Mechanical Allodynia Threshold Research Articles

Quercetin, Main Active Ingredient of Moutan Cortex, Alleviates Chronic Orofacial Pain via Block of Voltage-Gated Sodium Channel.

Chronic orofacial pain (COP) therapy is challenging, as current medical treatments are extremely lacking. Moutan Cortex (MC) is a traditional Chinese medicine herb widely used for chronic inflammatory diseases. However, the mechanism behind MC in COP therapy has not been well-established. The purpose of this study was to identify the active ingredients of MC and their specific underlying mechanisms in COP treatment. In this study, the main active ingredients and compound-target network of MC in COP therapy were identified through network pharmacology and bioinformatics analysis. Adult male Sprague-Dawley rats received oral mucosa lipopolysaccharide (LPS) injection to induce COP. Pain behaviors were evaluated by orofacial mechanical nociceptive assessment after intraganglionar injection. In vitro inflammatory cytokines in LPS-pretreated human periodontal ligament stem cells (hPDLSCs) and rat primary cultural trigeminal ganglion (TG) neurons were quantified by real-time quantitative polymerase chain reaction (RT-qPCR). Schrödinger software was used to verify the molecular docking of quercetin and critical targets. Whole-cell recording electrophysiology was used to evaluate the effect of quercetin on voltage-gated sodium (Na v ) channel in rat TG neurons. The assembled compound-target network consisted of 4 compounds and 46 targets. As 1 of the active components of MC correlated with most related targets, quercetin alleviated mechanical allodynia in LPS-induced rat model of COP (mechanical allodynia threshold median [interquartile range (IQR) 0.5 hours after drug administration: vehicle 1.3 [0.6-2.0] g vs quercetin 7.0 [6.0-8.5] g, P = .002). Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that immune response and membrane functions play essential roles in MC-COP therapy. Five of the related targets were identified as core targets by protein-protein interaction analysis. Quercetin exerted an analgesic effect, possibly through blocking Na v channel in TG sensory neurons (peak current density median [IQR]: LPS -850.2 [-983.6 to -660.7] mV vs LPS + quercetin -589.6 [-711.0 to -147.8] mV, P = .006) while downregulating the expression level of proinflammatory cytokines-FOS (normalized messenger RNA [mRNA] level mean ± standard error of mean [SEM]: LPS [2. 22 ± 0.33] vs LPS + quercetin [1. 33 ± 0.14], P = .034) and TNF-α (normalized mRNA level mean ± SEM: LPS [8. 93 ± 0.78] vs LPS + quercetin [3. 77 ± 0.49], P < .0001). Identifying Na v as the molecular target of quercetin clarifies the analgesic mechanism of MC, and provides ideas for the development of novel selective and efficient chronic pain relievers.

Exploring gene signatures and regulatory networks in a rat model of sciatica: implications and validation in neuropathic pain.

Sciatica (neuropathic pain [NP]) is a common disease characterized by pain from radiation along the sciatic nerve. The aim of this study was to study the genes associated with chronic systolic injury of sciatic nerve (SCN-CCI) in rats by RNA-Seq technique, and to explore their potential as therapeutic targets. Sciatic nerve rat model was obtained by ligation of sciatic nerve and divided into two groups: SCN-CCI group and Sham group. Behavioral assessments were performed to evaluate pain sensitivity, following which their spinal cord dorsal horn were resected and RNA sequencing was conducted to identify differentially expressed genes (DEGs). Bioinformatics and functional enrichment analysis was performed to identify promising DEGs and their related biological processes and pathways associated with SCN-CCI. PPI network analysis and hub gene identification were conducted. QRT-PCR, western blot, ELISA, and immunofluorescence staining were performed on rat models to validate the expression of these hub genes and investigate related proteins and inflammatory markers. The SCN-CCI rat model was successfully obtained, exhibiting increased pain sensitivity compared to the Sham group, as indicated by decreased mechanical allodynia thresholds, thermal latencies, and increased paw withdrawals. RNA-Seq analysis identified 117 DEGs in the SCN-CCI rat model, involved in various biological processes and pathways related to sciatica. PPI network analysis revealed hub genes, including Ly6g6e, which exhibited significant differential expression. QRT-PCR and Western blot analysis confirmed the expression patterns of these hub genes. Pain behavior assessment demonstrated reduced pain thresholds and increased paw flinching responses in the SCN-CCI group. Furthermore, the SCN-CCI group showed upregulated expression of Ly6g6e, increased protein levels of Ly6g6e, CGRP, and NGF, as well as elevated levels of IL-1β, MCP-1, and IL-6, and microglial cell activation in the spinal dorsal horn. ELISA results confirmed the increased levels of IL-1β, MCP-1, and IL-6 in the spinal dorsal horn. These comprehensive findings provide valuable insights into the SCN-CCI rat model, DEGs associated with sciatica, hub genes (Ly6g6e as promising targets), pain behavior changes and molecular alterations.

