once a day. The gait score and mechanical pain threshold were observed before treatment and 7 days into treatment; after the treatment, the expressions of mRNA and protein of Beclin-1 in spinal dorsal horn were detected by real-time fluorescence quantitative PCR and immunohistochemistry; the expression of GRP78 protein in spinal dorsal horn was detected by Western blot method; the autophagosomes and ultrastructure in spinal dorsal horn neurons were observed by electron microscope. RESULTS: After the treatment, compared with the sham operation group, in the model group, the gait score was increased and the mechanical pain threshold was decreased (P<0.01), and the expression of GRP78 protein in spinal dorsal horn was increased (P<0.01). Compared with the model group and the wheat-grain moxibustion+3-MA group, in the wheat-grain moxibustion group, the gait score was decreased and mechanical pain threshold was increased (P<0.01), and the expression of GRP78 protein in spinal dorsal horn was decreased, and the expressions of mRNA and protein of Beclin-1 were increased (P<0.01). Under electron microscope, the ultrastructure of spinal dorsal horn neurons in the wheat-grain moxibustion group was not significantly damaged, and its structure was basically close to normal, and the number of autophagosomes was more than the other three groups. CONCLUSION: Wheat-grain moxibustion at "Dazhui" (GV 14) has analgesic effect on CSR rats. The mechanism may be related to moderately up-regulate the expression of Beclin-1, enhance autophagy and reduce endoplasmic reticulum stress.

Direct moxibustion exerts an analgesic effect on cervical spondylotic radiculopathy by increasing autophagy via the Act A/Smads signaling pathway.

BACKGROUND: Direct moxibustion (DM) is reported to be useful for cervical spondylotic radiculopathy (CSR), but the analgesic mechanism remains unknown. Autophagy plays a protective role in neuronal apoptosis, Act A/Smads signaling pathway has been confirmed to be associated with the activation of autophagy. The study aimed to explore the effect of DM on autophagy in rats with CSR and the involvement of Act A/Smads signaling pathway. METHODS: Rats were randomly divided into Sham, CSR, CSR + DM, CSR + DM + 3-MA (PI3K inhibitor), and CSR + DM + SB (Act A inhibitor) group. Three days after establishment of CSR model with a fish line inserted under the axilla of the nerve roots, DM at Dazhui (GV14) was performed six times once for seven consecutive days. Western blot and immunofluorescence staining were used to observe the expression of the neuronal autophagy molecule LC3II/I, Atg7, and Act A/Smads signaling molecule Act A, p-Smad2, and p-Smad3. Bcl-2/Bax mRNA expression was measured by real time PCR. RESULTS: DM improved the pain threshold and motor function of CSR rats and promoted the expression of Act A, p-Smad2, p-Smad3, LC3II/I, and Atg7 in the entrapped-nerve root spinal dorsal horn. DM reduced the expression of Bax mRNA and decreased the number of apoptotic neurons. 3-MA and Act A inhibitor SB suppressed the expression of above-mentioned proteins and reduced the protective effect of DM on apoptotic neurons. CONCLUSION: DM exerts analgesic effects by regulating the autophagy to reduce cell apoptosis and repair nerve injury, and this feature may be related to the Act A/Smads signaling pathway.

[Effect of mild moxibustion on pain and expression of LC3 and Bax in spinal cord in cervical spondylotic radiculopathy rats].