Maternal immune activation alters placental histone-3 lysine-9 tri-methylation, offspring sensorimotor processing, and hypothalamic transposable element expression in a sex-specific manner

Animal models of maternal immune activation (MIA) are central to identifying the biological mechanisms that underly the association between prenatal infection and neuropsychiatric disorder susceptibility. Many studies, however, have limited their scope to protein coding genes and their role in mediating this inherent risk, while much less attention has been directed towards exploring the roles of the epigenome and transposable elements (TEs). In Experiment 1, we demonstrate the ability of MIA to alter the chromatin landscape of the placenta. We induced MIA by injecting 200 μg/kg (i.p.) of lipopolysaccharide (LPS) on gestational day 15 in Sprague-Dawley rats. We found a sex-specific rearrangement of heterochromatin 24-h after exposure to MIA, as evidenced by an increase in histone-3 lysine-9 trimethylation (H3K9me3). In Experiment 2, MIA was associated with long-term sensorimotor processing deficits as indicated by reduced prepulse inhibition (PPI) of the acoustic startle reflex in adult male and female offspring and an increased mechanical allodynia threshold in males. Analyses of gene expression within the hypothalamus-chosen for its involvement in the sex-specific pathogenesis of schizophrenia and the stress response-revealed significantly higher levels of the stress-sensitive genes Gr and Fkbp5. Deleterious TE expression is often a hallmark of neuropsychiatric disease and we found sex-specific increases in the expression of several TEs including IAP, B2 SINE, and LINE-1 ORF1. The data from this study warrant the future consideration of chromatin stability and TEs as part of the mechanism that drives MIA-associated changes in the brain and behavior.

Blood-nerve barrier disruption and coagulation system activation induced by mechanical compression injury participate in the peripheral sensitization of trigeminal neuralgia.

The aim of this study was to investigate the effect and possible mechanisms of the blood-nerve barrier (BNB) and the coagulation-anticoagulation system in modulating the mechanical allodynia in a trigeminal neuralgia (TN) rat model induced by chronic compression of the trigeminal root entry zone (TREZ). Von Frey filaments were applied to determine the orofacial mechanical allodynia threshold. The BNB permeability was evaluated by Evans blue extravasation test. Immunohistochemical staining and laser confocal microscopy were used to measure the length of the depletion zones of the nodes of Ranvier in the TREZ, the diameter of nerve fibers and the length of the nodal gap. The transcriptional levels of prothrombin and endogenous thrombin inhibitor protease nexin-1 (PN-1) in the TREZ of TN rats were assessed by RT-qPCR. A Western blotting assay was performed to detect the expression of paranodal proteins neurofascin-155 (NF155) and neurofascin-125 (NF125) in the TREZ. The spatiotemporal expression pattern of thrombin activated receptor (i.e. protease activated receptor 1, PAR1) in TREZ were defined by immunostaining and immunoblotting assays. PAR1 receptor inhibitors SCH79797 were administrated to TN rats to analyze the effect of thrombin-PAR1 on orofacial hyperalgesia. A compression injury of a rat's TREZ successfully induced TN-like behavior and was accompanied by the destruction of the permeability of the BNB and the promotion of prothrombin and thrombin inhibitor protease nexin-1 (PN-1) expression. The expression of the paranodal proteins neurofascin-155 (NF155) and neurofascin-125 (NF125) was increased, while the nodal gap length of the nodes of Ranvier was widened and the length of node-depleted zones was shortened. Moreover, the expression of PAR1 within the TREZ was upregulated at an early stage of TN, and administration of the PAR1 antagonist SCH79797 effectively ameliorated orofacial mechanical allodynia. A compression injury of the TREZ increased the permeability of the BNB and induced disturbances in the local coagulation-anticoagulation system, concomitant with the structural changes in the nodes of Ranvier, thrombin-PAR1 may play a critical role in modulating orofacial mechanical hyperalgesia in a TN rat model.