OBJECTIVE: To observe the effect of mild moxibustion (Moxi) at "Dazhui" (GV14) on neuropathic pain, expression of autophagy and apoptosis factor LC3 and Bax proteins and mRNAs in the spinal cord tissue in rats with cervical spondylotic radiculopathy (CSR), so as to explore its underlying mechanism underlying relief of CSR-induced pain. METHODS: Forty rats (half male half female) were randomly divided into blank control, model, Moxi, Moxi+autophagy inhibitor 3-methyladenine (3-MA, Moxi+3-MA) groups, with 10 rats in each group. The CSR model was established by loose ligature of the local cervical nerve roots. Three days after modeling, mild Moxi was applied to GV14 for 10 min, once daily for 7 days. Rats of the Moxi+3-MA group received intraperitoneal injection of 3-MA(1 mL, 15 mg/kg+ saline) before Moxi, once daily for 7 consecutive days. Rats of the model and Moxi groups were also given normal saline (i.p., 1 mL), once daily for 7 days. The gait behavior score (1-3 points) was scaled according to the rats' pain reaction and foot paw contracture produced walking disorder and the mechanical pain threshold (MPT) was detected before and after the treatment. The expression of spinal cord LC3 and Bax proteins and mRNAs were detected by immunohistochemistry and quantitative RT-PCR, respectively. RESULTS: Compared with the blank control group, the gait disorder score, and percentage of Bax positive cells and expression of Bax mRNA were significantly increased (P<0.01, P<0.05), and MPT was markedly decreased in the model group (P<0.01). After the treatment, the gait disorder score, percentage of Bax positive cells and Bax mRNA expression were significantly down-regulated (P<0.01, P<0.05), while the MPT and percentage of LC3 positive cells and LC3 mRNA expression were considerably increased (P<0.01, P<0.05) in both Moxi and Moxi+3-MA groups. The therapeutic effects of mild Moxi were remarkably superior to those of Moxi+3-MA in downregulating gait disorder score, Bax positive cell percentage and Bax mRNA expression, and in up-regulating MPT, LC3 positive cell percentage and LC3 mRNA expression (P<0.05), suggesting a reduction of the function of mild Moxi after administration of 3-MA. CONCLUSION: Mild Moxi at GV14 can relieve neuropathic pain in CSR rats, which may be related to its functions in up-regulating LC3 autophagy, thereby inhibiting the expression of Bax pro-apoptotic protein in spinal cord to reduce apoptosis and to repair nerve injury.

Immediate inhibition of spinal secretory phospholipase A2 prevents the pain and elevated spinal neuronal hyperexcitability and neuroimmune regulatory genes that develop with nerve root compression.

Cervical nerve root injury induces a host of inflammatory mediators in the spinal cord that initiate and maintain neuronal hyperexcitability and pain. Secretory phospholipase A2 (sPLA2) is an enzyme that has been implicated as a mediator of pain onset and maintenance in inflammation and neural injury. Although sPLA2 modulates nociception and excitatory neuronal signaling in vitro, its effects on neuronal activity and central sensitization early after painful nerve root injury are unknown. This study investigated whether inhibiting spinal sPLA2 at the time of nerve root compression (NRC) modulates the pain, dorsal horn hyperexcitability, and spinal genes involved in glutamate signaling, nociception, and inflammation that are seen early after injury. Rats underwent a painful C7 NRC injury with immediate intrathecal administration of the sPLA2 inhibitor thioetheramide-phosphorlycholine. Additional groups underwent either injury alone or sham surgery. One day after injury, behavioral sensitivity, spinal neuronal excitability, and spinal cord gene expression for glutamate receptors (mGluR5 and NR1) and transporters (GLT1 and EAAC1), the neuropeptide substance P, and pro-inflammatory cytokines (TNFα, IL1α, and IL1ß) were assessed. Treatment with the sPLA2 inhibitor prevented mechanical allodynia, attenuated neuronal hyperexcitability in the spinal dorsal horn, restored the proportion of spinal neurons classified as wide dynamic range, and reduced genes for mGluR5, substance P, IL1α, and IL1ß to sham levels. These findings indicate spinal regulation of central sensitization after painful neuropathy and suggest that spinal sPLA2 is implicated in those early spinal mechanisms of neuronal excitability, perhaps via glutamate signaling, neurotransmitters, or inflammatory cascades.

[Effects of mild moxibustion on Beclin-1/Bcl-2 expression in spinal cord of rats with cervical spondylotic radiculopathy].

OBJECTIVE: To observe the effects of mild moxibustion on the expression of autophagy and apoptosis factors Beclin-1, Bcl-2 mRNA and protein in spinal cord (including nerve root tissues) of cervical spondylotic radiculopathy (CSR) rats, so as to explore the analgesic mechanism of mild moxibustion at "Dazhui" (GV14) on CSR. METHODS: SD rats were randomly divided into blank group, model group, mild moxibustion group and mild moxibustion+3-methyladenine(3-MA) group, with 10 rats in each group. CSR model was established by inserting the wire into the cervical nerve root. The rats in the blank group were only fed normally without any intervention.The rats in the mild moxibustion group and mild moxibustion+3-MA group were given mild moxibustion at GV14 for 10 min each time,and intraperitoneal injection of 1 mL 0.9% normal saline and 1 mL 3-MA(15 mg/kg)separately. Rats in the model group were given 0.9% normal saline every day. All the three interventions were started from the 3rd day after modeling for 7 days. The rat's behavioral reaction of gait was scored and the pain threshold of rat was measured with a pain analyzer; the expressions of Beclin-1 and Bcl-2 mRNA and protein in the spinal cord (including nerve root) were detected by fluorescence quantitative PCR and immunohistochemistry, separately. The autophagosome and ultrastructure of the spinal nerve root tissue were observed by transmission electron microscope. RESULTS: After modeling, the gait score was significantly increased (P<0.01) and the pain threshold significantly decreased (P<0.01) in the model group in comparison with the blank group. There was no statistical difference in Beclin-1 and Bcl-2 mRNA and protein expression between the blank and the model groups. After intervention, compared with the model group, the gait scores were significantly reduced (P<0.01), the pain threshold and the expressions of Beclin-1 and Bcl-2 mRNA and protein were significantly increased (P<0.01，P<0.05) in the mild moxibustion and mild moxibustion+3-MA groups. The improvement of the above indicators as more significant in the mild moxibustion group than that in the mild moxibustion+3-MA group (P<0.05). After modeling, the organelles in the spinal nerve root tissue cells of the model group were damaged and there were a small amount of autophagosomes. Compared with the model group, the ultrastructure of the spinal nerve root tissue cells in the mild moxibustion group were relatively complete, and the number of autophagosomes increased. CONCLUSION: Mild moxibustion at GV14 has a good analgesic effect on CSR rats, which may be related to its effects in up-regulation of Beclin-1 and Bcl-2 expressions and activation of autophagy and inhibition of apoptosis.

MicroRNA-204 silencing relieves pain of cervical spondylotic radiculopathy by targeting GDNF.

Cervical spondylosis may cause chronic neck pain, radiculopathy and/or myelopathy, and consequently results in severe brain damage. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for motoneurons. Accumulating microRNAs (miRNAs) have highlighted as critical regulators of GDNF signaling in the mediation of neuroinflammation and neuropathic pain. Hence, we performed this study to investigate the potential role of miR-204 in the neuropathic pain of cervical spondylotic radiculopathy (CSR) by targeting GDNF. A rat model of spinal cord compression (SCC) was established to stimulate a pathologic lesion. RT-qPCR and western blot assays characterized the downregulation of GDNF and the upregulation of miR-204 in spinal cord tissues of rats under the conditions of SCC. Moreover, miR-204 could directly target GDNF, as evidenced by dual-luciferase reporter gene assay. In order to elucidate the roles of miR-204 and GDNF in SCC-induced neuropathic pain, miR-204 sponge, GDNF, or shRNA against GDNF was introduced to the rats, followed by measurements for SCC-induced neuroinflammation and neuropathic pain. GDNF upregulation or miR-204 silencing was identified to reduce the spontaneous pain score, gait scores and cell apoptosis. Furthermore, GDNF upregulation or miR-204 silencing resulted in elevated amplitude of sensory-evoked potentials (SEPs), number of motoneurons, release of pro-inflammatory factors, TNF-α, and IL-1ß in addition to an increase in the anti-inflammatory factor BDNF. Taken together, upregulation of GDNF induced by miR-204 silencing confers protection against SCC-induced pain in rat models, suggesting a potential therapeutic target for CSR treatment.

The temporal pattern of brachial plexus root avulsion-induced lncRNA and mRNA expression prior to the motoneuron loss in the injured spinal cord segments.

The neuronal mechanisms underlying brachial plexus roots avulsion-induced motoneuron death are unknown. Our previous studies showed that the avulsion induced obvious temporal and spatial expression of both degenerative and regenerative genes in the injured spinal cord tissue. Therefore, we hypothesized that lncRNAs (responsible for epigenetic molecular mechanisms) are altered (resulting in altered gene expression patterns) at days 3 and 14 after avulsion. In the present microarray study, 121 lncRNAs (83 up/38 down) and 844 mRNAs (726 up/118 down) were differentially expressed (ipsilateral vs contralateral) after avulsion. We further used qRT-PCR as a validation tool to confirm the expression patterns of 5 lncRNAs and 5 mRNAs randomly selected from our microarray analysis data. The gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed to identify the critical biological processes and pathways. The noted downregulation of the AF128540 (which targets the nNOS gene) is consistent with the high expression of nNOS protein observed at day 14 post-avulsion. The downregulation of MRAK034299, whose target is the Adra1d gene, is consistent with the downregulation of Adra1d mRNA and protein at days 3 and 14 post avulsion. Immunofluorescence evaluation showed cytoplasmic translocation of ECEL1 after avulsion injury. Moreover, we also found that IL6 and Rac2 are the core genes at days 3 and 14 after unilateral brachial plexus roots avulsion, respectively. Overall, our present data suggest that the altered LncRNAs (avulsion-induced), via unknown epigenetic mechanisms, certainly contribute to the molecular mechanism underpinning motoneuron death or survival. Therefore, the avulsion-induced differentially expressed lncRNAs and mRNAs may offer potential diagnostic and therapeutic targets for BPRA.