Hyperpolarization-activated cyclic nucleotide-gated 2 contributes to electroacupuncture analgesia on lumbar disc herniation-induced radicular pain through activation of microglia in spinal dorsal horn.

To explore the mechanisms of dorsal root ganglia and spinal microglia cascade cross in electroacupuncture (EA) analgesia in the treatment of lumbar disc herniation. A rat model of lumbar disc herniation (LDH) was established, EA was administered at Huantiao (GB30) acupoint 30 min once a day, for 3 d. Before and after modeling, and after EA, mechanical allodynia thresholds were detected. Hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) in dorsal root ganglia was detected by quantitative polymerase chain reaction (qPCR) and Western blot. C-X3-C motif chemokine ligand 1 (CX3CL1) and activity of microglia in spinal cord was observed separately qPCR and immunofluorescence staining. The mechanical allodynia threshold of the right planta of model rats was significantly reduced ( < 0.01), EA increased the mechanical pain threshold of rats ( < 0.01), and decreased HCN2 mRNA, and protein expression, reduced the expression of CX3CL1 and the activation of microglia. ZD7288 (a blocker of HCN channel) reduced the analgesic effect of EA from 1.83 ± 0.84 to 0.74 ± 0.20 ( < 0.05), and the expression of CX3CL1 in the spinal cord decreased from 0.52 ± 0.11 to 0.15 ± 0.05 ( < 0.01). EA analgesia on the radicular pain of LDH is definite. EA reduced the expression of HCN2 channel in the dorsal root ganglion, thereby decreasing the noxious stimulation entered to microglia in spinal dorsal horn. Our work supports EA is an effective treatment for radicular pain of LDH.

The anti-nociceptive effect of BPC-157 on the incisional pain model in rats.

BackgroundThe pentadecapeptide BPC-157 has been shown to have anti-inflammatory and wound healing effects on multiple target tissues and organs. Peptides have potent anti-inflammatory effects on periodontal tissues in rats with periodontitis. Few studies have investigated the effect of BPC-157 on pain after dental procedures or oral surgeries. The purpose of the present study was to investigate the antinociceptive effects of BPC-157 on postoperative incisional pain in rats.MethodsSprague–Dawley rats were randomly divided into five groups: control (saline with the same volume), BPC10 (10 µg/kg of BPC-157), BPC20 (20 µg/kg of BPC-157), BPC40 (40 µg/kg of BPC-157), and morphine (5 mg/kg of morphine). A 1-cm longitudinal incision was made through the skin, fascia, and muscle of the plantar aspect of the hind paw in isoflurane-anesthetised rats. Withdrawal responses were measured using von Frey filaments at 0, 2, 6 h and 4, 7 d after incision. The formalin test was also performed to differentiate its anti-nociceptive effect from an inflammatory reaction or central sensitization. Pain behavior was quantified periodically in phases 1 and 2 by counting the number of flinches in the ipsilateral paw after injection with 30 µL of 5% formalin.ResultsThe threshold of mechanical allodynia was significantly increased in the BPC10, BPC20, BPC40 and morphine groups compared with that in the control group at 2 h. These increasing thresholds then returned to the levels of the control group. The BPC-157 group showed a much higher threshold at 4 days after incision than the control group. The thresholds of the BPC groups, except the morphine group, were normalized 7 days after incision.The flinching numbers of the BPC10, BPC20, BPC40 and morphine groups were significantly decreased in phase 1, but there was no decrease in the BPC-157 groups except the morphine group in phase 2.ConclusionsBPC-157 was effective only for a short period after incision. It was also effective during phase 1 but not during phase 2, as determined by the formalin test. BPC-157 might have a short antinociceptive effect, even though it has anti-inflammatory and wound healing effects.

Increased Anxiety-Like Behavior in the Acute Phase of a Preclinical Model of Periodontal Disease.