Upregulation of Ih expressed in IB4-negative Aδ nociceptive DRG neurons contributes to mechanical hypersensitivity associated with cervical radiculopathic pain.

Cervical radiculopathy represents aberrant mechanical hypersensitivity. Primary sensory neuron's ability to sense mechanical force forms mechanotransduction. However, whether this property undergoes activity-dependent plastic changes and underlies mechanical hypersensitivity associated with cervical radiculopathic pain (CRP) is not clear. Here we show a new CRP model producing stable mechanical compression of dorsal root ganglion (DRG), which induces dramatic behavioral mechanical hypersensitivity. Amongst nociceptive DRG neurons, a mechanically sensitive neuron, isolectin B4 negative Aδ-type (IB4(-) Aδ) DRG neuron displays spontaneous activity with hyperexcitability after chronic compression of cervical DRGs. Focal mechanical stimulation on somata of IB4(-) Aδ neuron induces abnormal hypersensitivity. Upregulated HCN1 and HCN3 channels and increased Ih current on this subset of primary nociceptors underlies the spontaneous activity together with neuronal mechanical hypersensitivity, which further contributes to the behavioral mechanical hypersensitivity associated with CRP. This study sheds new light on the functional plasticity of a specific subset of nociceptive DRG neurons to mechanical stimulation and reveals a novel mechanism that could underlie the mechanical hypersensitivity associated with cervical radiculopathy.

Subjective health complaints in patients with lumbar radicular pain and disc herniation are associated with a sex - OPRM1 A118G polymorphism interaction: a prospective 1-year observational study.

BACKGROUND: Earlier observations show that development of persistent pain may be associated with the genetic variability in the gene encoding for the µ-opioid receptor 1, the OPRM1 A118G (rs1799971). The aim of this study was to investigate the association between OPRM1 genotype and subjective health complaints in patients with radicular pain and disc herniation. METHODS: A prospective, 1-year observational study was conducted at a hospital back clinic, including 118 Caucasian patients with lumbar radicular pain and MRI confirmed disc herniation. Single nucleotide polymorphism genotyping regarding the OPRM1 A118G was performed. The data of individuals with AA versus AG or GG were analysed separately by linear mixed models. The Subjective Health Complaints Inventory (0-81) including 27 common complaints experienced the previous month on a scale from not at all (0) to severe (3) was used as outcome. Pain, prior duration of leg pain, age, smoking status, and lumbar disc surgery were considered as covariates. RESULTS: In total 23 of 118 patients were carriers of the OPRM1 G-allele. All patients except female carriers of the G-allele reported a decrease in pain from baseline to 1 year. Female carriers of the G-allele reported significantly higher subjective health complaints score during the study time span than male carriers of the G-allele when controlling for pain and pain duration. CONCLUSION: The present data indicate that, when controlling for pain intensity and duration, subjective health complaints are associated with a sex - OPRM1 A118G polymorphism interaction in patients with radicular pain.

Genetic variability in the rat Aplec C-type lectin gene cluster regulates lymphocyte trafficking and motor neuron survival after traumatic nerve root injury.