Periodontal disease (PD) is an infectious-inflammatory oral disease that is highly prevalent among adolescence and adulthood and can lead to chronic orofacial pain and be associated with anxiety, stress and depression. This study aimed to identify anxiety-like behaviors in the ligature-induced murine preclinical model of PD in different phases of the disease (i.e., acute vs. chronic). Also, we investigated orofacial mechanical allodynia thresholds and superficial cortical plasticity along the orofacial motor cortex in both disease phases. To this aim, 25 male Wistar rats were randomly allocated in acute (14 days) or chronic (28 days) ligature-induced-PD groups and further divided into active-PD or sham-PD. Anxiety-like behavior was evaluated using the elevated plus maze, mechanical allodynia assessed using the von Frey filaments test and superficial motor cortex mapping was performed with electrical transdural stimulation. We observed increased anxiety-like behavior in active-PD animals in the acute phase, characterized by decreased number of entries into the open arm extremities [t(1,7) = 2.42, p = 0.04], and reduced time spent in the open arms [t(1,7) = 3.56, p = 0.01] and in the open arm extremities [t(1,7) = 2.75, p = 0.03]. There was also a reduction in the mechanical allodynia threshold in all active-PD animals [Acute: t(1,7) = 8.81, p < 0.001; Chronic: t(1,6) = 60.0, p < 0.001], that was positively correlated with anxiety-like behaviors in the acute group. No differences were observed in motor cortex mapping. Thus, our findings show the presence of anxiety-like behaviors in the acute phase of PD making this a suitable model to study the impact of anxiety in treatment response and treatment efficacy.

Effects of palmatine on BDNF/TrkB-mediated trigeminal neuralgia

Trigeminal neuralgia (TN), a sudden, needle-like pain in the distribution area of the trigeminal nerve, can seriously affect the physical and mental health of patients. In chronic pain conditions including TN, increased levels of brain-derived neurotrophic factor (BDNF) may enhance pain transmission. This study compares the effect of palmatine administration on the expression of BDNF and its receptor TrkB (tropomyosin receptor kinase B) in trigeminal ganglion cells of Sprague-Dawley rats in a sham versus TN model group. Within 14 days of surgery, the mechanical allodynia threshold of the TN group was significantly lower than that of the sham group, while the TN + palmatine group had a higher mechanical pain sensitivity threshold than the TN group (p < 0.05). Real-time quantitative PCR, immunohistochemistry, and immunofluorescence showed that BDNF and TrkB expression in the TN group was higher than that in the sham group, while palmatine treatment could reverse these changes. Western blotting showed that palmatine treatment could reduce the elevated phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) in TN rats. Thus, the BDNF/TrkB pathway may be involved in the pain transmission process of TN, and palmatine treatment may reduce pain transmission by inhibiting the BDNF/TrkB pathway and suppressing ERK1/2 phosphorylation.

Indomethacin attenuates mechanical allodynia during the organization but not the maintenance of the peripheral neuropathic pain induced by nervus ischiadicus chronic constriction injury.

The neurochemical mechanisms underlying neuropathic pain (NP) are related to peripheral and central sensitization caused by the release of inflammatory mediators in the peripheral damaged tissue and ectopic discharges from the injured nerve, leading to a hyperexcitable state of spinal dorsal horn neurons. The aim of this work was to clarify the role played by cyclooxygenase (COX) in the lesioned peripheral nerve in the development and maintenance of NP by evaluating at which moment the non-steroidal anti-inflammatory drug indomethacin, a non-selective COX inhibitor, attenuated mechanical allodynia after placing one loose ligature around the nervus ischiadicus, an adaptation of Bennett and Xie's model in rodents. NP was induced in male Wistar rats by subjecting them to chronic constriction injury (CCI) of the nervus ischiadicus, placing one loose ligature around the peripheral nerve, and a sham surgery (without CCI) was used as control. Indomethacin (2 mg/kg) or vehicle was intraperitoneally and acutely administered in each group of rats and at different time windows (1, 2, 4, 7, 14, 21, and 28 days) after the CCI or sham surgical procedures, followed by von Frey's test for 30 min. The data showed that indomethacin decreased the mechanical allodynia threshold of rats on the first, second, and fourth days after CCI (P<0.05). These findings suggested that inflammatory mechanisms are involved in the induction of NP and that COX-1 and COX-2 are involved in the induction but not in the maintenance of NP.

Evaluating a sensitive issue: reliability of a clinical evaluation for allodynia severity

Purpose/Aim: Allodynia is a common feature of neuropathic pain with few validated clinical evaluation options. We identified a need to estimate the measurement properties of the standardised evaluation procedure for static mechanical allodynia severity popularised by the somatosensory rehabilitation of pain method, known as the rainbow pain scale. This study (www.clinicaltrials.gov. NCT02070367) undertook preliminary investigation of the inter-rater and test-retest reliability of the rainbow pain scale.Methods: Persons with pain in one upper extremity after Complex Regional Pain Syndrome, a peripheral nerve injury or a recent hand fracture were recruited for assessment of static mechanical allodynia threshold using calibrated monofilaments by two raters at baseline, and repeated assessment one week later.Results: Single measures estimates suggested inter-rater reliability was substantial for the rainbow pain scale [intra-class correlation coefficient = 0.78 (n = 31), p < 0.001]. Test-retest reliability was also excellent at with an intraclass correlation coefficient of 0.87 [n = 28, p < 0.001]. However, confidence intervals suggest the true values could be more moderate, with lower bounds of the 95% confidence interval at 0.60 and 0.74, respectively.Conclusions: This pilot study has generated preliminary support for the inter-rater and test-retest reliability of the rainbow pain scale. Future studies should seek to increase confidence in estimates of reliability, and estimate validity and responsiveness to change in persons with somatosensory disorders.

Interleukin-6 Serum Levels Are Elevated in Individuals with Degenerative Cervical Myelopathy and Are Correlated with Symptom Severity.

BackgroundFew studies have investigated systemic inflammation levels in degenerative cervical myelopathy (DCM). This study evaluated the concentration of inflammatory cytokines in DCM patients and assessed whether they can predict symptom severity.Material/MethodsA total of 40 consecutive DCM patients and 10 healthy volunteers were included in this study. Concentrations of interleukin (IL)-1β, IL-6, interferon-γ, and tumor necrosis factor-α were compared between DCM patients and normal controls. Spearman’s correlation coefficient was used to examine relationships of cytokines with age, body mass index (BMI), symptom duration, and symptom severity. A DCM compression rat model was established to examine the levels of inflammatory cytokines in serum and cerebrospinal fluid (CSF).ResultsSerum level of IL-6 is significantly higher in DCM patients compared with normal people (0.8±0.5 pg/ml vs. 0.5±0.3 pg/ml, P=0.036). Positive correlations were found between IL-6 levels with BMI (ρ=0.519; P=0.001) and symptom severity (ρ=−0.556, P<0.001). In DCM compression model rats, IL-6 was elevated in CSF (40.5±3.3 vs. 13.2±2.4 pg/ml, P<0.001) and serum (7.1±1.7 vs. 2.9±1.6 pg/ml, P<0.001) samples from rats in the compression operation group compared with the sham operation group. Infusion of IL-6 in rats receiving the sham operation also led to motor function damage and mechanical allodynia threshold decline.ConclusionsSerum IL-6 level was elevated in DCM patients and its concentration can predict symptom severity. Local infusion of IL-6 led to myelopathy symptoms in model rats, which suggests that anti-inflammation can effectively treat DCM.

A new central post-stroke pain rat model: autologous blood injected thalamic hemorrhage involved increased expression of P2X4 receptor

Stroke is the leading cause of disability and death in the world. Central post-stroke pain (CPSP), a central neuropathic pain syndrome occurring after cerebral stroke, is a serious problem. But on account of the lack of reliable animal models, the mechanisms underlying CPSP remains poorly understood. To better understand of the pathophysiological basis of CPSP, we developed and characterized a new rat model of CPSP. This model is based on a hemorrhagic stroke lesion with intra-thalamic autologous blood (ITAB) injection in the ventral posterolateral nucleus of the thalamus. Behavioral analysis demonstrated that the animals displayed a significant decrease in mechanical allodynia threshold. We found a significant increase in P2 × 4 receptor expression in microglia in thalamic peri-lesion tissues post-hemorrhage. The mechanical allodynia in rats with CPSP were reversed by blocking P2 × 4 receptors. A significant alleviation of mechanical allodynia was achieved following the administration of adrenergic antidepressants and antiepileptics. Meanwhile, we found a significant decrease in P2 × 4 receptor expression after treatment with these drugs. Taken together, our results suggest that targeting P2 × 4 receptor may be effective in the treatment of CPSP.

Intrathecal Injection of Dual Zipper Kinase shRNA Alleviating the Neuropathic Pain in a Chronic Constrictive Nerve Injury Model.