BACKGROUND: C-type lectin (CLEC) receptors are important for initiating and shaping immune responses; however, their role in inflammatory reactions in the central nervous system after traumatic injuries is not known. The antigen-presenting lectin-like receptor gene complex (Aplec) contains a few CLEC genes, which differ genetically among inbred rat strains. It was originally thought to be a region that regulates susceptibility to autoimmune arthritis, autoimmune neuroinflammation and infection. METHODS: The inbred rat strains DA and PVG differ substantially in degree of spinal cord motor neuron death following ventral root avulsion (VRA), which is a reproducible model of localized nerve root injury. A large F2 (DAxPVG) intercross was bred and genotyped after which global expressional profiling was performed on spinal cords from F2 rats subjected to VRA. A congenic strain, Aplec, created by transferring a small PVG segment containing only seven genes, all C-type lectins, ontoDA background, was used for further experiments together with the parental strains. RESULTS: Global expressional profiling of F2 (DAxPVG) spinal cords after VRA and genome-wide eQTL mapping identified a strong cis-regulated difference in the expression of Clec4a3 (Dcir3), a C-type lectin gene that is a part of the Aplec cluster. Second, we demonstrate significantly improved motor neuron survival and also increased T-cell infiltration into the spinal cord of congenic rats carrying Aplec from PVG on DA background compared to the parental DA strain. In vitro studies demonstrate that the Aplec genes are expressed on microglia and upregulated upon inflammatory stimuli. However, there were no differences in expression of general microglial activation markers between Aplec and parental DA rats, suggesting that the Aplec genes are involved in the signaling events rather than the primary activation of microglia occurring upon nerve root injury. CONCLUSIONS: In summary, we demonstrate that a genetic variation in Aplec occurring among inbred strains regulates both survival of axotomized motor neurons and the degree of lymphocyte infiltration. These results demonstrate a hitherto unknown role for CLECs for intercellular communication that occurs after damage to the nervous system, which is relevant for neuronal survival.

Cytokine expression in the epidural space: a model of noncompressive disc herniation-induced inflammation.

STUDY DESIGN: Animal study. OBJECTIVE: Development of an animal model for the study of biochemical changes that occur in the epidural space after intervertebral disc herniation. SUMMARY OF BACKGROUND DATA: Although strong evidence for an inflammatory component exists, the biochemical processes underlying pain after disc herniation remain unknown. METHODS: Epidural lavage was performed in 48 rats after L5 dorsal root ganglion exposure at baseline and 3, 6, or 24 hours after placement of autologous nucleus pulposus (NP) (N = 15), saline (N = 15), or NP + an interferon-γ antibody (anti-IFN-γ; N = 18) directly onto the dorsal root ganglion. Multiplex assays quantifying interleukin (IL)-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, tumor necrosis factor α (TNF-α), IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were performed. NP (N = 7) was also analyzed for these cytokines by placing NP into saline and measuring the relative concentration. RESULTS: Cytokines measured low at baseline (0-100 pg/mL) in all groups. Compared with saline, NP application caused IL-6 elevation, peaking at T = 3 hours, that was prevented by anti-IFN-γ. NP induced elevation of TNF-α, peaking at T = 24 hours and was prevented by anti-IFN-γ. IFN-γ was elevated after NP at T = 3 hours and T = 24 hours. IL-1α was similar after saline versus NP. The concentrations of IL-1ß and IL-10 were elevated at T = 3 hours, 6 hours, and 24 hours in all groups without between-groups difference. The level of IL-4 peaked at T = 3 hours in the NP group and was different than saline and NP + anti-IFN-γ groups, but the time effect was insignificant. There was no change for GM-CSF. The concentration of cytokines measured in normal NP was less than 2 pg/mL for all cytokines except TNF-α. CONCLUSION: In this model of acute noncompressive disc herniation, NP caused the elevation of epidural IL-6, TNF-α, and IFN-γ--all attenuated by IFN-γ blockade. IL-1ß and IL-10 were both significantly elevated by saline alone and their response was not prevented by IFN-γ blockade. This model may prove useful for the study of the biochemical processes by which NP induces inflammation-induced nerve root irritation and radiculopathic pain.

The red wine polyphenol resveratrol shows promising potential for the treatment of nucleus pulposus-mediated pain in vitro and in vivo.