Dual leucine zipper kinase (DLK) is a member of mitogen-activated protein kinase kinase kinase (MAP3K) family mainly involved in neuronal degeneration. However, the role of DLK signaling in the neuropathic pain has not yet been fully determined. Chronic constrictive injury (CCI) was conducted by four 3-0 chromic gut ligatures loosely ligated around the sciatic nerve. Escalated DLK expression over the dorsal root ganglion was observed from one to four rings of CCI. Remarkable expression of DLK was observed in primary dorsal root ganglion cells culture subjected to electrical stimulation and attenuated by DLK short hairpin RNA (shRNA) treatment. Intrathecal injection of DLK shRNA attenuates the expression of DLK over the dorsal root ganglion and hippocampus neurons and increased the threshold of mechanical allodynia and decreased thermal hyperalgesia. In CatWalk gait analysis, significant decreases of print area, maximum contact maximum intensity, stand phase, single stance, and regular index by CCI were alleviated by the DLK shRNA administration. In conclusion, the expression of DLK was up-regulated in chronic constrictive injury and attenuated by the administration of DLK shRNA, which paralleled the improvement of neurobehavior of neuropathic pain. The modulation of DLK expression is a potential clinic treatment option for neuropathic pain.

Amitriptyline inhibits the MAPK/ERK and CREB pathways and proinflammatory cytokines through A3AR activation in rat neuropathic pain models.

BackgroundThe pain-relief properties of tricyclic antidepressants can be attributed to several actions. Recent observations suggest that adenosine is involved in the antinociceptive effect of amitriptyline. The A3 adenosine receptor (A3AR) is the only adenosine subtype overexpressed in inflammatory and cancer cells. This study was performed to investigate the role of A3AR in the anti-nociceptive effect of amitriptyline.MethodsSpinal nerve-ligated neuropathic pain was induced by ligating the L5 and L6 spinal nerves of male Sprague-Dawley rats. The neuropathic rats were randomly assigned to one of the following three groups (8 per group): a neuropathic pain with normal saline group, a neuropathic pain with amitriptyline group, and a neuropathic pain with amitriptyline and 3-ethyl-5-benzyl- 2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS) group. Amitriptyline or saline was administered intraperitoneally and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS-1191), an A3AR antagonist, was injected subcutaneously immediately before amitriptyline administration. The level of extracellular signal-regulated kinase P44/42 (ERK1/2), cyclic AMP response element-binding protein (CREB), and proinflammatory cytokines were assessed using immunoblotting or reverse-transciption polymerase chain reaction.ResultsAmitriptyline increased the mechanical withdrawal threshold of the neuropathic rats. The level of phospho-ERK1/2 and phospho-CREB proteins, and proinflammatory cytokines produced by spinal nerve ligation were significantly reduced by amitriptyline administration. However, the use of MRS-1191 before amitriptyline administration not only reduced the threshold of mechanical allodynia, but also increased the signaling protein and proinflammatory cytokine levels, which were reduced by amitriptyline.ConclusionsThe results of this study suggest that the anti-nociceptive effect of amitriptyline involves the suppression of ERK1/2 and CREB signaling proteins, and A3AR activation also affects the alleviation of the inflammatory response.

Increased Deacetylation of Proteins in Sensory Neurons by Sirtuin 1 Protein Overexpression Reverses T2D Peripheral Neuropathy

Sirtuin1 (SIRT1) protein uses NAD+ as a substrate to deacetylate transcription factors, cofactors, and histones to enhance mitochondrial function. We tested the hypothesis whether increased expression of SIRT1 protein in dorsal root ganglion (DRG) neurons would rescue mice from peripheral neuropathy induced by a high fat diet (HFD). Neuron-specific, doxycycline (DOX)-inducible, SIRT1 protein over expressing C57BL6 transgenic mouse (nSIRT1OE Tg) were generated. Expression of SIRT1 protein was observed in small to medium sized DRG neurons. nSIRT1OE was shut off by feeding, weaned Tg mice, with DOX in the diet for 12 weeks. The Tg mice were divided into 3 groups. Group # 1: nSIRT1OE Tg mice fed with standard diet (STD) plus DOX for 4 months; Group # 2: nSIRT1OE Tg mice fed with HFD plus DOX for 4 months; Group # 3: nSIRT1OE Tg mice fed with HFD plus DOX for 2 months until they develop neuropathy and then switched to HFD minus DOX for additional 2 months. Neuropathy was determined by mechanical allodynia thresholds (MAT) and nerve conduction velocity (NCV) at 0, 2 and 4 months. Intraepidermal nerve fiber density (IENFD) was measured at 4 months. MAT, NCV and IENFD were decreased in HFD-fed (Group # 2) mice compared to STD-fed mice (Group # 1) at 2 and 4 months. In Group # 3 mice, 2 months after turning on nSIRT1OE, we observed a reversal of mechanical allodynia, NCV and attenuation of IENFD (Group # 3 compared to Group # 2). Western blot of protein extracts from DRG neurons showed that HFD increased acetylation of proteins and SIRT1OE abolished the HFD-induced acetylation of proteins. The mitochondrial bioenergetics profile of cultured adult DRG neurons showed that hyperglycemia induced a decrease in the spare respiratory capacity in wild type (WT) DRG neurons. This was corrected in nSIRT1OE Tg DRG neurons. In type 2 diabetic neuropathy, altered acetylation of proteins and reduced mitochondrial function via a defective SIRT1 pathway may contribute to developing distal axonopathy. Disclosure K. Chandrasekaran: None. C. Ho: None. M. Salimian: None. P.H. Kumar: None. S. Konduru: None. J.W. Russell: None.

황금 추출물의 신경병증성 통증 유발 흰쥐에 미치는 영향

TRPA1 and TRPV1 are members of the TRP superfamily of structurally related, nonselective cation channels. TRPA1 and TRPV1 are often co-expressed in sensory neurons and play an important role in mechanical hyperalgesia and allodynia during neuropathic pain. Scutellariae Radix was reported to possess anti-inflammation properties and similar patterns of therapeutic action against different diseases. also Baicalin(a known principal constituent of Scutellaria Radix) was shown to down-regulate the mRNA expression levels of TRPV1. In this study, we observed the effects of Scutellariae Radix extract(SRE) in neuropathic pain induced SD rats via modulation of TRPV1 and TRPA1. Oral administration of a Scutellaria Radix extract(in doses of 300mg/㎏, SRE(300)) showed a meaningful increase in the withdrawal threshold of mechanical allodynia and showed a meaningful decrease in the expression of c-fos compared to the control group. SRE(100) and SRE(300) showed a meaningful decrease in the expression of TRPV1 level compared to the control group. These results suggest that Scutellariae Radix extract could decrease mechanical allodynia by down-regulate the TRPV1 on the model of neuropathic pain.

The effect of restraint stress on masseter mechanical hyperalgesia and the activity of neurons and astrocytes in the spinal trigeminal nucleus caudalis in rats

Objective: To investigate the effect of restraint stress on masseter mechanical hyperalgesia and the activity of neurons and astrocytes in the spinal trigeminal nucleus caudalis (Vc). Methods: The animals were randomly divided into the control group, 1-, 3-, 5-, 7-, 9-, 11-and 14-day stress groups, with 10 rats in each group. The body weight increase and behavior tests were used to testify the animal model. The mechanical sensitivity of masseter of the rat before and after the stress was measured with Von Frey filaments. Histological examinations were used to evaluate the expression of neuronal c-fos and astrocytic glial fibrillary acidic protein (GFAP). Results: Restraint stress resulted in remarkable mechanical allodynia in the masseter muscle. The head withdrawal threshold was significantly lower in the 7-, 9-, 11-and 14-day stress groups ([0.071±0.011], [0.059±0.020], [0.052±0.011], [0.033±0.011] N) than that in the control group ([0.120±0.025] N) (P<0.05). Compared to the control group, the rats in the 1-day stress group showed a significant increase of c-fos in neurons of the Vc and then declined to normal level after 1 week gradually. The GFAP expression in astrocytes of the Vc was significantly increased in the 7-, 9-, 11-and 14-day stress groups (4.3±1.0, 4.5±0.6, 4.6±0.5, 4.8±1.3) compared with the control group (2.0±0.8) (P<0.05). Conclusions: Chronic restraint stress could lower the threshold of mechanical allodynia in the masseter muscle and activate the neurons and astrocytes in Vc. The activation of neurons and astrocytes plays an important role in the masseter hyperalgesia induced by restraint stress in rats.

The Therapeutic Effect of Vitamin C in an Animal Model of Complex Regional Pain Syndrome Produced by Prolonged Hindpaw Ischemia-Reperfusion in Rats.