STUDY DESIGN: Descriptive and mechanistic investigation of the anti-inflammatory and anticatabolic effect of resveratrol in intervertebral discs (IVDs) in vitro and of the analgetic effect in vivo. OBJECTIVE: To determine whether resveratrol may be useful in treating nucleus pulposus (NP)-mediated pain. SUMMARY OF BACKGROUND DATA: Proinflammatory cytokines seem to be key mediators in the development of NP-mediated pain. Patients with discogenic or radiculopathic pain may substantially benefit from anti-inflammatory substances that could be used in a minimal-invasive treatment approach. Resveratrol, a polyphenolic phytoalexin found in red wine exhibits anti-inflammatory effects in various cell types and tissues, but no data exists so far with regards to the IVD in the context of low back and leg pain. METHODS: In part 1, the anti-inflammatory and anticatabolic effect of resveratrol was investigated in a cell culture model on interleukin 1ß (IL-1ß) prestimulated human IVD cells on the gene and protein expression level. In part 2, the molecular mechanisms underlying the effects observed upon resveratrol treatment were investigated (toll-like receptors, nuclear factor κB, sirtuin 1 (SIRT1), mitogen-activated protein (MAP) kinases p38/ERK/JNK). In part 3, the analgetic effects of resveratrol were investigated in vivo using a rodent model of radiculopathy and von Frey filament testing. All quantitative data were statistically evaluated either by Mann-Whitney U test or by one-way analysis of variance and Bonferroni post hoc testing (P < 0.05). RESULTS: In vitro, resveratrol exhibited an anti-inflammatory and anticatabolic effect on the messenger RNA and protein level for IL-6, IL-8, MMP1, MMP3 and MMP13. This effect does not seem to be mediated via the MAP kinase pathways (p38, ERK, JNK) or via the NF-κB/SIRT1 pathway, although toll-like receptor 2 was regulated to a minor extent. In vivo, resveratrol significantly reduced pain behavior triggered by application of NP tissue on the dorsal root ganglion for up to 14 days. CONCLUSION: Resveratrol was able to reduce levels of proinflammatory cytokines in vitro and showed analgetic potential in vivo. A decrease in proinflammatory cytokines may possibly be the underlying mechanism of pain reduction observed in vivo. Resveratrol seems to have considerable potential for the treatment of NP-mediated pain and may thus be an alternative to other currently discussed (biological) treatment options.

[Effect of the heat shock protein 27 on NOS expression of spinal cord anterior hornmotoneurons after brachial plexus avulsion].

AIM: To observe the effect of heat shock protein 27 (Hsp27) on nitric oxide synthase (NOS) of spinal cord anterior horn after brachial plexus roots avulsion. METHODS: Sixty male adult Wistar rats were divided into control and experiment groups at random. The experiment group subjected to heat shock under 45 degrees C for 15 min, and maintained under 42 degrees C for 20 min subsequently. After recovering 24 h under the room temperature, the nerves of brachial plexus were avulsion with microhemostatic forcep. In a span from 12 h to 7 d, these animals were killed at different time. But the control group only received the surgery of the nerve roots of brachial plexus avulsion. The freeze sections of spinal cord were stained by NADPH-d histochemistry, HSP27 immunohistochemical. RESULTS: (1) In experiment group, the motoneuron began to express NOS abundantly at 12 h after avulsion (A=0.13625). Then the NOS-positive neurons declined quickly, but in control group, the motoneuron began to express NOS at the 5th day after lesion. (2) Hsp27 begin to show the peak at 1 d in experiment and control groups, but the experiment group were more strong than the control group. CONCLUSION: Hsp27 inhibited NOS of motoneuron after avulsion and brought into full play the cytoprotection.

Cytokine mRNA expression in painful radiculopathy.

UNLABELLED: Inflammatory cytokines contribute to lumbar radiculopathy. Regulation of cytokines for transient cervical injuries, with or without longer-lasting inflammation, remains to be defined. The C7 root in the rat underwent compression (10gf), chromic gut suture exposure (chr), or their combination (10gf+chr). Ipsilateral C7 spinal cord and dorsal root ganglia (DRG) were harvested at 1 hour after injury for real-time PCR analysis of IL-1beta, IL-6, and TNF-alpha. Cytokine mRNA increased after all 3 injuries. TNF-alpha mRNA in the DRG was significantly increased over sham after 10gf+chr (P = .026). Spinal IL-1beta was significantly increased over sham after 10gf and 10gf+chr (P < .024); IL-6 was significantly increased after 10gf+chr (P < .024). In separate studies, the soluble TNF-alpha receptor was administered at injury and again at 6 hours in all injury paradigms. Allodynia was assessed and tissue samples were harvested for cytokine PCR. Allodynia significantly decreased with receptor administration for 10gf and 10gf+chr (P < .005). Treatment also significantly decreased IL-1beta and TNF-alpha mRNA in the DRG for 10gf+chr (P < .028) at day 1. Results indicate an acute, robust cytokine response in cervical nerve root injury with varying patterns, dependent on injury type, and that early increases in TNF-alpha mRNA in the DRG may drive pain-related signaling for transient cervical injuries. PERSPECTIVE: Inflammatory cytokine mRNA in the DRG and spinal cord are defined after painful cervical nerve root injury. Studies describe a role for TNF-alpha in mediating behavioral sensitivity and inflammatory cytokines in transient painful radiculopathy. Results outline an early response of inflammatory cytokine upregulation in cervical pain.

Gene expression profile of dorsal root ganglion in a lumbar radiculopathy model.

STUDY DESIGN: DNA array analysis of dorsal root ganglion (DRG) using a rat model with nerve root constriction. OBJECTIVE: To determine the molecular changes in the DRG adjacent to the injured nerve root in a lumbar radiculopathy model. SUMMARY OF BACKGROUND DATA: DNA array analysis in lumbar radiculopathy model has so far focused on the spinal dorsal horn. The molecular changes in the DRG adjacent to the injured nerve root in lumbar radiculopathy remain to be determined. METHODS: Bilateral L5 DRGs were removed from 12 Sprague-Dawley rats on days 2, 7, 14, and 21 after nerve root ligation and on day 7 from 3 rats with sham operation. The aRNAs from the DRGs with nerve root ligation were labeled with Cy5 dye and those from the opposite side DRG (control) were labeled with Cy3 dye, and then hybridized to a 7793-spot Panorama Micro Array. It was considered to be significantly upregulated, when an average expression ratio of Cy5 to Cy3 was 2 or more. Genes upregulated were classified into early phase group (upregulated on day 2), midphase group (upregulated on days 7 and 14), and continuous group (upregulated from day 2 to 21). Seventeen genes were subjected to validation analysis with real-time quantitative PCR. RESULTS: There were 16 upregulated genes in the early phase group, 56 genes in the midphase group, and 17 genes in the continuous group. Functional categorization revealed dominantly upregulated gene categories in each group; transcription/translation in the early phase group, enzyme/metabolism in the midphase group, and structure in the continuous group. Validation analysis of 17 genes demonstrated mean relative expression of 2.0 or more in all but 1 gene in the DRGs with nerve root ligation and none of them in the DRGs with sham operation. CONCLUSION: The genes identified in this study, especially those involved in pain signaling and inflammation, serve as potential targets for molecular-based therapy for lumbar radiculopathy.

Tumor necrosis factor-alpha in the nucleus pulposus mediates radicular pain, but not increase of inflammatory peptide, associated with nerve damage in mice.

STUDY DESIGN: Changes in behavior and the immunohistochemistry of dorsal root ganglion (DRG) neurons were examined using a mouse model of radicular pain. OBJECTIVE: To examine the effects of TNF-alpha in the nucleus pulposus (NP) on nerve roots. SUMMARY OF BACKGROUND DATA: Radicular pain is induced by mechanical compression and inflammation of nerve roots. Many authors have reported that following disc herniation, producing TNF-alpha plays a major role in neuropathic pain. Their findings suggest that TNF-alpha contained in the NP is significant in the development of pain and nerve root degeneration, but it has not been clearly demonstrated. METHODS: Wild-type NPs or TNF-KO NPs, which were harvested from C57BL/6 mice (wild-type NP) or TNF-knock-out mice (TNF-KO NP), were applied to the left sciatic nerves of 30 wild-type mice, and the nerves were pinched. Production of hind paw mechanical allodynia, activating transcription factor 3, and calcitonin gene- related peptide (CGRP) were assessed. RESULTS: Animals receiving a NP application demonstrated significant mechanical allodynia compared to the pinch-only and the control groups. The degree of mechanical allodynia was greater in the wild-type than in the TNF-KO group. The number of activating transcription factor 3 immunoreactive neurons was significantly higher in the wild-type than in the TNF-KO group. The number of CGRP-immunoreactive neurons was higher in the wild-type and TNF-KO than in the control groups. However, no significant difference in activity was observed between both CGRP positive groups. CONCLUSION: In this study TNF-alpha contained in the NP was important for the production of radicular pain accompanied by long-lasting degeneration of DRG neurons. However, other cytokines in the NP and nerve compression may also play important roles in pain transmission. In this model system, TNF-alpha in the NP appears to mediate pain, but not cause an increase in CGRP in the DRG neurons.