Objectives: It is known that increased free radicals from oxidative stress are one of the major causes of complex regional pain syndrome (CRPS). In this study, we tested the hypothesis that vitamin C has a dose-related treatment effect in a chronic post-ischemic pain (CPIP) model.Methods: A total of 49 male rats weighing 250 to 350 g were used. The 4 treatment groups were control (no medication), group 1.0 (administration of 1 mg/day for vitamin C for 5 days), group 2.5 (administration of 2.5 mg/day vitamin C for 5 days), and group 7.5 (administration of 7.5 mg/day vitamin C for 5 days). The 50% mechanical withdrawal threshold and total blood antioxidant status (TAS) were measured before and after administration of vitamin C.Results: Twenty-eight CPIP model rats were generated from 49 rats. Seven rats were randomly allocated to each group. The 50% mechanical withdrawal threshold of group 2.5 (after the administration of vitamin C) was higher than that of the control group and group 1.0 (P < 0.05). At 1 day of the administration of vitamin C, the 50% mechanical withdrawal threshold of group 1.0 was higher than that of the control group and the blood levels of TAS in groups 2.5 and 7.5 were higher than that in control group (P < 0.05). Twelve days after the administration of vitamin C, the blood levels of TAS in groups 2.5 and 7.5 were lower than that of the control group (P < 0.05).Discussion: The administration of a proper dose of vitamin C can reduce oxidative stress, increase antioxidants, and recover the threshold for mechanical allodynia in the CPIP model.

Intravenous Administration of Substance P Attenuates Mechanical Allodynia Following Nerve Injury by Regulating Neuropathic Pain-Related Factors.

This study aimed to investigate the analgesic effect of substance P (SP) in an animal model of neuropathic pain. An experimental model of neuropathic pain, the chronic constriction injury (CCI) model, was established using ICR mice. An intravenous (i.v.) injection of SP (1 nmole/kg) was administered to the mice to examine the analgesic effects of systemic SP on neuropathic pain. Behavioral testing and immunostaining was performed following treatment of the CCI model with SP. SP attenuated mechanical allodynia in a time-dependent manner, beginning at 1 h following administration, peaking at 1 day post-injection, and decaying by 3 days post-injection. The second injection of SP also increased the threshold of mechanical allodynia, with the effects peaking on day 1 and decaying by day 3. A reduction in phospho-ERK and glial fibrillary acidic protein (GFAP) accompanied the attenuation of mechanical allodynia. We have shown for the first time that i.v. administration of substance P attenuated mechanical allodynia in the maintenance phase of neuropathic pain using von Frey’s test, and simultaneously reduced levels of phospho-ERK and GFAP, which are representative biochemical markers of neuropathic pain. Importantly, glial cells in the dorsal horn of the spinal cord (L4–L5) of SP-treated CCI mice, expressed the anti-inflammatory cytokine, IL-10, which was not seen in vehicle saline-treated mice. Thus, i.v. administration of substance P may be beneficial for improving the treatment of patients with neuropathic pain, since it decreases the activity of nociceptive factors and increases the expression of anti-nociceptive factors.

Feasibility of Human Amniotic Fluid Derived Stem Cells in Alleviation of Neuropathic Pain in Chronic Constrictive Injury Nerve Model.

PurposeThe neurobehavior of neuropathic pain by chronic constriction injury (CCI) of sciatic nerve is very similar to that in humans, and it is accompanied by a profound local inflammation response. In this study, we assess the potentiality of human amniotic fluid derived mesenchymal stem cells (hAFMSCs) for alleviating the neuropathic pain in a chronic constriction nerve injury model.Methods and MethodsThis neuropathic pain animal model was conducted by four 3–0 chromic gut ligatures loosely ligated around the left sciatic nerve in Sprague—Dawley rats. The intravenous administration of hAFMSCs with 5x105 cells was conducted for three consecutive days.ResultsThe expression IL-1β, TNF-α and synaptophysin in dorsal root ganglion cell culture was remarkably attenuated when co-cultured with hAFMSCs. The significant decrease of PGP 9.5 in the skin after CCI was restored by administration of hAFMSCs. Remarkably increased expression of CD 68 and TNF-α and decreased S-100 and neurofilament expression in injured nerve were rescued by hAFMSCs administration. Increases in synaptophysin and TNF-α over the dorsal root ganglion were attenuated by hAFMSCs. Significant expression of TNF-α and OX-42 over the dorsal spinal cord was substantially attenuated by hAFMSCs. The increased amplitude of sensory evoked potential as well as expression of synaptophysin and TNF-α expression was alleviated by hAFMSCs. Human AFMSCs significantly improved the threshold of mechanical allodynia and thermal hyperalgesia as well as various parameters of CatWalk XT gait analysis.ConclusionHuman AFMSCs administration could alleviate the neuropathic pain demonstrated in histomorphological alteration and neurobehavior possibly through the modulation of the inflammatory response.